In Vivo Assessment of Anti-Inflammatory Effects of Aqueous Extracts of Nepeta nuda ssp. nuda L. in Experimental Model of Peripheral Inflammation in Male Long Evans Rats

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the article submitted for review, the authors investigated the possible anti-inflammatory and analgesic effects of aqueous extracts from the flowers of the medicinal plant Nepeta nuda ssp. nuda L. (naked catnip) in a model of acute peripheral inflammation induced by subcutaneous injection of λ-carrageenan in the hind paw of Long Evans rats. Currently available anti-inflammatory drugs, particularly non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids, have many side effects, including the risk of stomach, liver, or kidney damage with prolonged use. Therefore, there is a significant need to develop new, safer anti-inflammatory therapies, particularly those derived from natural sources. In this context, the study of plant extracts such as Nepeta nuda is an interesting alternative.

For the article to be further processed, I suggest the following corrections:

1. Please provide the exact number of rats in the study and how they were divided into experimental groups.
2. Please include a diagram showing the course of the experiment, groups, doses, and routes of administration of the extract.
3. Please specify the AUC units in the graphs (Figures 1b, 2b).
4. Discuss both the strengths and limitations of the study.
5. In the conclusions section, please indicate the optimal doses, the possibilities for extrapolating the results to clinical trials, and specify directions for further research.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The Introduction repeats the same concepts multiple times: Acute vs chronic inflammation explained twice, Mediators (histamine, bradykinin, prostaglandins) described repeatedly, Nepeta metabolites and their activities are listed, then listed again in the Discussion.

No justification for why 2.5/25 mg/kg and 50/200 mg/kg were chosen.
The authors say “based on in vitro studies”, but provide zero rationale linking in vitro concentrations to in vivo mg/kg doses. 

Only male rats used. Sex bias is not justified. No explanation why females were excluded.

For behavioral studies, n=6 is underpowered and weak for statistical claims.

The study compares multiple groups at multiple time points, but uses t-tests instead of ANOVA + post-hoc correction. This inflates false positives - major statistical flaw.

Reporting p-values is inconsistent - Sometimes the authirs p-values, sometimes only symbols.
Sometimes “not shown in figures” is written - this is unacceptable.

Figures are overloaded and hard to read

Claim: “effect evident at 1st hour” lacks statistical strength. In systemic group, authors claim early effect, but the data seems marginal. Statistical robustness is questionable.

“suggesting central mechanisms” - but no central mechanism experiment was performed. No antagonists, no CNS biomarkers. Pure speculation.

The authors dump huge lists of metabolites and their possible actions (terpenes, flavonoids, iridoids), but do not link these to actual concentrations in their extract.

Comments on the Quality of English Language

There are many grammatical errors, inconsistent tenses, and awkward formulations. The English requires major, full-manuscript editing by someone fluent.

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript presents an in vivo study evaluating the anti-inflammatory effects of aqueous extracts of Nepeta nuda ssp. nuda L. (naked catmint) in a model of acute peripheral inflammation induced by carrageenan in male Long Evans rats. The authors test two routes of administration at different dosages, measuring paw oedema and spontaneous locomotor activity. The results show significant anti-inflammatory effects with different temporal patterns compared to diclofenac (positive control), suggesting different mechanisms of action between the routes of administration. An analgesic effect on local administration is also reported.

- (Line 159): It is stated that the rats were ‘randomly divided into nine groups of six animals each’, but the total number should be 54 animals considering that the ‘control group with intraplantar injection of vehicle’ is ‘common to both routes of administration’ (lines 162-164). This needs to be clarified. I suggest specifying exactly how many animals were used in total.

- line 312 says ‘Ne. nuda’ instead of ‘N. nuda’

- Lines 153-157: Intraplantar route: 0.25 mg/rat; Intraperitoneal route: 25 mg/kg. These doses are not equipotent or comparable. For a rat weighing approximately 360 g (0.36 kg), 25 mg/kg = ~9 mg, which is very different from 0.25 mg. This compromises the comparison. I suggest justifying the doses chosen and/or reconsidering the comparison doses to make them more equipotent.

- All data on the metabolic composition of the extract (GC-MS, UHPLC-LTQ OrbiTrap MS, NMR, total phenolics and flavonoids) are relegated to supplementary materials. I would recommend including at least one table in the main text summarising the main active components with their percentage concentrations. This is essential for the understanding and reproducibility of the study.

- The extract preparation protocol is described, but critical information is missing: Extraction yield (%); Extract stability during the study period, etc.

- The entire discussion on mechanisms (histamine, prostaglandins, neutrophils, free radicals) is purely speculative. I suggest either conducting further experiments, such as measuring cytokine levels (TNF-α, IL-6, IL-1β) or quantifying prostaglandins (PGE2) in plasma or paw tissue, or making these statements in the text less robust and more like a future prospect.

- A ‘Limitations’ section is completely absent.

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Although the revised manuscript is improved in presentation, several major scientific and methodological concerns remain unresolved.

The Introduction continues to repeat the same concepts multiple times: 

- Acute vs. chronic inflammation is explained at least twice (lines 41–56 and again in lines 46–54)

- Early mediators (histamine, bradykinin, leukotrienes, complement, substance P) are described repeatedly (lines 57–61 and again in Discussion)

- The catalogue of Nepeta metabolites and their activities (phenolics, iridoids, nepetalactones, polyphenols) appears in the Introduction (lines 72–82) and is again discussed in detail in the Discussion (lines 369–383 and 416–462).

The authors state that the doses (2.5/25 mg/kg intraplantar and 50/200 mg/kg intraperitoneal) were chosen: “based on previously conducted in vitro studies, showing 100% inhibitory activity at 2 mg/mL” (lines 154–159, 387–389) - However, no pharmacological rationale is provided for translating in vitro concentrations to in vivo mg/kg doses. No references, scaling assumptions, or prior in vivo data are cited.

Only male rats were included (lines 129–136), with no scientific justification for excluding females. Current ARRIVE guidelines and NIH policies require justification when a study uses a single sex.

All behavioral and locomotor activity analyses are based on n = 6 per group (e.g., Figures 4–5). For behavioral work, especially with multi-group comparison n = 6 is too low to support strong statistical claims and increases the risk of Type I/II error.

The authors use repeated t-tests for intergroup and time-point comparisons (lines 219–226). This approach inflates false positives and violates assumptions of independence. Without proper ANOVA, many of the reported “significant” effects are not statistically credible. This remains the most critical issue to address before acceptance.

Reporting of p-values is inconsistent and unclear.

Figures contain multiple panels, symbols, percentages, AUC values, and multiple layers of annotation. Legibility is reduced, especially in Figure 2 and Figure 3.

“the anti-inflammatory effect… was evident even at the 1st hour” (lines 287–289). ? Toner down claims or provide mechanistic explanation plus correct statistical validation.

The manuscript presents interesting preliminary findings, but significant methodological and interpretive issues remain unaddressed. The most urgent problems are the inadequate statistical analysis, unjustified dosing, sex bias, and overinterpretation of results.

The manuscript presents interesting preliminary findings, but significant methodological and interpretive issues remain unaddressed.

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The authors responded clearly to all requests made. 

Author Response

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Author Response File: Author Response.docx

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript shows some improvement in presentation and organization compared to the previous versions. The authors have made efforts to respond to several of my comments, including adding methodological clarifications and revising parts of the Discussion. However, several major scientific and methodological concerns remain insufficiently addressed, and these issues substantially limit the strength and credibility of the conclusions.

Further streamline the Introduction to focus strictly on study rationale and objectives. General background material should be reduced, and mechanistic discussion should be confined to interpretation of the presented results.

The authors have expanded the explanation of dose selection; however, the rationale remains qualitative rather than pharmacologically grounded. The link between in vitro concentrations and in vivo doses is still weak, and no scaling assumptions, exposure considerations, or bioavailability arguments are provided.

Behavioral data are based on n = 6 animals per group, which is low for multi-group behavioral comparisons. While this sample size is commonly used in screening studies, it limits statistical power and increases the risk of both Type I and Type II errors. The authors should clearly frame the behavioral findings as exploratory and avoid strong claims regarding analgesic efficacy. This limitation should be explicitly acknowledged.

The authors state that one-way ANOVA with post hoc testing was applied; however, the statistical approach remains insufficiently transparent, particularly given the number of groups and repeated time-point comparisons. It is not always clear which comparisons were included in each analysis, and how multiplicity was controlled across time points.

Claims regarding anti-inflammatory effects at the first hour post-carrageenan injection have been moderated but remain borderline overstated given the statistical strength and variability of the data.

Further reduce speculative metabolite-based interpretations or clearly frame them as hypotheses for future work.

Comments on the Quality of English Language

The manuscript is generally understandable; however, the quality of the English language requires small improvement. The text contains numerous grammatical inconsistencies, awkward sentence constructions, incorrect verb tenses, missing articles, and non-native phrasing. In several sections, particularly in the Results and Discussion, overly long and complex sentences reduce clarity and readability.

Author Response

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