Melampyrum nemorosum L. Herb Extracts: Phytochemical Composition and Screening of Pharmacological Activities
, Ain Raal 5 and Oleh Koshovyi 5,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Authors,
The article "Melampyrum nemorosum L. herb extracts: phytochemical composition and screening of pharmacological activities" is undoubtedly interesting, as it presents experimental data on phytochemical and pharmacological studies of extracts that have certain therapeutic potential.
It should be noted that the level of obtaining and presenting experimental data is quite high.
However, there are some debatable questions.
In the “Materials and Methods” section or in the “Results” section, it is necessary to indicate the yield of the extract.
In the experimental part, the preparation of a liquid extract is described, and apparently, all studies were carried out with liquid extracts. In this case, the logic of calculating biologically active substances in Table 1 (Content in the dry residue of the extract, μg/g) is not clear.
In my opinion, if the extract is intended to be used in liquid form, it is necessary to calculate the content per 1 mL of extract. If the composition of the liquid extract varies and dry extracts are more stable, then the technology of their preparation should be described, and all experiments should be carried out with dry extracts (this is much more convenient from a technological point of view, solving issues of stability and standardization).
In the “Materials and Methods” section, indicate the series and manufacturers of all reagents, reference standards used in the phytochemical studies, as well as reagents, comparison drugs, and strains of microorganisms used in pharmacological tests.
Line 221: “solution of novocaine” – possibly procaine hydrochloride? In any case, it is necessary to indicate the manufacturer and batch number for the substance or product used.
In the Wound healing and haemostatic activity section, the method of application and the amount of drugs/substances used are not specified.
On what basis are the standardization data presented? For this, at least 6 batches of extracts are needed. How were they obtained? From the same type of raw material on different days? Or from different samples of collected material? This should be described in the “Materials and Methods” section.
The indicators of a single extract cannot be presented as standardization results, even preliminary ones. Rather, this subsection should be renamed as quality indicators of the obtained extracts. They are better presented in a table, and the data should be statistically processed. In the “Materials and Methods” section, it is also necessary to provide references to the methods used (e.g., pharmacopoeial) for selecting and measuring/evaluating the standardization parameters of the obtained extracts.
The citations of sources 20, 25–27, 32, 33, 41 look like a self-citation. I would recommend removing these sources from the references list.
I think that manuscript needs minor revisions
Author Response
The manuscript's authors are grateful to the reviewer and editors for their useful and inspiring comments and suggestions. We have taken them into account and hope that this improved the quality of our article and that it meets the high scientific standards of the journal and allows us to publish the results of our research.
The article "Melampyrum nemorosum L. herb extracts: phytochemical composition and screening of pharmacological activities" is undoubtedly interesting, as it presents experimental data on phytochemical and pharmacological studies of extracts that have certain therapeutic potential.
It should be noted that the level of obtaining and presenting experimental data is quite high.
However, there are some debatable questions.
Comment 1. In the “Materials and Methods” section or in the “Results” section, it is necessary to indicate the yield of the extract. In the experimental part, the preparation of a liquid extract is described, and apparently, all studies were carried out with liquid extracts. In this case, the logic of calculating biologically active substances in Table 1 (Content in the dry residue of the extract, μg/g) is not clear.
Response 1. Thank you for your comment. We prepared 1:1 liquid extracts. This means, for example, that 1 kg of raw material yields 1 kg of a liquid extract (after appropriate concentration), so discussing the yield in this case is not quite accurate. It is more appropriate to indicate the dry residue content in the liquid extract (MN40 – 13,65%; MN70 – 12,22%). This information is provided in the manuscript. We recalculated the content of the determined substances in the liquid extracts and updated Table 1 accordingly.
Comment 2. In my opinion, if the extract is intended to be used in liquid form, it is necessary to calculate the content per 1 mL of extract. If the composition of the liquid extract varies and dry extracts are more stable, then the technology of their preparation should be described, and all experiments should be carried out with dry extracts (this is much more convenient from a technological point of view, solving issues of stability and standardization).
Response 2. Thank you for your comment. Liquid extracts are a widely used and popular dosage form in Ukraine (and in many other countries). Domestic manufacturers produce liquid extracts of valerian, echinacea, eleutherococcus, Rhodiola rosea, and other medicinal plants, typically packaged in 30–50 mL vials ready for use. Considering this, we selected liquid extracts for our study, as more pharmaceutical companies possess the necessary manufacturing capacity and licenses for this form. We recalculated the content of active substances per 1 mL of the extracts. While we acknowledge that dry extracts are generally more convenient and stable, liquid extracts are preferable in this case due to their ease of external application in wound healing.
Comment 3. In the “Materials and Methods” section, indicate the series and manufacturers of all reagents, reference standards used in the phytochemical studies, as well as reagents, comparison drugs, and strains of microorganisms used in pharmacological tests.
Line 221: “solution of novocaine” – possibly procaine hydrochloride? In any case, it is necessary to indicate the manufacturer and batch number for the substance or product used.
Response 3. Thank you for the comments. The information has been added. We used a 2% solution of novocaine (batch number 2308-011B, Novocaine-Zdorovye, LLC "Pharmaceutical Company Zdorovye", Kharkiv, Ukraine), in which the active ingredient is procaine hydrochloride. Clinical strains of microorganisms were isolated from patient samples and subsequently identified using standardized microbiological methods.
Comment 4. In the Wound healing and haemostatic activity section, the method of application and the amount of drugs/substances used are not specified.
Response 4. Thank you for this comment. All extracts were applied topically directly onto the wound area once daily. Each wound was treated with 0.2 mL of the respective extract per application. The information was added to the manuscript.
Comment 5. On what basis are the standardization data presented? For this, at least 6 batches of extracts are needed. How were they obtained? From the same type of raw material on different days? Or from different samples of collected material? This should be described in the “Materials and Methods” section.
The indicators of a single extract cannot be presented as standardization results, even preliminary ones. Rather, this subsection should be renamed as quality indicators of the obtained extracts. They are better presented in a table, and the data should be statistically processed. In the “Materials and Methods” section, it is also necessary to provide references to the methods used (e.g., pharmacopoeial) for selecting and measuring/evaluating the standardization parameters of the obtained extracts.
Response 5. Thank you for the comments. Three batches of extracts from different raw materials harvested in the same year were prepared to determine their quality indicators. The relevant information and table have been added to the manuscript. The title of the subsection has been changed. The applied methods are presented in the European Pharmacopoeia, and the corresponding references have been included.
Comment 6. The citations of sources 20, 25–27, 32, 33, 41 look like a self-citation. I would recommend removing these sources from the references list.
Response 6. Thank you for the comments. We have reviewed the references and removed several of them (25-27, 33 and 41). However, we would like to emphasize that the level of self-citation remains within the acceptable limits.
Comment 7. I think that manuscript needs minor revisions
Response 7. Thanks once again. The manuscript passed the minor revisions by us accordingly to your useful comments.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe paper of Reznik et al. “Melampyrum nemorosum L. herb extracts: phytochemical …” aimed to study chemical composition and bioactivity of Melampyrum nemorosum extracts. The presented data have rather weak scientific novelty, although they have practical significance.
Highlights and strengths of the manuscript are:
New data about Melampyrum nemorosum may further increase the interest in this species as new source of bioactive drugs.
Specific comments and suggested revisions:
- It is not clear to me why 40% and 70% ethanol were chosen as the extractant. Have there been any preliminary studies to show the effectiveness of the extraction? and why are we talking about two extracts from one object? Are you suggesting two extracts for use? How will one differ from the other?
- The results of HPLC characterization and quantification were already published in https://doi.org/10.11603/2312-0967.2024.4.14982
- Neither the earlier work nor the present manuscript provides a method for identifying the compounds. The HPLC-DAD assay you used does not allow for reliable structural identification of metabolites.
- The results of flavonoid quantification by HPLC and spectrophotometry do not correspond to each other. I understand that they should not match, but it is desirable that the values are close. HPLC values only for rutin were 3.4% (~33885 mcg/g) and 4.1% (~40992 mcg/g) but spectrophotometry gave total content values 0.86% and 1.01%. Such differences indicate a basic error in the analysis and unreliability of the results.
- Section 3.2 is a jumbled list of facts without any experimental process. The data presented does not contain information on standard deviation. In this regard, it is unclear how reliable they are.
- Line 319: “which corresponds to substances considered practically non-toxic (LDâ‚…â‚€ ≥ 5000 mg/kg) [46]” Can’t find this ref. [Reznik, V.V.; Grytsyk, A.R. STUDY OF ACUTE TOXICITY OF EXTRACTS FROM WOOD COW-WHEAT (MELAMPYRUM NEMOROSUM L.). Pharmaceutical Review 2025, 2, doi:https://doi.org/10.11603/2312-0967.2025.2.15392]. Google couldn't help me with this.
- Table 2. According to your data, it turns out that quercetin at a dose of 5 mg/kg is more effective than diclofenac at a dose of 8 mg/kg. Are you sure this isn't a typo?
- Table 3. I can't find the data table for the drug Rotokan. But there is a strange group of Liquid extract of Polygonum hydropiper. Please, correct. Why do you use non-standardized extract as a reference drug?
With all due respect to authors, I see no possibility to recommend paper for publication. Authors should clearly formulate the purpose of the work, remove unnecessary material, add correct and reliable data and a clear discussion.
Author Response
The manuscript's authors are grateful to the reviewer and editors for their useful and inspiring comments and suggestions. We have taken them into account and hope that this improved the quality of our article and that it meets the high scientific standards of the journal and allows us to publish the results of our research.
Comment 1. The paper of Reznik et al. “Melampyrum nemorosum L. herb extracts: phytochemical …” aimed to study chemical composition and bioactivity of Melampyrum nemorosum extracts. The presented data have rather weak scientific novelty, although they have practical significance.
Response 1. Thank you for the comment. This plant is widely distributed across the European zone; however, there is a notable lack of publications specifically dedicated to the species Melampyrum nemorosum L. In most cases, it is mentioned only briefly within broader studies on the genus. For instance, a search in the Scopus database using this species as a keyword yields only 25 publications, of which merely 5 focus on its phytochemical and pharmacological aspects, and even those date back to 2000–2010. Moreover, the hemostatic, wound-healing, and antimicrobial activities of extracts from this species have not been investigated in the accessible literature. Therefore, the presented data possess clear scientific novelty.
Highlights and strengths of the manuscript are:
New data about Melampyrum nemorosum may further increase the interest in this species as a new source of bioactive drugs.
Specific comments and suggested revisions:
Comment 2. It is not clear to me why 40% and 70% ethanol were chosen as the extractant. Have there been any preliminary studies to show the effectiveness of the extraction? and why are we talking about two extracts from one object? Are you suggesting two extracts for use? How will one differ from the other?
Response 2. Thank you for the comment. We based our approach on the traditions of folk medicine. Typically, folk remedies involve infusions prepared with vodka (40% ethanol), whereas official pharmaceutical practice favours tinctures made using a 70% ethanol solution. Therefore, these two solvents were selected to obtain liquid extracts in order to scientifically validate the ethnopharmacological use of this raw material. The analysis results, as well as our previous experiences, indicate that 70% ethanol provides greater extraction of flavonoids and phenolic compounds overall. Furthermore, in all conducted pharmacological studies, this extract proved to be more effective. These findings are discussed in the Discussion section.
Comment 3. The results of HPLC characterization and quantification were already published in https://doi.org/10.11603/2312-0967.2024.4.14982
Response 3. Thank you for the comment. Yes, this is a publication by one of our co-authors (Reznik V, a PhD student), but it presents a completely different research subject. The study focused on the herb Melampyrum nemorosum, and extraction was performed using 80% ethanol in accordance with the method for quantitative determination of phenolic compounds in the herb. The primary focus of that article was the investigation of the herb as a medicinal raw material. The article was published in a local Ukrainian journal in the Ukrainian language. In contrast, the object of study in our current work was the liquid extracts derived from the same raw material, obtained using 40% and 70% ethanol solutions. Although we employed the same equipment and analytical approaches, the research subjects are fundamentally different. Moreover, it is not entirely appropriate to discuss the pharmacological activity of extracts without presenting phytochemical data. Therefore, we conducted a detailed analysis of the phenolic compounds in our extracts and reported the results in this manuscript.
Comment 3. Neither the earlier work nor the present manuscript provides a method for identifying the compounds. The HPLC-DAD assay you used does not allow for reliable structural identification of metabolites.
Response 3. Thank you for the comment. We identified these compounds by comparison with reference standards available to us. This information is presented in lines 181–187.
Comment 4. The results of flavonoid quantification by HPLC and spectrophotometry do not correspond to each other. I understand that they should not match, but it is desirable that the values are close. HPLC values only for rutin were 3.4% (~33885 mcg/g) and 4.1% (~40992 mcg/g), but spectrophotometry gave total content values 0.86% and 1.01%. Such differences indicate a basic error in the analysis and unreliability of the results.
Response 4. Thank you very much for your valuable comment. Regarding the individual compounds, in the previous version of the manuscript, we presented their contents in the dry residue of the liquid extract, while the spectrophotometric measurements were carried out on the liquid extract itself. This discrepancy resulted in the inconsistency. We have now recalculated our results and expressed all compound contents relative to the liquid extract. The corresponding changes have been made in Table 1.
Comment 5. Section 3.2 is a jumbled list of facts without any experimental process. The data presented does not contain information on standard deviation. In this regard, it is unclear how reliable they are.
Response 5. Thank you for the comments. Three batches of extracts from different raw materials harvested in the same year were prepared to determine their quality indicators. The relevant information and Table 2 have been added to the manuscript.
Comment 6. Line 319: “which corresponds to substances considered practically non-toxic (LDâ‚…â‚€ ≥ 5000 mg/kg) [46]” Can’t find this ref. [Reznik, V.V.; Grytsyk, A.R. STUDY OF ACUTE TOXICITY OF EXTRACTS FROM WOOD COW-WHEAT (MELAMPYRUM NEMOROSUM L.). Pharmaceutical Review 2025, 2, doi:https://doi.org/10.11603/2312-0967.2025.2.15392]. Google couldn't help me with this.
Response 6. Thank you for the comment. In this article, our co-authors describe a study on the toxicity of the investigated extracts. The manuscript has been accepted for publication, and the editorial office has provided us with the citation details. It is scheduled to appear in the second issue of the journal (https://ojs.tdmu.edu.ua/index.php/pharm-chas/issue/archive). The publication was expected by the end of June; however, it appears that technical delays or the summer holiday period may have affected the timeline. We believe the article will be published in the coming days, and the DOI will become active shortly. Nevertheless, due to time constraints, we have removed the sentence and the corresponding reference.
Comment 7. Table 2. According to your data, it turns out that quercetin at a dose of 5 mg/kg is more effective than diclofenac at a dose of 8 mg/kg. Are you sure this isn't a typo?
Response 7. Thank you very much for your attentive observation. We understand the concern regarding the apparent efficacy of quercetin at a dose of 5 mg/kg compared to diclofenac sodium at 8 mg/kg. However, this is not a typo. While diclofenac demonstrated a stronger suppression index across all time points, quercetin also showed significant anti-inflammatory activity, particularly at the 3- and 5-hour marks. Diclofenac reduces swelling by 60.69%, while quercetin by 37.3%, and this trend holds for all parameters. We attribute this effect to the pharmacological properties of quercetin, including its antioxidant and modulatory actions on inflammatory pathways. Nevertheless, we have clarified this point in the revised version of the manuscript to avoid potential misunderstanding and have emphasized that diclofenac remains more potent overall based on the suppression index values.
Comment 8. Table 3. I can't find the data table for the drug Rotokan. But there is a strange group of Liquid extract of Polygonum hydropiper. Please, correct. Why do you use non-standardized extract as a reference drug?
Response 8. Thank you for your comment. Liquid extracts are a widely used and popular dosage form in Ukraine. Domestic manufacturers produce liquid extracts of valerian, echinacea, eleutherococcus, Rhodiola rosea, and other medicinal plants, typically packaged in 30–50 mL vials ready for use. Rotokan is the Liquid extract (1:1) from medicinal plant raw materials: chamomile flowers (flores chamomillae), calendula flowers (flores calendulae), and yarrow herb (herba millefolii) in a ratio of 2:1:1. These two herbal preparations are produced by several Ukrainian manufacturers and are freely available in pharmacies in Ukraine, so they are considered standardized.
Rotokan: https://compendium.com.ua/info/6090/rotokan/
Liquid extract of Polygonum hydropiper: https://compendium.com.ua/info/59170/21283/
Comment 8. With all due respect to authors, I see no possibility to recommend paper for publication. Authors should clearly formulate the purpose of the work, remove unnecessary material, add correct and reliable data and a clear discussion.
Response 8. The authors of the manuscript thank you very much for this recommendation.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript titled “Melampyrum nemorosum L. herb extracts: phytochemical composition and screening of pharmacological activities” presents a novel in vivo pharmacological application of the Melampyrum nemorosum herb. However, I have included several suggestions in the attached document to help improve the scientific rigor and clarity of the work.
Comments for author File: 
Comments.pdf
Author Response
The manuscript's authors are grateful to the reviewer and editors for their useful and inspiring comments and suggestions. We have taken them into account and hope that this improved the quality of our article and that it meets the high scientific standards of the journal and allows us to publish the results of our research.
Comment 1. Table 1: Please include a statistical comparison (e.g., t-test or Tukey Kramer test) of the mean phenolic compound content between both samples.
Response 1. Thank you for your comment. The comparison has been conducted, and the relevant information has been added to Table 1.
Comment 2. Section 3.2 – Preliminary standardization of the extracts: This section contains methodological details that would be more appropriately placed in Section 2 (materials and methods). In section 3, the authors should focus on presenting and interpreting the observed results more precisely—for instance, by providing specific numerical values and describing the types and quantities of microorganisms found in the extracts.
Response 2. Thank you very much for your recommendations. We have revised the manuscript accordingly and added Table 2 titled “Results of Determining Quality Indicators in the Liquid Extracts of Melampyrum nemorosum Herb.”
Comment 3. The methodological content in this section should be relocated to Section 2.4.2, where experimental procedures are described.
Response 3. Thank you for your comment. This was corrected.
Comment 4. Table 4: It would be helpful to report either the range or the mean wound healing area for animals in each group at the different time points.
Response 4. Thank you for your comment. We have revised the title to “Percentage (%) of Wound Area Healing Compared to the Initial State Over Time (Days),” which more clearly describes the data presented.
Comment 5. Section 3.6: Please include a descriptive summary of the most significant results from Table 5.
Response 5. Thank you for your comment. This part was added.
Comment 6. Section 4 – Discussion (Lines 377–420): The discussion only qualitatively addresses the anti-inflammatory activity of the identified compounds. However, it lacks commentary on differences in the quantitative content of these compounds when compared to previous studies involving the same herb sourced from different regions or extracted using different methods.
Response 6. Thank you for your comment. We did not find similar data in the literature available to us. There is a notable lack of publications specifically dedicated to the species Melampyrum nemorosum L. In most cases, it is mentioned only briefly within broader studies on the genus. For instance, a search in the Scopus database using this species as a keyword yields only 25 publications, of which merely 5 focus on its phytochemical and pharmacological aspects, and even those date back to 2000–2010. Therefore, the presented data possess clear scientific novelty.
Comment 7. Line 437: This statement could be substantiated through a statistical comparison of the individual and total phenolic compound content between the two extracts analyzed in this study.
Response 7. Thank you for your comment. This part was added “Statistical analysis of the content of the main identified phenolic compounds (Table 1) revealed significant differences in the levels of chlorogenic acid, caffeic acid, p-coumaric acid, trans-ferulic acid, trans-cinnamic acid, rutin, naringin, neohesperidin, quercetin, naringenin, apigenin, rhamnetin, kaempferol, catechin, and gallocatechin in the extracts MN40 and MN70. Likewise, the content of flavonoids and total polyphenols in the liquid extract, as determined by spectrophotometry, differed significantly. Their concentrations were higher in the MN70 extract.”
Comment 8. Lines 457 and 459: When discussing the effect on bleeding time, please include the doses used in previous studies with extracts from other herbs.
Response 8. Thank you for your comment. This is a standard method. All extracts were applied topically to the wound area once daily. Each wound received 0.2 mL of the respective extract per application. This information has been added to the manuscript (lines 270–271).
Comment 9. Lines 483 and 486: Similarly, specify the doses employed in earlier studies investigating the wound healing effects of other herb extracts.
Response 9. Thank you for your comment. Following the determination of bleeding time, wound healing dynamics were examined using the same groups of animals (Table 5). Accordingly, the dosage and application method remained consistent. This information has been incorporated into the manuscript (lines 406–407).
Comment 10. Please ensure that the formatting of all references is consistent and adheres to the journal’s citation guidelines. Some entries differ in style, punctuation, or order of elements.
Response 10. Thank you for your comment. We used the automatic referencing program Zotero and have double-checked all the references.
Reviewer 4 Report
Comments and Suggestions for AuthorsGeneral Comment for the Authors:
The manuscript provides a strong multidisciplinary approach to evaluating M. nemorosum, combining phytochemical profiling with pharmacological assessments. The standardization of extracts and use of appropriate in vivo and in vitro models are commendable. The discussion effectively links compound classes with bioactivities, although it would benefit from added precision regarding dose relationships, statistical significance between extract types, and mechanistic insights into antimicrobial effects. A concise limitations section would further strengthen the work. Overall, the study is novel, well-executed, and of interest to the field.
Specific comments:
Abstract:
Please check the English language and grammar.
Introduction
Lines 57 – 62: The list of biologically active compounds is comprehensive, but the sentence structure is overly long and dense. Please break it into two or more sentences.
Lines 86 – 87: The absence of Melampyrum species from pharmacopeias is a strong point; consider moving this earlier in the paragraph discussing the lack of validated data.
Materials and Methods
Lines 98 – 99: What was the altitude (this can influence secondary metabolite content)?
Line 115 – The quantity of raw material (1000 g) is noted, but it's unclear whether this is fresh or dried weight—please clarify.
Results
Lines 301 – 310: Consider removing the clarification of methodology in Material and methodology section of the manuscript.
Table 2: Please add units for edema measurements (e.g., mL or % increase), a column for sample size (n) per group and clarification on whether values are means ± standard deviation or standard error.
Table 3: Please clarify whether values are means ± standard deviation or standard error.
Table 3 and 4: Mention the statistical test used and apply significance indicators to Table 4 as well, especially to compare different treatment groups.
Section 3.5.: Was the same set of animals used for both bleeding time and wound healing evaluation, or if they were conducted in parallel on different groups?
Section 3.6.
Table 5: Table 5 is dense and hard to navigate. Consider dividing it into two separate tables: one for antibacterial activity and one for antifungal activity.
Please clearly define the control used for comparison (solvent control? ethanol control?), as the effectiveness of extracts is assessed relative to this.
The differences between MN40 and MN70 are notable, particularly with MRSA and Candida albicans. Please give a brief interpretation of which compounds might be contributing to this enhanced effect (e.g., iridoids, flavonoids, phenolics) and briefly compare the results.
Discussion
Lines 421 – 433: Are the differences between MN70 and MN40 statistically significant? If so, mention the p-values or significance levels in the discussion text.
Lines 467 – 494: Were there any adverse reactions or irritation noted with topical application of extracts?
Lines 459 – 509: Group these pathogens into a summary (e.g., MRSA, ESBL-producing E. coli, fluconazole-resistant Candida, etc.) to avoid an overly long list.
Consider adding a short paragraph acknowledging limitations (e.g., no histological analysis, lack of chronic model, not yet tested in human cells).
Comments for author File: Comments.pdf
Author Response
The manuscript's authors are grateful to the reviewer and editors for their useful and inspiring comments and suggestions. We have taken them into account and hope that this improved the quality of our article and that it meets the high scientific standards of the journal and allows us to publish the results of our research.
General Comment for the Authors:
Comment 1. The manuscript provides a strong multidisciplinary approach to evaluating M. nemorosum, combining phytochemical profiling with pharmacological assessments. The standardization of extracts and use of appropriate in vivo and in vitro models are commendable. The discussion effectively links compound classes with bioactivities, although it would benefit from added precision regarding dose relationships, statistical significance between extract types, and mechanistic insights into antimicrobial effects. A concise limitations section would further strengthen the work. Overall, the study is novel, well-executed, and of interest to the field.
Response 1. Thank you for the valuable suggestions. We will take them into account in our future research. We have included a section addressing the limitations and incorporated it into the discussion.
Specific comments:
Abstract:
Comment 2. Please check the English language and grammar.
Response 2. Thank you very much. The English language and grammar of the abstract have been checked and corrected.
Introduction
Comment 3. Lines 57 – 62: The list of biologically active compounds is comprehensive, but the sentence structure is overly long and dense. Please break it into two or more sentences.
Response 3. Thank you for the comment. This was corrected.
Comment 4. Lines 86 – 87: The absence of Melampyrum species from pharmacopeias is a strong point; consider moving this earlier in the paragraph discussing the lack of validated data.
Response 4. Thank you. This was corrected.
Materials and Methods
Comment 5. Lines 98 – 99: What was the altitude (this can influence secondary metabolite content)?
Response 5. Thank you very much. The altitude was around 350 meters above sea level. This was added to the manuscript.
Comment 6. Line 115 – The quantity of raw material (1000 g) is noted, but it's unclear whether this is fresh or dried weight—please clarify.
Response 6. Thank you for the comment. We used dried raw material. The information was added to the manuscript.
Results
Comment 7. Lines 301 – 310: Consider removing the clarification of methodology in Material and methodology section of the manuscript.
Response 7. Thank you very much. The TLC methodology has been moved to the Materials and Methods section, and only one sentence presenting the results is included “TLC analysis of the extracts, conducted in comparison with authentic standards, confirmed the presence of hydroxycinnamic acids (caffeic and chlorogenic) and flavonoids (quercetin and rutin).”
Comment 8. Table 2: Please add units for edema measurements (e.g., mL or % increase), a column for sample size (n) per group and clarification on whether values are means ± standard deviation or standard error.
Response 8. Thank you for the comment. In subsection 2.4.1. Anti-inflammatory activity, it is stated that each group consisted of six animals. This information was included in the table. Units for oedema measurements are % increase, which is presented in Table 2. In subsection 2.5. Statistical Analysis, it is stated that the results are expressed as the mean ± standard deviation (SD).
Comment 9. Table 3: Please clarify whether values are means ± standard deviation or standard error.
Response 9. Thank tou for the comment. In subsection 2.5. Statistical Analysis, it is stated that the results are expressed as the average mean ± standard deviation (SD).
Comment 10. Tables 3 and 4: Mention the statistical test used and apply significance indicators to Table 4 as well, especially to compare different treatment groups.
Response 10. Thank you for the comments. The information was added. In Table 3 “The effect of the liquid extracts from Melampyrum nemorosum herb and water pepper on bleeding duration”. Values are presented as mean ± SD; n = 6. Statistical significance was assessed using one-way ANOVA followed by Tukey’s post hoc test. Table 4 “Dynamics of wound healing with liquid extracts from Melampyrum nemorosum herb and Rotokan.” Values are presented as percentage of wound area reduction over time (days). Statistical significance assessed using repeated measures ANOVA; asterisks indicate significant differences compared to the control group at each time point (P ≤ 0.05)
Comment 11. Section 3.5.: Was the same set of animals used for both bleeding time and wound healing evaluation, or if they were conducted in parallel on different groups?
Response 11. Thank you for the comment. We used the same groups of animals. First, we determined the bleeding time, and then we measured the wound healing time. This was clarified in the manuscript.
Comment 12. Section 3.6.
Table 5: Table 5 is dense and hard to navigate. Consider dividing it into two separate tables: one for antibacterial activity and one for antifungal activity.
Please clearly define the control used for comparison (solvent control? ethanol control?), as the effectiveness of extracts is assessed relative to this.
The differences between MN40 and MN70 are notable, particularly with MRSA and Candida albicans. Please give a brief interpretation of which compounds might be contributing to this enhanced effect (e.g., iridoids, flavonoids, phenolics) and briefly compare the results.
Response 12. Thank you for the comments. In the antimicrobial analysis, we used 40% and 70% ethanol solutions, corresponding to the extraction solvents of the studied liquid extracts, for comparison. This approach allows for a more accurate assessment of the extractant’s influence on antimicrobial activity. Splitting Table 5 into two parts would not significantly affect the perception of the material. On the contrary, we aimed to unify and consolidate the data in a single location. Additionally, we have included a section in the discussion addressing the impact of the main extract constituents on antimicrobial activity.
Discussion
Comment 13. Lines 421 – 433: Are the differences between MN70 and MN40 statistically significant? If so, mention the p-values or significance levels in the discussion text.
Response 13. Thank you for the comments. The information was added to the discussion part. p-values is < 0.05.
Comment 14. Lines 467 – 494: Were there any adverse reactions or irritation noted with topical application of extracts?
Response 14. Thank you for the comment. No signs of irritation or adverse reactions were observed following topical application of either MN40 or MN70 extracts, indicating a favourable safety profile for dermal use. The information was added to the manuscript.
Comment 15. Lines 459 – 509: Group these pathogens into a summary (e.g., MRSA, ESBL-producing E. coli, fluconazole-resistant Candida, etc.) to avoid an overly long list.
Response 15. Thank you for the comment. The correction was done.
Comment 16. Consider adding a short paragraph acknowledging limitations (e.g., no histological analysis, lack of chronic model, not yet tested in human cells).
Response 16. Thank you for the comment. This part was added.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have taken into account most of the reviewer's comments. The reviewer still has doubts about achieving all the stated goals, but overall the work looks good. If the editor finds it suitable for publication, I will not spoil everyone's mood and will agree with his decision. Considering the explanations provided by the authors, the article after correction can be accepted for publication. The only thing that irritates the eye is the incorrect formatting of the bibliography. Please try to be attentive and do everything as required.
Author Response
Comment. The authors have taken into account most of the reviewer's comments. The reviewer still has doubts about achieving all the stated goals, but overall the work looks good. If the editor finds it suitable for publication, I will not spoil everyone's mood and will agree with his decision. Considering the explanations provided by the authors, the article after correction can be accepted for publication. The only thing that irritates the eye is the incorrect formatting of the bibliography. Please try to be attentive and do everything as required.
Response. Thank you very much for your kind and constructive feedback. We sincerely appreciate your positive evaluation and thoughtful remarks. Regarding the bibliography, we have carefully reviewed the formatting and made the necessary corrections in accordance with the journal’s requirements. Please rest assured that we have paid close attention to this aspect.
With gratitude and respect,
The Authors
