Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This article discusses the extraction of bioactive compounds from hop cone residue using microwave-assisted and ultrasound-assisted solvent extraction. The chemical composition of the extract was determined by GC-MS, and biological assays were performed. The results indicated antioxidant, antiproliferative, and antimicrobial activities, as well as positive results in inhibiting NO production and protecting the skin against UV radiation. These results are reported for the first time for hop cone residue from the brewing industry. My opinion is that the article should be published after some adjustments based on the observations I have submitted.

1) What type of alcohol is used in hydroalcoholic solvent?

2) Why was this alcohol chosen?

3) Why use the solvent in solution form? What is the justification for using a 70% concentration?

4) Lines: 183 - 184, E. cloacae and S. aureus should be in italics.

5) In Table 3, Remove the bold text from A. btasiliensis.

6) To me, Table 5 became unformatted.

7) I would like the authors to comment further on the antitumor activity results, comparing the results against tumor cells and normal cells. What further studies would be needed to categorize the extract as an antitumor agent for future use in humans?

8) The overall extract was used in the biological assays. Since the extract is composed of several components, was the action of any of these components individually investigated in the biological activities studied? Given that beta-pinene and cis-beta-farnesene are the major components, are there reports of the activity of these components in studies available in the literature? I suggest including a more extensive discussion on this.

9) Is GC-MS capable of identifying all the components present in the extract? Discuss this in the text.

10) Ultrasound and microwave-assisted solvent extractions are usually accompanied by an increase in temperature. Given the presence of thermolabile compounds, it is important to control this temperature. A temperature of 35 °C was reported for the ultrasound-assisted extraction. Was this temperature maintained constant throughout the process? If so, what procedure was used to maintain this constant value? And what was the temperature value for the microwave-assisted extraction?

 

 

 

Author Response

Os autores agradecem sinceramente ao revisor pela avaliação positiva do nosso trabalho e pelos comentários e sugestões construtivas. Agradecemos o tempo e o esforço dedicados à revisão do manuscrito. Em resposta às observações feitas, revisamos cuidadosamente o manuscrito e abordamos cada ponto em detalhes. Anexamos o documento intitulado “Resposta aos Comentários e Sugestões dos Autores” , onde todos os comentários foram abordados individualmente. Acreditamos que as revisões melhoraram a clareza e a qualidade do manuscrito.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

See the attached file

Comments for author File: Comments.docx

Author Response

The authors sincerely thank the reviewer for the positive evaluation of our work and for the constructive comments and suggestions provided. We appreciate the time and effort dedicated to reviewing the manuscript. In response to the observations raised, we have carefully revised the manuscript and addressed each point in detail. Please find attached the document entitled “Response to Comments and Suggestions for Authors”, where all comments have been addressed individually. We believe that the revisions have improved the clarity and quality of the manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

This article discusses the evaluation of Humulus lupulus cone byproduct from the beer industry using environmentally friendly extraction techniques (UAE and MAE), presenting both chemical characterization and multifaceted biological activity analyses. The study is comprehensive in its assessment of antioxidant, antimicrobial, antiproliferative, NO inhibition, and photoprotective activities, and is current and important from a circular economy perspective. While the study has publishable potential, it requires significant revisions in its current form.

Below are my suggestions for revisions to the article:

1- In the GC-MS analysis, compounds are reported only as relative area (%); absolute quantification is not performed, and validation parameters (LOD, LOQ, linearity, reproducibility) are not presented (pp. 3–4). Furthermore, the determination of only volatile compounds does not adequately reflect the phenolic content of the hydroalcoholic extract. The phenolic profile should be supported by LC-MS/MS or HPLC-DAD.

2- Although major compounds such as β-pinene and caryophyllene are mentioned (Table 1), the relationship between these compounds and the observed biological activities has not been statistically or mechanistically discussed. Despite claims of multiple activities, the effect-component relationship is lacking.

3- MIC values ​​range from 1–10 mg/mL (Table 3), which are relatively high even for crude extracts. Despite this, strong expressions such as "potent antimicrobial activity" have been used. Clinical significance should be discussed more cautiously.

4- Although GI50 values ​​are given, mechanistic evaluations such as apoptotic pathways, cell cycle analysis, or ROS relationship have not been performed. Furthermore, only the SRB test was used; the type of cell death was not determined.

5- EH-MA is written twice consecutively in the text (p. 9, lines 254–255). Also, the discussion of the anti-inflammatory mechanism remains superficial.

6- SPF values ​​were calculated spectrophotometrically (pp. 10–13), but validation was not performed using UVA/UVB ratio, photostability, or cellular UV damage models. Therefore, the claim of cosmetic application appears premature.

7- ANOVA and Tukey tests are mentioned, but effect size, confidence interval, or multiple comparison corrections are not provided. Furthermore, correlation analysis between chemical and biological data has not been performed.

8- The phrase "reported for the first time" is used in several places; however, previous studies on hop byproducts exist in the literature (e.g., reference 12). Novelty should be positioned more carefully.

9- No comparison has been made with the conventional extraction method. The superiority of UAE and MAE is defended solely based on the literature; there is no experimental comparison.

10- There are some typographical errors, repetitions, and table formatting problems (e.g., "MB" instead of "MBC," line breaks, punctuation errors). The text requires major editing in terms of language.

Comments on the Quality of English Language

There are some typographical errors, repetitions, and table formatting problems (e.g., "MB" instead of "MBC," line breaks, punctuation errors). The text requires major editing in terms of language.

Author Response

The authors sincerely thank the reviewer for the positive evaluation of our work and for the constructive comments and suggestions provided. We appreciate the time and effort dedicated to reviewing the manuscript. In response to the observations raised, we have carefully revised the manuscript and addressed each point in detail. Please find attached the document entitled “Response to Comments and Suggestions for Authors”, where all comments have been addressed individually. We believe that the revisions have improved the clarity and quality of the manuscript.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors made modifications based on the questions previously asked. In my opinion, the modifications improved the article and it is now ready for publication in the journal.

Author Response

The authors thank the reviewer for the positive comments about the work. We greatly appreciate the constructive comments provided throughout the review process, which have significantly contributed to improving the quality and clarity of the work.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

See the attached file

Comments for author File: Comments.pdf

Author Response

Os autores agradecem ao revisor pelos comentários positivos sobre o trabalho. Agradecemos imensamente os comentários construtivos fornecidos ao longo do processo de revisão, que contribuíram significativamente para a melhoria da qualidade e clareza do trabalho.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have made the corrections I suggested in the first version. It is now suitable for publication.

Author Response

The authors thank the reviewer for the positive comments about the work. We greatly appreciate the constructive comments provided throughout the review process, which have significantly contributed to improving the quality and clarity of the work.

Author Response File: Author Response.pdf