Alpinia zerumbet Extract Mitigates PCB 126-Induced Neurotoxicity and Locomotor Impairment in Adult Male Mice

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have mainly focused on the effect of Alpinia zerumbet extract to cure PCB 126-induced neurotoxi-2 city and locomotor impairment in adult male mice . In the previous communication the authors have tested vasodilator potential of the same extract. To extend the study further they have tested their neuroprotective effect which is interesting and  seems to be original.

1. The background of the abstract is confusing; simplify it. It is also advisable to write the abstract in the past tense.
2. In the introduction, it is difficult to understand the motive of the study. Rewrite the introduction part starting with neurotoxicity, its treatment with synthetic chemicals, their side effects and the remedy.
3. Supplement the manuscript with current UHPLC/ESI-QTOF-MS chromatograms and spectra (as supplementary data) rather than referencing prior work, ensuring self-contained transparency It seems to be a repletion of work.
4. Provide precise details for methodology  mentioning all the necessary conditions and concentrations to ensure reproducibility instead of simply giving reference..
5. Clarify the statistical significance in Figures to strengthen the interpretation of results.
6. Rewrite discussion part as indicated in the introduction.
7. Supplement references list by adding latest references.
8. the conclusions presented is not consistent with the evidence and arguments presented as further studies are needed to evaluate the mechanism of neuroprotective effect of the extract.
9. Write future prospective of the study.

File attached

Author Response

1. Reviewer comment: The background of the abstract is confusing; simplify it. It is also advisable to write the abstract in the past tense. 

   Authors’ response: As recommended by the reviewer, we rewrote the background of the abstract, highlighting the neurotoxic effect of PCB-126 and the neuroprotective potential of polyphenols, such as those present in our extract, which was the research objective of the study. We also modified the wording of the text so that it was in the past tense, except for the first line, which deals with information about the characteristics of polychlorinated biphenyls (Page 1, lines 22 and 23).

2. Reviewer comment: In the introduction, it is difficult to understand the motive of the study. Rewrite the introduction part starting with neurotoxicity, its treatment with synthetic chemicals, their side effects and the remedy. 

   Authors’ Response: As recommended by the reviewer, we have added information about the difficulties in treating neurotoxicity and neurodegeneration to the introduction's initial part to highlight the importance of studying new preventive and therapeutic strategies for these conditions induced by PCBs.

   Treating neurotoxicity is a significant challenge for pharmacology. Riluzole, for example, is a drug with potential use to treat neurodegeneration. However, its mechanism of action is related to blocking the release of glutamate to reduce excitotoxicity (Daverey and Agrawal. Neurosci Lett. 2020; 1: 738:135351). Therefore, it does not act directly on oxidative stress, which plays a key role in the effects of PCBs. Additionally, it causes adverse effects such as asthenia, nausea, and changes in liver function (Miller et al. Cochrane Database Syst Rev. 2012; 3: CD001447). In light of this explanation, the levels of human exposure to PCBs due to their persistence in the environment and bioaccumulation remain a growing concern, particularly considering the lack of effective treatments to mitigate their toxic effects, especially in terms of neurotoxicity and the associated systemic dysfunctions (Gupta et al. Drug Deliv. and Transl. Res. 2018, 8, 740–759) (Page 2, lines 75 to 83).

3. Reviewer comment: Supplement the manuscript with current UHPLC/ESI-QTOF-MS chromatograms and spectra (as supplementary data) rather than referencing prior work, ensuring self-contained transparency It seems to be a repletion of work. 

   Authors’ Response: We greatly appreciate your feedback and the importance you place on this point. Your input allows us to clarify our work and engage in further dialogue.

   The extract used in this study to investigate the neuroprotective effect was prepared from the same collection that originated the published work characterizing the chemical composition (Da Silva, M.A et al. Med Chem Res. 2021). Therefore, since the extracts were prepared from the same collection, in the same period, and under the same conditions, the main chemical compounds present in the characterization of the chemical composition in the study cited are the same for the extract used in this study. We meticulously detailed the methodology used to analyze the chemical composition of the extract (S1) and added its chromatogram (S2) to the supplementary material, ensuring the reliability and reproducibility of our data.

4. Reviewer comment: Provide precise details for methodology mentioning all the necessary conditions and concentrations to ensure reproducibility instead of simply giving reference. 

   Authors’ Response: As recommended by the reviewer, we improved the description of the methods used in the study.

   Oxidative damage

   The left cerebral cortex was homogenized in phosphate buffer, and 50 microliters of the samples were mixed with 100 microliters of 10 % trichloroacetic acid and centrifuged at 2000 rpm for 10 minutes. Subsequently, we collected 100 microliters of the supernatant, to which we added 100 microliters of 0.67 % thiobarbituric acid and then heated them in a boiling water bath for 30 minutes. The absorbance of the organic phase containing the pink chromogen was measured spectrophotometrically at 532 nm. MDA equivalents were expressed in nanomoles per milligram protein (Page 5, lines 224 to 231).

   SOD activity

   For the assay, cortex samples homogenates were used in 10, 30, and 50 μL concentrations. These samples were incubated in separate cuvettes containing 2 x 970 μL of glycine buffer, 40 μL of norepinephrine, and 20 μL of enzyme to remove hydrogen peroxide generated by the reaction catalyzed by SOD. The adrenochrome concentration was determined by spectrophotometry at 480 nm, with measurements every 10 seconds for 180 seconds (Page 5, lines 236 to 242).

   Ultra-high performance liquid chromatography

   Tissue preparation

   We used the right cortex samples for UHPLC analysis. The tissue was placed in previously labeled Precellys® tubes, to which added 500 μL of ascorbic acid solution (1 mM) and formic acid (2%). Subsequently, we added an isoprenaline standard (1 mM) in each tube. The samples were processed in the Precellys tissue homogenizer (Berlin Technologies – Precellys 24 DUAL) at a temperature of 12°C. After homogenization, the samples were centrifuged (Eppendorf® 5427R) at 8000 rpm / 4°C / 2 minutes to reduce the foam formed. The homogenate was transferred to 1.5 mL microtubes and diluted in 500 μL of LC-MS water. The samples were centrifuged at 1400 rpm at 4°C for 30 minutes. For protein precipitation, we transferred 200 μL of the supernatant to new 1.5 mL microtubes containing 800 μL of acetonitrile, and the samples were centrifuged again at 1400 rpm, at 4°C for 30 minutes. In the next step, 500 μL of the supernatant was transferred to vials and mixed with 500 μL of diluent. The final sample dilution was 10x.

   Neurotransmitter quantification

   We evaluated the tyrosine, dopamine (DA), levodopa (L-DOPA), and 3,4-dihydroxyphenylacetic acid (DOPAC) levels by UHPLC with electrospray ionization, operated in a positive mode in right cerebral cortex homogenates. The MassLynx software, v4.1 (Waters Corp., Manchester, United Kingdom), was used for data collection associated with the TargetLyx program (Waters Corp., Manchester, United Kingdom). Additionally, the DA turnover was estimated to be the ratio DOPAC/DA (Page 7, lines 292 to 312).

   Thank you once again for your valuable feedback.

5. Reviewer comment: Clarify the statistical significance in Figures to strengthen the interpretation of results. 

   Authors’ Response: We sincerely thank the reviewer for their insightful observation regarding clarifying the paper's statistical significance. Their feedback is invaluable to us. 

   As a result, we have chosen to include the different P values ​​obtained in the statistical analyses in the description of the results to strengthen their interpretation. We appreciate the reviewer's suggestion to add them to the figure, but we believe that doing so would introduce confusion, as most figures contain more than one graph (Results, Pages 7 to 11).

6. Reviewer comment: Rewrite discussion part as indicated in the introduction. 

   Authors’ Response: As recommended by the reviewer, we highlighted the PCB's neurotoxic effects and treatment complexity in the first paragraph of the discussion.

   Thus, despite regulatory efforts, PCBs persist in the environment and human tissue and are still routinely detected, inducing neurotoxicity and neurodegeneration, for which no drugs are capable of adequately preventing or treating (Page 12, lines 439 and 440).

   Thank you once again for your valuable feedback.

7. Reviewer comment: Line 119: Supplement references list by adding latest references. 

   Authors’ Response: As recommended by the reviewer, we added studies about PCB environmental accumulation and human damage latest references to the discussion as follows:

   - Dreyer and Minkos. Environ Pollut. 2023; 316: 120511.

   - Megson et al. Chemosphere. 2022; 288: 132639.

   - Lemaitre et al. Environ Res. 2021; 197: 111005.

   - Bandow et al. Int J Hyg Environ Health. 2020; 224: 113426.

   - Lan et al. Sci Rep. 2023; 13: 18322.

   - Frederiksen et al. Int J Hyg Environ Health. 2020; 224: 113430.

   - Ermler and Kortenkamp. Environ Health. 2022; 21: 94.

   - Quitete et al. PLoS One. 2024; 19: e0308334.

   - Li et al. Ecotoxicol Environ Saf. 2022; 241: 113726.

   - Sethi et al. Front Neurosci. 2021; 15: 766826.

8. Reviewer comment: The conclusions presented is not consistent with the evidence and arguments presented as further studies are needed to evaluate the mechanism of neuroprotective effect of the extract. 

   Authors’ Response: We thank the reviewer for their insightful comments, which have helped us improve the conclusion. We agree with the reviewer that further preclinical and clinical studies are required to enhance our understanding of the neuroprotective potential of ALE. Therefore, we made the following modifications to the conclusion:

   Therefore, this preclinical study suggests the possibility of using ALE to prevent the neurotoxicity induced by PCB 126. However, further studies are needed to deepen our knowledge about the mechanisms involved in ALE's neuroprotective properties against PCB-induced damage (Page 15, lines 586 to 589).

9. Reviewer comment: Write future prospective of the study. 

   Authors’ Response: As recommended by the reviewer, we added a future perspective of the study as follows:

   Therefore, further studies are needed to assess ALE's potential for chronic use and better understand its therapeutic prospects in managing PCB-induced neurotoxicity and sex differences (Page 15, lines 577 to 579).

   We sincerely appreciate the valuable comments of the reviewer, which have improved our paper. We have carefully responded to each comment and hope the paper will be suitable for publication in Scientia Pharmaceutica.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Review Report

Dear Authors,

Thank you for submitting your manuscript on the neuroprotective properties of Alpinia zerumbet extract. Below, I present detailed comments, the incorporation of which will substantially enhance the scientific and editorial value of your work.

General Assessment

The study addresses an important and timely toxicological issue. The animal model employed enables the evaluation of the potential neuroprotective effects of Alpinia zerumbet extract. However, in its current form, the manuscript does not fully comply with the editorial standards of Sci. Pharm. and requires substantial scientific and linguistic revisions prior to potential acceptance.

Abstract (Line 24)

The abstract includes a sequence of abbreviations, lacks a clear aim and methods, and introduces terms such as PCB 126, ALE, and EPM/OF without explanation. Abbreviations must be defined upon first mention, especially in the abstract, as their use otherwise misleads the reader. The abbreviation following the species epithet is incorrect. The correct form is Alpinia zerumbet (Pers.) B.L.Burtt & R.M.Sm., italicized on first mention. The abbreviation of the species introduces ambiguity. Additionally, PCB 126 is mentioned without explanation—is it a chemical compound, gene, or clinical abbreviation?

Similar issues arise in line 26 and subsequent lines, which contain excessive and undefined abbreviations. The entire abstract needs to be rewritten according to scientific writing conventions. The aim, methods, and results should be clearly and concisely stated.

Recommendations:

• Avoid custom abbreviations of taxon names (e.g., “A. zerumbet”); always provide the full name with authorship.
• Define PCB 126 at first mention to clarify its nature.
• Follow the structure: Aim → Methods → Key Results → Conclusion; limit to approximately 200 words.

1. Introduction

The introduction is generally well-written, but lacks justification for selecting a plant species that is non-native to Brazil. While the authors mention that it has been introduced to Brazil, further details are necessary—is the species cultivated, ornamental, or naturalized?

Line 81:
The species name must be cited in accordance with scientific standards and nomenclatural codes: Alpinia zerumbet (Pers.) B.L.Burtt & R.M.Sm.

Additionally, the introduction lacks detailed information on the medicinal and biological properties of the species. Please include a summary of its most important secondary metabolites.

As in the abstract, taxon abbreviations (e.g., line 94) should be avoided.

Recommendations:

• Clarify the introduction and status of this non-native plant in Brazil (introduced, cultivated, or naturalized).
• Add information on phytochemistry and pharmacological background.
• Ensure consistency in citing the literature; four references (e.g., [19]–[22]) are listed but not discussed.

2. Materials and Methods

2.1 Preparation of Alpinia zerumbet Leaf Extract

Remove the abbreviation (ALE) from the section title. Specify the plant material source—was it collected from a naturalized population? According to POWO (https://powo.science.kew.org/taxon/urn:lsid:ipni.org:names:872083-1), the species is not reported in the collection region. This information must be clarified.

2.2 Chemical Composition Analysis

Essential experimental details are missing. The LC-MS methodology is insufficiently described. For reproducibility, a detailed description of the chromatographic system (mobile phases, columns, separation parameters) must be provided. Was the method validated? If so, by whom? Include citations. If not, validation is required and must be documented.

Also, chromatographic results presented here should be moved to the Results section.

2.3 Experimental Model

This section should be rewritten in a stepwise and simplified manner to clearly guide the reader through the procedures. The rationale behind selected extract and PCB concentrations must be explicitly justified. The current text only provides fragmented explanations.

2.4 Behavioral Tests

Clearly define all abbreviations. Although EPM and OF are explained in subsections 2.4.1 and 2.4.2, a general overview of both tests and their purpose should precede the detailed procedures.

The rationale for the dosages used (PCB 126: 2 mg/kg i.p.; ALE: 50 mg/kg p.o.) is insufficient—references [18, 29] are not adequate justification.

2.5 Assessment of Oxidative Damage

This section lacks sufficient description of analytical methods. All information regarding methodology, reagents, concentrations, and execution must be included. The same applies to section 2.6 on SOD activity determination.

2.9 Liquid Chromatography

No methodological details are provided for the quantification of tyrosine, dopamine, levodopa, and DOPAC. These must be specified.

3. Results

PCB significantly reduced locomotor activity and DOPAC/DA turnover; ALE reversed this effect (Figures 2, 6).
An unexpected decrease in DOPAC levels in the Control + ALE group requires discussion—this suggests potential effects of the extract alone.
Tyrosine ↑, DA ↔ – the mechanism behind unchanged dopamine despite increased precursor availability needs to be clarified.
Neuroinflammation (iNOS/IL-6/TNF): no changes observed. While the authors hypothesize a time-dependent expression, this should be validated with serum cytokine measurements.
Apoptosis: BAX ↑, Bcl-2 ↔; ALE normalized BAX levels—these findings are consistent.

4. Discussion

The authors successfully integrate their findings with the literature on PCB and polyphenols. However:

• Sex differences are not addressed (only male animals were used).
• The hypothesis involving cytochrome P4501A1 requires experimental confirmation or should be clearly presented as a hypothesis.
• The effect of ALE on D2 receptor expression must be clarified—indicate the brain region (cortex vs. striatum) and the proposed mechanism.

5. References

No missing references were detected, but authors are advised to use automated tools (e.g., EndNote, Zotero) for consistency. Standardize DOIs and punctuation (e.g., fix “Fundamemntal Clinical Pharma” in reference [20]).

6. Suggested Revisions

Major:

• Use more conservative post-hoc statistical tests or adjust p-values accordingly (e.g., Tukey, Bonferroni).
• Provide chromatographic analysis of the extract batch used (or include it in supplementary material).
• Expand the discussion to address:
• Sex as a biological variable.
• Effect of ALE in the control group (dopamine metabolism).
• Possible PCB × ALE interactions at the pharmacokinetic level.

Minor:

• Correct language and grammatical errors.
• Standardize unit formatting (°C, mg·kg⁻¹).
• Update figure legends to include statistical test information and alpha values.
• Consider adding a graphical abstract per MDPI guidelines.

7. Reviewer’s Conclusion

This study presents compelling results that highlight the potential neuroprotective effect of Alpinia zerumbet against PCB 126-induced toxicity. Following substantial improvements in methodology (chromatography, protocol details, statistical approach, references) and language, the manuscript may be considered for publication.

Yours sincerely

reviewer

Author Response

1. Reviewer comment: The study addresses an important and timely toxicological issue. The animal model employed enables the evaluation of the potential neuroprotective effects of Alpinia zerumbet extract. However, in its current form, the manuscript does not fully comply with the editorial standards of Sci. Pharm. and requires substantial scientific and linguistic revisions prior to potential acceptance. 

Authors’ Response: We are deeply grateful for the reviewer's contributions, which have significantly enhanced the scientific content of our paper. We have carefully reviewed each point raised in the review and sent the manuscript for English editing to make our paper suitable for publication in Scientia Pharmaceutica.

2. Reviewer comment: The abstract includes a sequence of abbreviations, lacks a clear aim and methods, and introduces terms such as PCB 126, ALE, and EPM/OF without explanation. Abbreviations must be defined upon first mention, especially in the abstract, as their use otherwise misleads the reader. The abbreviation following the species epithet is incorrect. The correct form is Alpinia zerumbet (Pers.) B.L.Burtt & R.M.Sm., italicized on first mention. The abbreviation of the species introduces ambiguity. Additionally, PCB 126 is mentioned without explanation—is it a chemical compound, gene, or clinical abbreviation? (Line 24) 

Authors’ Response: We are grateful to the reviewer for their insightful comments, which have significantly improved our abstract. Their feedback is invaluable to us. 

As the reviewer's recommendation, we added the meaning of PCB 126, OF, and EPM in the abstract, enhancing its clarity. We also made it explicit that PCBs are chemical compounds and refined the study's objective as follows:

Background: Polychlorinated biphenyls (PCBs) are synthetic chemical compounds that have bioaccumulated and contaminated the entire global ecosystem, causing neurotoxic effects. However, polyphenols may have protective effects against this neurotoxicity. We aimed to investigate the neuroprotective effect of a hydroalcoholic extract of fresh leaves of Alpinia zerumbet (ALE), which is rich in polyphenols, on the neurobehavioral changes induced by 3,3',4,4',5-pentachlorobiphenyl (PCB 126). Methods: We divided C57BL/6 male mice into four groups (n=40): Control, Control+ALE, PCB, and PCB+ALE. We administered the ALE extract (50 mg/kg/day) through drinking water and PCB 126 (2 mg/kg/once a week) intraperitoneally for four weeks. The mice were subjected to the elevated plus maze (EPM) and open field (OF) tests in the last week of treatment (Page 1, lines 21 to 30).

We did not include Alpinia zerumbet's botanical classification in the abstract since the section that discusses the plant refers to the extract used, referring to the acronym (ALE), and due to the restricted word count (200), which limits the amount of information we can include. However, we corrected its classification in the introduction when the medicinal plant was first mentioned (Page 2, lines 86 and 87).

3. Reviewer comment: Similar issues arise in line 26 and subsequent lines, which contain excessive and undefined abbreviations. The entire abstract needs to be rewritten according to scientific writing conventions. The aim, methods, and results should be clearly and concisely stated.

Recommendations:

• Avoid custom abbreviations of taxon names (e.g., “A. zerumbet”); always provide the full name with authorship.
• Define PCB 126 at first mention to clarify its nature.
• Follow the structure: Aim → Methods → Key Results → Conclusion; limit to approximately 200 words. 

Authors’ Response: As the reviewer's recommendation, we added the meaning of PCB 126 in the abstract, enhancing its clarity. We also made it explicit that PCBs are chemical compounds and refined the study's conclusion as follows:

Background: Polychlorinated biphenyls (PCBs) are synthetic chemical compounds that have bioaccumulated and contaminated the entire global ecosystem, causing neurotoxic effects. However, polyphenols may have protective effects against this neurotoxicity. We aimed to investigate the neuroprotective effect of a hydroalcoholic extract of fresh leaves of Alpinia zerumbet (ALE), which is rich in polyphenols, on the neurobehavioral changes in-duced by 3,3',4,4',5-pentachlorobiphenyl (PCB 126). Methods: We divided C57BL/6 male mice into four groups (n=40): Control, Control+ALE, PCB, and PCB+ALE. We adminis-tered the ALE extract (50 mg/kg/day) through drinking water and PCB 126 (2 mg/kg/once a week) intraperitoneally for four weeks. The mice were subjected to the elevated plus maze (EPM) and open field (OF) tests in the last week of treatment. Results: PCB 126 re-duced locomotor activity, DOPAC levels, dopamine turnover, and D2 receptor expression. This compound also increased lipid peroxidation, tyrosine levels, and BAX expression in the cerebral cortex. Notably, ALE treatment prevented locomotor activity reduction and increased DOPAC levels, dopamine turnover, and D2 receptor expression. Moreover, the extract prevented the PCB-induced increases in BAX expression and lipid peroxidation. Finally, the ALE increased SOD antioxidant activity. Conclusion: Our investigation high-lights that the ALE may serve as a therapeutic strategy against PCB-induced neurotoxicity (Page 1, lines 21 to 38).

We did not include Alpinia zerumbet's botanical classification in the abstract since the section that discusses the plant refers to the extract used, referring to the acronym (ALE), and due to the restricted word count (200), which limits the amount of information we can include. However, we corrected its classification in the introduction when the medicinal plant was first mentioned (Page 2, lines 86 and 87).

Finally, we revised the abstract to 200 words, as requested.

Thank you once again for your valuable feedback.

4. Reviewer comment: The introduction is generally well-written, but lacks justification for selecting a plant species that is non-native to Brazil. While the authors mention that it has been introduced to Brazil, further details are necessary—is the species cultivated, ornamental, or naturalized?

Authors’ Response: We are delighted to know that you enjoyed our introduction. Thank you for asking us about the medicinal plant, which allows us to highlight the reason for its choice as a therapeutic tool for the paper's study.

Although native to East Asia, Alpinia zerumbet has become naturalized in Brazil. It was introduced to the country in the 19th century and initially cultivated in the Rio de Janeiro Botanical Garden (Nishidono and Tanaka. Molecules. 2024; 29: 2845; Batista et al. Braz. J. Biol. 2021; 84: e253616). Since then, it has spread and adapted to several regions, including the northeast and southeast of Brazil (Faculty of Applied Sciences. J.Chin.Pharm.Sci. 2017; 26). This plant is commonly grown in gardens in the country for ornamental and medicinal use, such as antihypertensive, antiulcerogenic, and diuretic effects (Lahlou et al., Fundamemntal Clinical Pharma 2003; 17: 323–330; Santos et al. Phytomedicine 2011; 18: 539–543).

Our group was interested in studying the effects of Alpinia zerumbet on the cardiovascular system due to widespread reports of its diuretic effects in traditional use. Thus, preclinical studies conducted by our group have demonstrated that ALE presents vasodilatory, antihypertensive, and antioxidant effects in a model of spontaneous hypertension (Menezes et al. Braz J Med Biol Res. 2025; 58: e14210). Moreover, data from the literature have also shown that Alpinia zerumbet causes anxiolytic (Murakami et al. Nat Prod Commun. 2009; 4: 129–132; Satou et al. Nat Prod Commun. 2010; 5: 143–146), antidepressant (Bevilaqua et al. Pharmaceutical Biology. 2016; 54: 151–156), and antipsychotic effects (de Araújo et al. Metab Brain Dis. 2021; 36: 2283–2297), proving its potential to act in diseases that affect the CNS (Ferreira et al. Ann. Res. Rev. Biol. 2024; 39: 36–51). Given the antioxidant effect of Alpinia zerumbet found by our group and its properties on the CNS, demonstrated by other researchers, we were interested in investigating its neuroprotective potential.

 

Therefore, as recommended by the reviewer, we rewrote this introduction point as follows:

Alpinia zerumbet (Pers.) B.L.Burtt & R.M.Sm. is a medicinal plant rich in polyphenols, popularly known as cologne due to its aroma. Although native to East Asia, Alpinia zerumbet has become naturalized in Brazil. It was introduced to the country in the 19th century and initially cultivated in the Rio de Janeiro Botanical Garden (Nishidono and Tanaka. Molecules. 2024; 29: 2845; Batista et al. Braz. J. Biol. 2021; 84: e253616). Since then, it has spread and adapted to several regions, including the northeast and southeast of Brazil (Faculty of Applied Sciences. J.Chin.Pharm.Sci. 2017; 26). This plant is commonly grown in gardens in the country for ornamental and medicinal use, such as antihypertensive, antiulcerogenic, and diuretic effects (Lahlou et al., Fundamemntal Clinical Pharma 2003; 17: 323–330; Santos et al. Phytomedicine 2011; 18: 539–543) (Page 2, lines 88 to 93).

Given the antioxidant effect of Alpinia zerumbet found by our group and its properties on the CNS, demonstrated by other researchers, we were interested in investigating its neuroprotective potential (Page 3, lines 105 to 107).

5. Reviewer comment: The species name must be cited in accordance with scientific standards and nomenclatural codes: Alpinia zerumbet (Pers.) B.L.Burtt & R.M.Sm (Line 81).

Authors’ Response: We thank the reviewer for pointing out that the plant species' names should follow scientific standards and nomenclatural codes. As recommended by the reviewer, we rewrote the plant species' names in the introduction as follows:

Alpinia zerumbet (Pers.) B.L.Burtt & R.M.Sm. is a medicinal plant rich in polyphenols, popularly known as cologne due to its aroma (Page 2, lines 86 and 87).

6. Reviewer comment: Additionally, the introduction lacks detailed information on the medicinal and biological properties of the species. Please include a summary of its most important secondary metabolites. 

As recommended by the reviewer, we added in the introduction the main biological effects of Alpinia zerumbet, which are antihypertensive, antiulcerogenic, diuretic, anxiolytic, antidepressant, and antipsychotic, to contextualize our group's previous studies and the investigation of the neuroprotective effect in the present study (Page 2, lines 92 and 93; Page 3, line 104).

We also included the Alpinia zerumbet secondary metabolites as follows:

Moreover, previous findings have demonstrated that Alpinia zerumbet has more than 100 secondary metabolites isolated from its leaves, flowers, rhizomes, seeds, pericarps, and fruits, such as kavalactones, chalcones, flavonoids, diterpenoids, sesquiterpenoids, monoterpenoids, meroterpenoids, steroids, diarylheptanoids, neolignans, glucoside esters, and phenolic compounds, among others (Nishidono and Tanaka. Molecules. 2024; 29: 2845; Ohtsuki et al. Bioorg. Med. Chem. 2009; 17: 6748–6754; You et al. Nat. Prod. Res. 2022; 36: 5740–5746; Xiong et al. J. Agric. Food Chem. 2023; 72: 424–436; Xiao et al. Fitoterapia. 2024; 172: 105753; Mpalantinos et al. Phytother. Res. 1998; 12: 442–444; Zhang et al. J. Agric. Food. Chem. 2021; 69: 9229–9237) (Page 2 and 3, lines 93 to 98).

Thank you once again for your valuable feedback.

7. Reviewer comment: As in the abstract, taxon abbreviations (e.g., line 94) should be avoided.

Recommendations:

Clarify the introduction and status of this non-native plant in Brazil (introduced, cultivated, or naturalized).

Add information on phytochemistry and pharmacological background.

Ensure consistency in citing the literature; four references (e.g., [19]–[22]) are listed but not discussed. 

Authors’ Response: We thank the reviewer for his valuable suggestions for improving the abstract and introduction. We have carefully reviewed these sections and followed all the reviewer's recommendations, as described in the previous comments. Moreover, we have revised references 19 to 22, ensuring their appropriate use in the text to highlight the antioxidant, anti-apoptotic, and anti-inflammatory effects of polyphenols present in medicinal plants.

8. Reviewer comment: 2.1 Preparation of Alpinia zerumbet Leaf Extract

Remove the abbreviation (ALE) from the section title. Specify the plant material source—was it collected from a naturalized population? According to POWO (https://powo.science.kew.org/taxon/urn:lsid:ipni.org:names:872083-1), the species is not reported in the collection region. This information must be clarified.

Authors’ Response: As recommended by the reviewer, we removed the abbreviation ALE from the subtitle, referring to the preparation of the extract.

As previously mentioned, Alpinia zerumbet is a plant that became naturalized in Brazil after its insertion in the 19th century in the Catete Palace in Rio de Janeiro, where we collected the plant leaves to prepare the extract. We have meticulously detailed the methodology, including the precise location of the collection at the Catete Palace, according to the coordinates previously added to the method (Page 3, line 113).

Thank you once again for your valuable feedback.

9. Reviewer comment: 2.2 Chemical Composition Analysis

Essential experimental details are missing. The LC-MS methodology is insufficiently described. For reproducibility, a detailed description of the chromatographic system (mobile phases, columns, separation parameters) must be provided. Was the method validated? If so, by whom? Include citations. If not, validation is required and must be documented.  Also, chromatographic results presented here should be moved to the Results section. 

Authors’ Response: We greatly appreciate your feedback and the importance you place on this point. Your input allows us to clarify our work.

The extract used in this study to investigate the neuroprotective effect was prepared from the same collection that originated the published work characterizing the chemical composition (Da Silva, M.A et al. Med Chem Res. 2021). Therefore, since the extracts were prepared from the same collection, in the same period, and under the same conditions, the main chemical compounds present in the characterization of the chemical composition in the study cited are the same for the extract used in this study. We meticulously detailed the methodology used to analyze the chemical composition of the extract (S1) and added its chromatogram (S2) to the supplementary material, ensuring the reliability and reproducibility of our data. However, since we published the characterization of ALE mentioned in the methodology in a previous study, it cannot be included in the results of the present study.

10. Reviewer comment: 2.3 Experimental Model

This section should be rewritten in a stepwise and simplified manner to clearly guide the reader through the procedures. The rationale behind selected extract and PCB concentrations must be explicitly justified. The current text only provides fragmented explanations. 

Authors’ Response: We sincerely appreciate the reviewer's insightful feedback on the experimental model. Their suggestions for improvements will enhance the clarity and reproducibility of our research. Therefore, we rewrite the experimental as follows:

The experimental protocol lasted for four weeks. During this period, the animals received administration of PCB 126 (2 mg/kg, Sigma-Aldrich, San Luis, MO, EUA) or vehicle via the intraperitoneal route once a week and ALE (50 mg/kg) in the drinking water daily. The vehicle used to dilute the PCB 126 was corn oil, and the ALE was changed every two days. The bottles containing the extract were carefully protected from light to prevent oxidation. As we intended to investigate the preventive neuroprotective effect of ALE, the extract administration started simultaneously with PCB 126 and lasted for four weeks. In the last week of the protocol, the animals carried out the elevated plus maze (EPM) and open field (OF) behavioral tests. Finally, we sacrificed the mice by decapitation, isolating the cerebral cortex, and freezing it at -80 °C for analysis (Figure 1) (Page 4, lines 146 to 155).

We used PCB 126 in our study because it has a high binding affinity with the AHR receptor, leading to CYP activation and subsequent oxidative stress and inflammation. Therefore, this chemical compound is considered a highly toxic congener (Leece et al. J. Toxicol. Environ. Health. 1985; 16, 379-388; Silva et al. Oecologia Brasiliensis. 2007; 11: 179-187). The dose of PCB 126 used (2 mg/kg) and the intraperitoneal route of administration was chosen based on its proven neurotoxicity in previous studies with a similar dosage of PCB congeners in adult animals (Selvakumar et al. Toxicol Lett. 2013; 222: 45-54; Venkatarama et al. Neurotoxicology. 2007; 28: 490-498). Regarding ALE, we used 50 mg/kg in drinking water (Menezes et al. Braz J Med Biol Res. 2025; 58: e14210; de Moura et al. Journal of Cardiovascular Pharmacology. 2005; 46: 288-294), as this concentration demonstrated beneficial biological effects in vivo and relevant antioxidant properties, which suggests its potential beneficial action on neurotoxicity induced by PCB 126 (Page 4, lines 181 to 189).

11. Reviewer comment: 2.4 Behavioral Tests

Clearly define all abbreviations. Although EPM and OF are explained in subsections 2.4.1 and 2.4.2, a general overview of both tests and their purpose should precede the detailed procedures. 

Authors’ Response: We greatly appreciate your feedback and the importance you place on this point. As suggested by the reviewer, we added the full names of abbreviations in the behavioral tests subsection (Pages 4 and 5, lines 192, 197, 198, 210, and 211) .

12. Reviewer comment: The rationale for the dosages used (PCB 126: 2 mg/kg i.p.; ALE: 50 mg/kg p.o.) is insufficient—references [18, 29] are not adequate justification. 

Authors’ Response: As recommended by the reviewer, we justify the choice of the concentration of PCB 126 and ALE used in the study as follows:

We used PCB 126 in our study because it has a high binding affinity with the AHR receptor, leading to CYP activation and subsequent oxidative stress and inflammation. Therefore, this chemical compound is considered a highly toxic congener (Wu et al. Toxicol. Sci. 2016; 152: 309-322; Leece et al. J. Toxicol. Environ. Health. 1985; 16, 379-388). The dose of PCB 126 used (2 mg/kg) and the intraperitoneal route of administration was chosen based on its proven neurotoxicity in previous studies with a similar dosage of PCB congeners in adult animals (Selvakumar et al. Toxicol Lett. 2013; 222: 45-54; Venkatarama et al. Neurotoxicology. 2007; 28: 490-498). Regarding ALE, we used 50 mg/kg in drinking water (Menezes et al. Braz J Med Biol Res. 2025; 58: e14210; de Moura et al. Journal of Cardiovascular Pharmacology. 2005; 46: 288-294), as this concentration demonstrated beneficial biological effects in vivo and relevant antioxidant properties, which suggests its potential beneficial action on neurotoxicity induced by PCB 126 (Page 4, lines 181 to 189).

Thank you once again for your valuable feedback.

13. Reviewer comment: 2.5 Assessment of Oxidative Damage

This section lacks sufficient description of analytical methods. All information regarding methodology, reagents, concentrations, and execution must be included. The same applies to section 2.6 on SOD activity determination. 

Authors’ Response: We thank the reviewer for his valuable suggestion to detail the experiments used to assess oxidative stress. As recommended by the reviewer, we improved the description of the oxidative damage and SOD activity methods as follows:

Oxidative damage

The left cerebral cortex was homogenized in phosphate buffer, and 50 microliters of the samples were mixed with 100 microliters of 10 % trichloroacetic acid and centrifuged at 2000 rpm for 10 minutes. Subsequently, we collected 100 microliters of the supernatant, to which we added 100 microliters of 0.67 % thiobarbituric acid and then heated them in a boiling water bath for 30 minutes. The absorbance of the organic phase containing the pink chromogen was measured spectrophotometrically at 532 nm. MDA equivalents were expressed in nanomoles per milligram protein (Page 5, lines 224 to 231).

SOD activity

For the assay, cortex samples homogenates were used in 10, 30, and 50 μL concentrations. These samples were incubated in separate cuvettes containing 2 x 970 μL of glycine buffer, 40 μL of norepinephrine, and 20 μL of enzyme to remove hydrogen peroxide generated by the reaction catalyzed by SOD. The adrenochrome concentration was determined by spectrophotometry at 480 nm, with measurements every 10 seconds for 180 seconds (Page 5, lines 236 to 241).

14. Reviewer comment: 2.9 Liquid Chromatography

No methodological details are provided for the quantification of tyrosine, dopamine, levodopa, and DOPAC. These must be specified. 

Authors’ Response: We greatly appreciate your feedback and the importance you place on the detail of the methodology used in the study. Your input allows us to clarify our work. As recommended by the reviewer, we improved the description of the tyrosine, dopamine, levodopa, and DOPAC quantification methods as follows:

Ultra-high performance liquid chromatography

Tissue preparation

We used the right cortex samples for UHPLC analysis. The tissue was placed in previously labeled Precellys® tubes, to which added 500 μL of ascorbic acid solution (1 mM) and formic acid (2%). Subsequently, we added an isoprenaline standard (1 mM) in each tube. The samples were processed in the Precellys tissue homogenizer (Berlin Technologies – Precellys 24 DUAL) at a temperature of 12°C. After homogenization, the samples were centrifuged (Eppendorf® 5427R) at 8000 rpm / 4°C / 2 minutes to reduce the foam formed. The homogenate was transferred to 1.5 mL microtubes and diluted in 500 μL of LC-MS water. The samples were centrifuged at 1400 rpm at 4°C for 30 minutes. For protein precipitation, we transferred 200 μL of the supernatant to new 1.5 mL microtubes containing 800 μL of acetonitrile, and the samples were centrifuged again at 1400 rpm, at 4°C for 30 minutes. In the next step, 500 μL of the supernatant was transferred to vials and mixed with 500 μL of diluent. The final sample dilution was 10x.

Neurotransmitter quantification

We evaluated the tyrosine, dopamine (DA), levodopa (L-DOPA), and 3,4-dihydroxyphenylacetic acid (DOPAC) levels by UHPLC with electrospray ionization, operated in a positive mode in right cerebral cortex homogenates. The MassLynx software, v4.1 (Waters Corp., Manchester, United Kingdom), was used for data collection associated with the TargetLyx program (Waters Corp., Manchester, United Kingdom). Additionally, the DA turnover was estimated to be the ratio DOPAC/DA (Page 7, lines 292 to 312).

15. Reviewer comment: PCB significantly reduced locomotor activity and DOPAC/DA turnover; ALE reversed this effect (Figures 2, 6).

An unexpected decrease in DOPAC levels in the Control + ALE group requires discussion—this suggests potential effects of the extract alone.

Tyrosine ↑, DA ↔ – the mechanism behind unchanged dopamine despite increased precursor availability needs to be clarified. 

Authors’ Response: As recommended by the reviewer, we discussed these points to clarify some of the results found as follows:

A previous study has shown that PCBs can reduce tyrosine hydroxylase activity, minimizing dopamine synthesis and increasing tyrosine levels (Choksi. Fundamental and Applied Toxicology. 1997; 39: 76–80). Moreover, other data have shown that 3-O-methyldopa administration did not alter DA levels but significantly decreased DOPAC, DA turnover, and locomotor activity (Onzawa et al. Biological & Pharmaceutical Bulletin. 2012; 35: 1244–1248), similar to our data. These findings can explain the increase in tyrosine found in the PCB groups in our study, with no change in dopamine synthesis but a reduction in this neurotransmitter's bioavailability. We suggested that the polyphenols present in ALE reduce neurotoxicity and neurodegeneration and might counteract the actions of PCB 126, promoting the availability of DA. Additionally, another investigation demonstrated that quercetin, a chemical compound present in ALE, causes the inhibition of monoamine oxidase-B (Lee et al. J. Agric. Food Chem. 2001; 49: 5551–5555) and A (van Diermen et al. J. Ethnopharmacol. 2009; 122: 397–401). This inhibition may contribute to ALE's effect on DOPAC levels in animals in the Control+ALE group since the reduction in dopamine metabolism results in lower DOPAC levels, as found in the present study (Page 14, lines 535 to 547).

Thank you once again for your valuable feedback.

16. Reviewer comment: Neuroinflammation (iNOS/IL-6/TNF): no changes observed. While the authors hypothesize a time-dependent expression, this should be validated with serum cytokine measurements.

Apoptosis: BAX ↑, Bcl-2 ↔; ALE normalized BAX levels—these findings are consistent.

Authors’ Response: We concur with the reviewer that measuring plasma cytokine levels could significantly enhance our understanding of PCB 126's pro-inflammatory effects. Our original plan was to evaluate plasma levels of cytokines and thyroid hormones, which are closely linked to locomotor activity and altered by exposure to PCBs. Unfortunately, due to the significant hemolysis of the collected plasma, we could not perform the intended plasma analyses. However, we believe that recent investigations into PCB 126's pro-inflammatory actions could provide valuable insights that could potentially impact your research in the following ways:

A recent study has shown that PCB 126 can increase TNF and IL-6 plasma levels (Quitete et al. PLoS One. 2024; 19: e0308334). Additionally, another study found elevated pro-inflammatory cytokines in the colons of mice exposed to this chemical compound. These findings suggest that the influence of intestinal inflammation and microbiota changes induced by PCB 126 could be a significant factor in neuroinflammation (Li et al. Ecotoxicol Environ Saf. 2022; 241: 113726). Therefore, we hypothesize that neuroinflammation is time-dependent and may be developing in our experimental model.

Thank you once again for your valuable feedback (Pages 13 and 14, lines 508 to 513).

17. Reviewer comment: The authors successfully integrate their findings with the literature on PCB and polyphenols. However:

Sex differences are not addressed (only male animals were used).

Authors’ Response: We thank the reviewer for highlighting the importance of discussing the effects of PCBs between the sexes. As recommended by the reviewer, we discuss this point as follows:

Exposure to PCBs can cause distinct neurobehavioral effects between the sexes. Literature reported that social interaction and grooming behavior are affected in males but not females exposed to PCBs during development (Sethi et al. Front Neurosci. 2021; 15: 766826), highlighting the importance of studies investigating the neurotoxic properties of these chemicals between the sexes not only during development but also in adult animals. Despite the promising findings in our study, the research using only male mice is a limitation. Therefore, further studies are needed to assess ALE's potential for chronic use and better understand its therapeutic prospects in managing PCB-induced neurotoxicity and sex differences (Page 15, lines 572 to 579).

18. Reviewer comment: • The hypothesis involving cytochrome P4501A1 requires experimental confirmation or should be clearly presented as a hypothesis. 

Authors’ Response: As recommended by the reviewer, we present clearly as a hypothesis the uncoupling of the cytochrome P4501A1 in neurotoxicity induced by PCB 126 as follows:

We hypothesize that the uncoupling of CYP1A1 and activation of the AhR receptor participates in ROS production and oxidative damage induced by PCB 126, causing the neurotoxicity observed in the present study (Page 13, lines 485 to 487).

Thank you once again for your valuable feedback.

19. Reviewer comment: • The effect of ALE on D2 receptor expression must be clarified—indicate the brain region (cortex vs. striatum) and the proposed mechanism. 

Authors’ Response: As recommended by the reviewer, we indicate the brain region chosen for performed D2 receptor expression and ALE proposed mechanism as follows:

The dorsolateral region of the prefrontal cortex actively participates in the control of voluntary movements and acts actively in the movement's control (Yogev-Seligmann et al. Mov. Disord. 2008; 23: 329-342). Previous findings highlighted the importance of the right hemisphere since they observed increased activity in this region in older adults during walking (Hoang et al. Int. J. Psychophysiol. 2022; 174 : 9-16). In line with these data, another study demonstrated increased blood flow in the right cerebral hemisphere, compared to the left hemisphere, during faster walking (Greenfield et al. Sensors (Basel). 2023; 23: 3986). Therefore, given the importance of this cerebral hemisphere in the movement's control, we used samples from the right cerebral cortex (which also contained the prefrontal cortex region) to evaluate the expression of dopaminergic D2 receptors and identified that PCB 126 administration reduced this receptor's expression, similar to a previous study (Bavithra et al. Neurotox Res. 2012; 21: 149-159) (Page 14, lines 548 to 556).

 

Notably, ALE caused the D2 receptor content to return to control expression levels. The polyphenols in the extract may participate in this effect, as a study using quercetin (one of the ALE compounds) reported normalized D2 receptor expression in animals exposed to PCB (Bavithra et al. Neurotox Res. 2012; 21: 149-159). Regarding the mechanisms of quercetin, this bioactive compound of ALE increases the phosphorylation of the proteins protein kinase A (PKA) and dopamine- and cAMP-regulated neuronal phosphoprotein (DARP-32) and reduces the phosphorylation of protein phosphatase 1α (PP1α). Highlighting a key role of this polyphenol in affecting motor function since phosphorylation of DARPP-32 at threonine- 34 by PKA activates the inhibitory function of DARPP-32 over PP1α (Gupta et al. Sci Rep. 2018; 8: 2528). Therefore, we propose that the preventive effect of our extract on locomotor changes induced by PCB 126 also involves action on dopaminergic D2 receptors, promoting the stimulation of movement (Page 15, lines 564 to 569).

Thank you once again for your valuable feedback.

20. Reviewer comment: No missing references were detected, but authors are advised to use automated tools (e.g., EndNote, Zotero) for consistency. Standardize DOIs and punctuation (e.g., fix “Fundamemntal Clinical Pharma” in reference [20]). 

Authors’ Response: We thank the reviewer for carefully reviewing the article and suggesting essential improvements. As recommended by the reviewer, we reviewed all references and formatted them using Zotero to avoid errors.

21. Reviewer comment: • Use more conservative post-hoc statistical tests or adjust p-values accordingly (e.g., Tukey, Bonferroni).

Authors’ Response: We sincerely thank the reviewer for their insightful observation regarding clarifying the paper's statistical significance. Their feedback is invaluable to us. 

Previous studies have highlighted the statistical power of Fisher's LSD post-hoc test to highlight statistical differences between different experimental groups (Agbangba et al. 2024. Heliyon. 2024; 10: e25131; Assaad et al. 2015. Springerplus. 2015; 4: 33) . In the last 20 years, approximately 18.15% of research works used the Fisher LSD post-hoc test, as follows:

- Sreelatha et al. Neurol Int. 2024; 16: 1094-1111.

- Dutra-Tavares et al. Brain Sci. 2024; 14: 855.

- Pardo-Giménez et al. Journal of the Science of Food and Agriculture. 2016; 96: 3838-3845.

- Liao et al. Biomedicines. 2021; 9: 542.

-  Hamed et al. Environmental Pollution. 2024; 349: 123912.

- Adde-Michel et al. Neurosci Lett. 2005; 374: 109-112.

- Xu et al. Int J Biol Macromol. 2024; 277: 134404.

- Qinwufeng et al. J Ethnopharmacol. 2022; 295: 115428.

- Dietrich et al. Comp Biochem Physiol C Toxicol Pharmacol. 2011; 153: 422-429.

- Du et al. Food Bioscience. 2023; 53, 102555.

It's crucial to note that for the LSD post-test to be used effectively, an analysis of variance (ANOVA) must be conducted to identify any statistical differences and reject the null hypothesis. Additionally, the samples must pass the normality test (Agbangba et al. 2024. Heliyon. 2024; 10: e25131). Therefore, before using the LSD post-hoc test, we performed the Shapiro-Wilk normality test, and the samples used were under a normal distribution (added in the Material and Methods section). Moreover, we performed ANOVA to reject the null hypothesis. Thus, we demonstrate our unwavering commitment to adhering to the necessary recommendations for using the post-hoc LSD test. Given this, since the post-hoc LSD test has statistical power to perform statistical comparisons between the groups and given the number of recently published articles that use it, we decided to keep it as a post-hoc test in our analyses. However, as recommended by the reviewer, we have included the different P values ​​obtained in the statistical analyses in the description of the results to strengthen their interpretation.

22. Reviewer comment: • Provide chromatographic analysis of the extract batch used (or include it in supplementary material). 

Authors’ Response: We greatly appreciate your feedback and the importance you place on this point. Your input allows us to clarify our work and engage in further dialogue.

The extract used in this study to investigate the neuroprotective effect was prepared from the same collection that originated the published work characterizing the chemical composition (Da Silva, M.A et al. Med Chem Res. 2021). Therefore, since the extracts were prepared from the same collection, in the same period, and under the same conditions, the main chemical compounds present in the characterization of the chemical composition in the study cited are the same for the extract used in this study. We meticulously detailed the methodology used to analyze the chemical composition of the extract (S1) and added its chromatogram (S2) to the supplementary material, ensuring the reliability and reproducibility of our data.

23. Reviewer comment: • Expand the discussion to address: • Sex as a biological variable. • Effect of ALE in the control group (dopamine metabolism). • Possible PCB × ALE interactions at the pharmacokinetic level.

Authors’ Response: As recommended by the reviewer, we expand the discussion as follows:

Exposure to PCBs can cause distinct neurobehavioral effects between the sexes. Literature reported that social interaction and grooming behavior are affected in males but not females exposed to PCBs during development (Sethi et al. Front Neurosci. 2021; 15: 766826), highlighting the importance of studies investigating the neurotoxic properties of these chemicals between the sexes not only during development but also in adult animals  Despite the promising findings in our study, the research using only male mice is a limitation. Therefore, further studies are needed to assess ALE's potential for chronic use and better understand its therapeutic prospects in managing PCB-induced neurotoxicity and sex differences (Page 15, lines 572 to 579).

Additionally, another investigation demonstrated that quercetin, a chemical compound present in ALE, causes the inhibition of monoamine oxidase-B (Lee et al. J. Agric. Food Chem. 2001; 49: 5551–5555) and A (van Diermen et al. J. Ethnopharmacol. 2009; 122: 397–401). This inhibition may contribute to ALE's effect on DOPAC levels in animals in the Control+ALE group since the reduction in dopamine metabolism results in lower DOPAC levels, as found in the present study (Page 14, lines 543 to 547).

Further studies are needed to deepen this discussion regarding the pharmacokinetic interaction between PCB 126 and ALE since our research group recently began studying ALE. The present study was the first to investigate its effects on the central nervous system. Therefore, further research is needed to understand its pharmacokinetic characteristics.

Thank you once again for your valuable feedback.

24. Reviewer comment: • Correct language and grammatical errors. 

Authors’ Response: We acknowledge and apologize for the grammatical errors in the paper. As per the reviewer's suggestion, we used the English editing service recommended by the journal to rectify these errors. We value the importance of clear and precise communication in scientific research and have made the necessary language and grammatical corrections to ensure the quality of our work.

25. Reviewer comment: • Standardize unit formatting (°C, mg·kg⁻¹).

Authors’ Response: As recommended by the reviewer, we standardize unit formatting. Thank you once again for your valuable feedback.

26. Reviewer comment: • Update figure legends to include statistical test information and alpha values. 

Authors’ Response: We sincerely thank the reviewer for their insightful observation regarding clarifying the paper's statistical significance. Their feedback is invaluable to us. 

As a result, we have chosen to include the different P values obtained in the statistical analyses in the description of the results to strengthen their interpretation (Results, Pages 7 to 11). We appreciate the reviewer's suggestion to add them to the figure, but we believe that doing so would introduce confusion, as most figures contain more than one graph.

27. Reviewer comment: • Consider adding a graphical abstract per MDPI guidelines.

Authors’ Response: As recommended by the reviewer, we added the study's graphical abstract. Thank you once again for your valuable feedback.

28. Reviewer comment: This study presents compelling results that highlight the potential neuroprotective effect of Alpinia zerumbet against PCB 126-induced toxicity. Following substantial improvements in methodology (chromatography, protocol details, statistical approach, references) and language, the manuscript may be considered for publication. 

Authors’ Response: We sincerely appreciate the valuable comments of the reviewer, which have improved our paper. We have carefully responded to each comment and hope the paper will be suitable for publication in Scientia Pharmaceutica.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

After a thorough reading of the publication, which is very interesting. However, in my opinion, the description of the methodology should be completed so that other researchers, can benefit from this publication. I kindly ask you to respond to the following points:

• please describe the method (Ultra performance liquid chromatography) in more detail so that it can be replicated by other researchers.
• which spectrophotometer and chromatograph were used in the determinations
• Please describe in more detail, the following methodology:  2.5. Oxidative damage determination, 2.6. Superoxide dismutase (SOD) activity,  2.9. Ultra performance liquid chromatography (UHPLC)

 

Having addressed the above questions and remedied methodological shortcomings, recommends this publication for publication in the journal Sci. Pharm.

Author Response

1. Reviewer comment: • please describe the method (Ultra performance liquid chromatography) in more detail so that it can be replicated by other researchers.

Authors’ Response: We greatly appreciate your feedback and the importance you place on this point. Your input allows us to clarify our work and engage in further dialogue.

The extract used in this study to investigate the neuroprotective effect was prepared from the same collection that originated the published work characterizing the chemical composition (Da Silva, M.A et al. Med Chem Res. 2021). Therefore, since the extracts were prepared from the same collection, in the same period, and under the same conditions, the main chemical compounds present in the characterization of the chemical composition in the study cited are the same for the extract used in this study. We meticulously detailed the methodology used to analyze the chemical composition of the extract (S1) and added its chromatogram (S2) to the supplementary material, ensuring the reliability and reproducibility of our data.

2. Reviewer comment: • which spectrophotometer and chromatograph were used in the determinations.

Authors’ Response: As recommended by the reviewer, we included the spectrophotometer and chromatograph used in the analyses as follows:

We used the Ultrospec 2100 pro spectrophotometer from Amersham Bioscience for oxidative stress analysis (Page 5, lines 230 and 231). Moreover, we used the Acquity UHPLC I class coupled with Triple Quadrupole TDQ from Waters to analyze dopamine levels, precursors, and metabolites (Pages 6 and 7, lines 289 and 290). Thank you once again for your valuable feedback.

3. Reviewer comment: • Please describe in more detail, the following methodology:  2.5. Oxidative damage determination, 2.6. Superoxide dismutase (SOD) activity, 2.9. Ultra performance liquid chromatography (UHPLC).

Authors’ Response: As recommended by the reviewer, we improved the description of the methods used in the study.

Oxidative damage

The left cerebral cortex was homogenized in phosphate buffer, and 50 microliters of the samples were mixed with 100 microliters of 10 % trichloroacetic acid and centrifuged at 2000 rpm for 10 minutes. Subsequently, we collected 100 microliters of the supernatant, to which we added 100 microliters of 0.67 % thiobarbituric acid and then heated them in a boiling water bath for 30 minutes. The absorbance of the organic phase containing the pink chromogen was measured spectrophotometrically at 532 nm. MDA equivalents were expressed in nanomoles per milligram protein.

SOD activity

For the assay, cortex samples homogenates were used in 10, 30, and 50 μL concentrations. These samples were incubated in separate cuvettes containing 2 x 970 μL of glycine buffer, 40 μL of norepinephrine, and 20 μL of enzyme to remove hydrogen peroxide generated by the reaction catalyzed by SOD. The adrenochrome concentration was determined by spectrophotometry at 480 nm, with measurements every 10 seconds for 180 seconds.

Ultra-high performance liquid chromatography

Tissue preparation

We used the right cortex samples for UHPLC analysis. The tissue was placed in previously labeled Precellys® tubes, to which added 500 μL of ascorbic acid solution (1 mM) and formic acid (2%). Subsequently, we added an isoprenaline standard (1 mM) in each tube. The samples were processed in the Precellys tissue homogenizer (Berlin Technologies – Precellys 24 DUAL) at a temperature of 12°C. After homogenization, the samples were centrifuged (Eppendorf® 5427R) at 8000 rpm / 4°C / 2 minutes to reduce the foam formed. The homogenate was transferred to 1.5 mL microtubes and diluted in 500 μL of LC-MS water. The samples were centrifuged at 1400 rpm at 4°C for 30 minutes. For protein precipitation, we transferred 200 μL of the supernatant to new 1.5 mL microtubes containing 800 μL of acetonitrile, and the samples were centrifuged again at 1400 rpm, at 4°C for 30 minutes. In the next step, 500 μL of the supernatant was transferred to vials and mixed with 500 μL of diluent. The final sample dilution was 10x.

Neurotransmitter quantification

We evaluated the tyrosine, dopamine (DA), levodopa (L-DOPA), and 3,4-dihydroxyphenylacetic acid (DOPAC) levels by UHPLC with electrospray ionization, operated in a positive mode in right cerebral cortex homogenates. The MassLynx software, v4.1 (Waters Corp., Manchester, United Kingdom), was used for data collection associated with the TargetLyx program (Waters Corp., Manchester, United Kingdom). Additionally, the DA turnover was estimated to be the ratio DOPAC/DA.

Thank you once again for your valuable feedback.

4. Reviewer comment: Having addressed the above questions and remedied methodological shortcomings, recommends this publication for publication in the journal Sci. Pharm.

Authors’ Response: We sincerely appreciate the valuable comments of the reviewer, which have improved our paper. We have carefully responded to each comment and hope the paper will be suitable for publication in Scientia Pharmaceutica.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

I recommend accepting this manuscript after minor revisions. Here are some minor comments for consideration before it is accepted for publication: The manuscript presents compelling findings on the neuroprotective properties of Alpinia zerumbet extract against PCB-induced toxicity. With targeted revisions and expanded discussion of dose-response relationships, this work could become a valuable contribution to neuropharmacology research.

Specific Recommendations

1. Dosage Formatting
• Abstract: Insert a space between numerals and units in all dosage expressions (e.g., revise "50mg/Kg/day" → "50 mg/kg/day").
• Figures/Tables: Apply this correction to dosage labels in Figures 2–7 and Table 2 (e.g., "50mg/Kg/day" → "50 mg/kg/day").

 

2. Would it be possible to include a lower dose group (e.g., 1 mg/kg/once a week) in your experimental design? This could help determine whether the observed effects are dose-dependent and provide additional insight into the sensitivity of your behavioral outcomes.

Author Response

1. Reviewer comment: Dosage Formatting. • Abstract: Insert a space between numerals and units in all dosage expressions (e.g., revise "50mg/Kg/day" → "50 mg/kg/day"). • Figures/Tables: Apply this correction to dosage labels in Figures 2–7 and Table 2 (e.g., "50mg/Kg/day" → "50 mg/kg/day").

Authors’ Response: As recommended by the reviewer, we standardize dosage formatting. Thank you once again for your valuable feedback.

2. Reviewer comment: Would it be possible to include a lower dose group (e.g., 1 mg/kg/once a week) in your experimental design? This could help determine whether the observed effects are dose-dependent and provide additional insight into the sensitivity of your behavioral outcomes.

Authors’ Response: A previous study reported the dose-dependent effect of PCB in inducing cell death in neuroblastoma SH-SY5Y cells (Formisano et al. J Pharmacol Exp Ther. 2011; 338: 997-1003). However, in our research, we chose the dose of PCB 126 used (2 mg/kg) and the intraperitoneal route of administration based on its proven neurotoxicity in previous studies with a similar dosage of PCB congeners in adult animals (Selvakumar et al. Toxicol Lett. 2013; 222: 45-54; Venkataraman et al. Neurotoxicology. 2007; 28: 490-498). As our work involves investigating the neuroprotective effect of Alpinia zerumbet, we chose a dose of PCB that could cause neurotoxicity, following previous studies. We agree with the reviewer that a dose-dependent study of PCB 126 would be fascinating and a significant contribution to the literature. Therefore, we will conduct such an investigation in future studies. Thank you once again for your valuable feedback.

3. Reviewer comment: One of the oldest methods for making post hoc comparisons is known as Fisher’s least significant difference (LSD) test (also known as Fisher’s protected t).

Authors’ Response: We sincerely thank the reviewer for their insightful observation regarding clarifying the paper's statistical significance. Their feedback is invaluable to us. 

Previous studies have highlighted the statistical power of Fisher's LSD post-hoc test to highlight statistical differences between different experimental groups (Agbangba et al. 2024. Heliyon. 2024; 10: e25131; Assaad et al. 2015. Springerplus. 2015; 4: 33) . In the last 20 years, approximately 18.15% of research works used the Fisher LSD post-hoc test, as follows:

- Sreelatha et al. Neurol Int. 2024; 16: 1094-1111.

- Dutra-Tavares et al. Brain Sci. 2024; 14: 855.

- Pardo-Giménez et al. Journal of the Science of Food and Agriculture. 2016; 96: 3838-3845.

- Liao et al. Biomedicines. 2021; 9: 542.

-  Hamed et al. Environmental Pollution. 2024; 349: 123912.

- Adde-Michel et al. Neurosci Lett. 2005; 374: 109-112.

- Xu et al. Int J Biol Macromol. 2024; 277: 134404.

- Qinwufeng et al. J Ethnopharmacol. 2022; 295: 115428.

- Dietrich et al. Comp Biochem Physiol C Toxicol Pharmacol. 2011; 153: 422-429.

- Du et al. Food Bioscience. 2023; 53, 102555.

It's crucial to note that for the LSD post-test to be used effectively, an analysis of variance (ANOVA) must be conducted to identify any statistical differences and reject the null hypothesis. Additionally, the samples must pass the normality test (Agbangba et al. 2024. Heliyon. 2024; 10: e25131). Therefore, before using the LSD post-hoc test, we performed the Shapiro-Wilk normality test, and the samples used were under a normal distribution (added in the Material and Methods section). Moreover, we performed ANOVA to reject the null hypothesis. Thus, we demonstrate our unwavering commitment to adhering to the necessary recommendations for using the post-hoc LSD test. Given this, since the post-hoc LSD test has statistical power to perform statistical comparisons between the groups and given the number of recently published articles that use it, we decided to keep it as a post-hoc test in our analyses.

 

We sincerely appreciate the valuable comments of the reviewer, which have improved our paper. We have carefully responded to each comment and hope the paper will be suitable for publication in Scientia Pharmaceutica.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have given response to all the comments raised by the reviewers very well. Hence, the manuscript may be accepted for publication.

Comments on the Quality of English Language

-

Author Response

The authors thank the reviewer for his/her time and valuable suggestions. We have addressed the comments of this reviewer as follows:

1. Reviewer comment: The authors have given response to all the comments raised by the reviewers very well. Hence, the manuscript may be accepted for publication.

Authors’ response: We thank the reviewer for his valuable contributions, which helped improve our paper. We are very pleased that we have answered his questions satisfactorily. As requested by the reviewer, we have sent our manuscript for English editing to improve the understanding of our research and correct spelling errors. I am attaching the language editing certificate.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for submitting the revised version (v2) of your manuscript and for providing a point-by-point reply to the review. Your revision has substantially improved the clarity, methodological transparency and linguistic quality of the work. Below I summarise the outcome of my reassessment and list the remaining issues that should be addressed before the paper can be accepted.

1.1 Strengths of the revision

Reviewer request	Your action in v2	Outcome
Concise 200-word abstract, expansion of all abbreviations, and correct botanical nomenclature	Abstract trimmed to 199 words; first mentions now carry fully expanded terms and italicised species name	✔ Fully satisfied
Rationale for using a non-native plant species	New paragraph outlines naturalisation history of Alpinia zerumbet in Brazil and ethnopharmacological relevance, supported by three additional references	✔ Satisfied
Detailed Methodology (plant material, extraction, UHPLC-ESI-QTOF-MS, TBARS, SOD assays)	GPS coordinates added; extraction protocol clarified; full LC-MS method and chromatogram moved to Supplementary S1-S2; oxidative-stress assays described step-by-step	✔ Satisfied (see comment 1 below)
Dose justification for PCB 126 and ALE	Added toxicological rationale citing recent in-vivo studies	✔ Satisfied
Expanded Discussion (sex differences, pharmacokinetics, baseline ALE effects)	Three new paragraphs cover these items	✔ Satisfied
Consistency in units, typography	Professional language edit performed; °C and mg kg⁻¹ unified	✔ Satisfied, minor typos remain
Figure legends: statistical test and α level	p values moved into Results text	△ Partially satisfied (see comment 2)
Post-hoc test choice	LSD defended with literature support	✔ Acceptable, though more conservative tests would add rigour

1.2 Issues still requiring attention

1. Analytical-method validation
   Please provide, in Supplementary Materials, a validation table for the UHPLC-ESI-QTOF-MS method (linearity, accuracy, precision, LOD, LOQ). This is essential for reproducibility.
2. Figure legends
   Each legend should explicitly state
• the statistical test used,
• sample size (n per group),
• significance threshold (α), and
• explanation of symbols.
  Readers should not need to consult the main text to identify the test.
3. Bibliographic polish
   A few typographical errors persist in the reference list (e.g., missing accent in “Pharmacología”, inconsistent DOI capitalisation). Please run an automated reference check.
4. Statistics transparency (optional but recommended)
   Consider adding a supplementary table with results from a multiple-comparison-corrected test (e.g., Tukey HSD) to demonstrate that the conclusions are robust to stricter criteria.
5. Limitations
   In the final paragraph of the Discussion, explicitly mention:
• lack of female animal data;
• possible pharmacokinetic differences between oral and i.p. administration;
• need for dose-response studies.

Addressing the above points should require only minor textual additions and will further strengthen the manuscript.

Yours sincerely,

Reviewer

Author Response

The authors thank the reviewer for his/her time and valuable suggestions. We have addressed the comments of this reviewer as follows:

Thank you for submitting the revised version (v2) of your manuscript and for providing a point-by-point reply to the review. Your revision has substantially improved the clarity, methodological transparency and linguistic quality of the work. Below I summarise the outcome of my reassessment and list the remaining issues that should be addressed before the paper can be accepted.

1. Reviewer comment: Analytical-method validation

Please provide, in Supplementary Materials, a validation table for the UHPLC-ESI-QTOF-MS method (linearity, accuracy, precision, LOD, LOQ). This is essential for reproducibility.

Authors’ response: We thank the reviewer for his attention and careful review of our manuscript. We understand the time and effort it takes to review a manuscript, and we are grateful for his thorough feedback. As recommended by the reviewer, we have added the article's reference in the supplementary material, which deals with the characterization of the chemical composition, and the table with the information requested for the experiment's reproducibility.

2. Reviewer comment: Figure legends

Each legend should explicitly state the statistical test used, sample size (n per group), significance threshold (α), and explanation of symbols. Readers should not need to consult the main text to identify the test.

Authors’ Response: As recommended by the reviewer, we revised the figure legends and added all requested information as follows:

Figure 2. Effects of treatment with ALE (50 mg/kg/day) on locomotor activity in EPM (A) and OF (B) tests in C57BL/6 adult male mice exposed to PCB 126. Data are means ± SEMs n = 10 for all groups. *Significantly different (p < 0.05) from the Control group. #Significantly different (p < 0.05) from the Control+ALE group. +Significantly different (p < 0.05) from the PCB group. Significant values between the pairs of groups were examined by one-way ANOVA in GraphPad Prism analysis of LSD variance statistically significant difference test.

Figure 3. Effects of treatment with ALE (50 mg/kg/day) on lipid peroxidation (A) and SOD antioxidant activity (B) in left cerebral cortex homogenates from C57BL/6 adult male mice exposed to PCB 126. Data are means ± SEMs (n = 6-7 per group). *Significantly different (p < 0.05) from the Control group. #Significantly different (p < 0.05) from the Control+ALE group. +Significantly different (p < 0.05) from the PCB group. Significant values between the pairs of groups were examined by one-way ANOVA in GraphPad Prism analysis of LSD variance statistically significant difference test.  

Figure 4. Effects of treatment with ALE (50 mg/kg/day) on iNOS (A), TNF (B), and IL6 (C) gene expression in right cerebral cortex homogenates from C57BL/6 adult male mice exposed to PCB 126. Data are means ± SEMs (n = 5-6 per group). Significant values between the pairs of groups were examined by one-way ANOVA in GraphPad Prism analysis of LSD variance statistically significant difference test.

Figure 5. Effects of treatment with ALE (50 mg/kg/day) on BAX (A) and Bcl-2 (C) expression in the right cerebral cortex homogenates from C57BL/6 adult male mice exposed to PCB 126. Data are mean ± SEM (n = 5-6 per group). #Significantly different (p < 0.05) from the Control+ALE group. +Significantly different (p < 0.05) from the PCB group. Significant values between the pairs of groups were examined by one-way ANOVA in GraphPad Prism analysis of LSD variance statistically significant difference test.

Figure 6. Effects of treatment with ALE (50 mg/kg/day) on tyrosine (A), L-DOPA (B), DA (C), DOPAC (D), and DA turnover (E) levels in right cerebral cortex homogenates from C57BL/6 adult male mice exposed to PCB 126. Data are means ± SEMs (n = 5-6 per group). *Significantly different (p < 0.05) from the Control group. +Significantly different (p < 0.05) from the PCB group. Significant values between the pairs of groups were examined by one-way ANOVA in GraphPad Prism analysis of LSD variance statistically significant difference test. 

Figure 7. Effects of treatment with ALE (50 mg/kg/day) on D2 receptor expression in right cerebral cortex homogenates from C57BL/6 adult male mice exposed to PCB 126. Data are means ± SEMs (n = 5-6 per group). #Significantly different (p < 0.05) from the Control+ALE group. Significant values between the pairs of groups were examined by one-way ANOVA in GraphPad Prism analysis of LSD variance statistically significant difference test.

Thank you once again for your valuable feedback.

3. Reviewer comment: Bibliographic polish

A few typographical errors persist in the reference list (e.g., missing accent in “Pharmacología”, inconsistent DOI capitalisation). Please run an automated reference check.

Authors’ Response: We thank the reviewer for carefully evaluating the references in the bibliographic paper's list. As mentioned in the first review submitted, we used Zotero to format the references to strictly follow all the rules and avoid mistakes with manual formatting. However, since the reviewer still found errors after using Zotero to format the references, we checked them individually. Therefore, we identified no DOI in reference 57, which was corrected using Zotero.

Moreover, references 6, 42, 43, 57, and 61 do not have DOIs because they are book citations. We cited these references following the Scientia Pharmaceutica requirements for chapter book citations.

Finally, we checked references 33 and 34, which do not have DOIs, and found that Natural Product Communication, the journal in which these articles were published and we found that it does not provide a DOI number. We used their PMID to add these references to Zotero.

The typographical errors mentioned are unrelated to manually inserting the references, as we use Zotero for formatting.

We hope that these modifications and explanations have improved our bibliographic references, as requested by the reviewer.

4. Reviewer comment: Statistics transparency (optional but recommended)

Consider adding a supplementary table with results from a multiple-comparison-corrected test (e.g., Tukey HSD) to demonstrate that the conclusions are robust to stricter criteria.

Authors’ Response: We agree with the reviewer that transparency in our publications is essential. We sent the raw data from our analyses during the submission process and all membranes used in the Western blotting assays to meet this need. Regarding the statistical test, we based ourselves on data from the literature to use one-way ANOVA with Fisher's post-hoc test. In the first review of the paper, we demonstrated that previous studies have highlighted the statistical power of Fisher's LSD post-hoc test to highlight statistical differences between different experimental groups (Agbangba et al. 2024. Heliyon. 2024; 10: e25131; Assaad et al. 2015. Springerplus. 2015; 4: 33). Moreover, we strictly followed the rules for using the LSD post-hoc test, which demonstrates that the statistical differences found are robust. Therefore, we relied on data from the literature to decide not to perform the Tucker post-test on our data and to make it available as supplementary material.

5. Reviewer comment: Limitations

In the final paragraph of the Discussion, explicitly mention: lack of female animal data; possible pharmacokinetic differences between oral and i.p. administration; need for dose-response studies.

Authors’ Response: We thank the reviewer for pointing out the need for improvement regarding the study's limitations. As recommended by the reviewer, we have improved this paragraph as follows:

Despite the promising findings in our research, the use of male mice is a limitation since there is a lack of female animal data. Another essential point of discussion is the striking differences between the intraperitoneal and oral administration routes. The intraperitoneal route used for the administration of PCB 126 in our study has the advantage of enabling chronic administration of substances in mice since continuous administration via the intravenous route is a challenge. In addition, compounds administered via the intraperitoneal route avoid passing through the gastrointestinal tract, during which degradation or modification may occur (Al Shoyaib et al. Pharm Res. 2019; 37: 12), a key characteristic for administering PCBs that promotes their toxic effects when they exceed the body's metabolization capacity. Thus, intraperitoneal administration of this chemical compound enables its arrival in the CNS and the development of its neurotoxic effects. In contrast, oral administration of ALE through drinking water, despite causing it to pass through the gastrointestinal tract and subjecting the extract to metabolism, also allows its consumption throughout the day, an essential characteristic for choosing oral administration since our group has not yet investigated the bioavailability and half-life of ALE.

Finally, a previous study reported the dose-dependent effect of PCB in inducing cell death in neuroblastoma SH-SY5Y cells (Formisano et al. J Pharmacol Exp Ther. 2011; 338: 997-1003). As our work involves investigating the neuroprotective effect of Alpinia zerumbet, we chose a dose of PCB that could cause neurotoxicity, following previous studies (Selvakumar et al. Toxicol Lett. 2013; 222: 45-54; Venkataraman et al. Neurotoxicology. 2007; 28: 490-498). However, a dose-dependent study of PCB 126 would be fascinating and a significant contribution to the literature. Therefore, further studies are needed to assess ALE's potential for chronic use and better understand its pharmacokinetics characteristics and therapeutic prospects in managing PCB-induced dose-dependent neurotoxicity and sex differences.

6. Reviewer comment: Addressing the above points should require only minor textual additions and will further strengthen the manuscript.

Authors’ Response: We sincerely appreciate the valuable comments of the reviewer, which have improved our paper. We have carefully responded to each comment and hope the paper will be suitable for publication in Scientia Pharmaceutica.

Author Response File: Author Response.pdf