Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

No clear justification is given for why the authors specifically chose Usnea barbata and not another lichen also containing biologically active metabolites.

The authors correctly point out the importance of secondary metabolites (primarily usnic acid) in U. barbata that can be extracted with oil. However, the manuscript lacks data on the content of these metabolites, which could be obtained using the HPLC method. Without these data, it is difficult to interpret many of the other results obtained by the authors.

The GC-MS method presents an analysis of the control oil and the oil with the lichen extract. Since derivatization was not used, only volatile terpenes and terpenoids contained in the peppermint oil and jojoba oil were detected. It is unclear how the identification was performed; it is not stated what internal standards were used, or whether retention index calculation and comparison with literature data were conducted (comparison of obtained mass spectra with database spectra alone is insufficient for terpenes and terpenoids identification). All of this needs to be thoroughly described in the Materials and Methods section.

For the heavy metal analysis, only two elements were chosen—arsenic and lead—while other heavy metals were ignored without explanation in the text.

It is unclear why PEG400 was added to the oil for the Total Phenolic Content analysis; no explanation is provided in the manuscript text.

What exactly are the differences in the IR spectra of UBPJO vs. PJO? Or, if there are no differences, this should be explicitly stated in the text.

The results of the statistical analysis are highly questionable, particularly the implausibly high correlation coefficients and R2 values. At the same time, this section is difficult to evaluate due to the lack of primary data. 

Author Response

Dear Reviewer 1, 

The authors are grateful for your time, professionalism, and accurate comments, which helped clarify the data provided and significantly improve the quality of our manuscript. 

Please find all responses in the attached document. 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled “Phytochemical Screening and Physicochemical Properties of Oil Extract of Usnea barbata L. F.H.Wigg from Călimani Mountains, Romania” addresses a relevant and timely topic that fits well within the scope of the International Journal of Molecular Sciences. Nevertheless, in its current form, the manuscript requires major revisions before it can be considered for publication. Several aspects related to the structure, clarity, and formal presentation of the manuscript should be substantially improved to ensure scientific rigor and full compliance with the journal’s guidelines. The following comments and suggestions are provided to assist the authors in strengthening the quality of the manuscript:

Comments 1: “The authors are recommended to strengthen and expand the introductory section of the manuscript by incorporating relevant recent studies published between 2020 and 2025, with particular emphasis on the pharmacological effects of Usnea barbata extracts, together with a critical evaluation of safety aspects and limitations associated with the therapeutic use of these extracts.”

Comments 2: “The authors mention the significance and characteristics of peppermint essential oil (PEO) in the Results and Discussion section only briefly. Considering that the study also includes a GC/MS analysis of this oil, it is recommended that the authors incorporate in the Introduction a concise yet informative overview of its key characteristics and biological properties, in order to provide clearer context for the interpretation of the obtained results.”

Comments 3: “It is recommended that the authors further expand the discussion section, particularly regarding the chemical composition of the oil extract of Usnea barbata as determined by GC/MS analysis and the total polyphenol content. This should include a comparison with recent literature (published in the last few years) addressing Usnea barbata oil extracts. Such a comparison would provide a more comprehensive context and a clearer explanation of the observed differences among the samples, while also highlighting the significance of this oil extracts due to its potential pharmacological and cosmetic properties.”

Comments 4: “The authors state in the conclusion that the identified bioactive phytochemicals have a positive effect on skin health due to their antioxidant and antimicrobial properties. However, the manuscript does not clearly indicate whether antimicrobial assays were performed for the investigated oil extracts, except for the statement in the introductory section that usnic acid, as one of the most active metabolites of Usnea barbata, exhibits antimicrobial activity. If antimicrobial tests were indeed conducted, it is recommended that the authors present and discuss these results in the manuscript in order to adequately support their conclusions.”

Final Recommendation: Major Revision

Although the study addresses an important and timely topic and provides a detailed analysis of the obtained results, the manuscript would benefit from further refinement to improve its overall clarity and scientific contribution. Specifically, a clearer organization and expansion of the manuscript content are recommended, along with the inclusion a more detailed and comprehensive review of recent relevant literature. Addressing these aspects would significantly enhance the manuscript’s readability and its overall readiness for pub

Author Response

Dear Reviewer 2, 

The authors are grateful for your time, professionalism, and accurate comments, which helped clarify the data provided and significantly improve the quality of our manuscript. 

Please find all responses in the attached document. 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

For reliable identification of essential oil components, comparing the obtained mass spectra with those in the NIST library is insufficient; Kovats retention indices must also be taken into account. The authors should address this point.

Figure 1. The FTIR spectra of PJO (A) and UBPJO (B) are recommended to be moved to the supplementary materials.

Figure 3. On the rheological properties plots (A and B), a legend must be added to indicate which color corresponds to Dynamic viscosity and which to Shear stress.

The section 2.9. Data Analysis . The results of the correlation analysis need to be discussed in detail. Simply stating the presence of a statistically significant correlation is not sufficient to establish a reliable cause-and-effect relationship. Furthermore, the manuscript does not specify which variables were compared in the correlation analysis. The attached file only indicates that there were six. This data (e.g., the list of variables) must be added to both the main text and the supplementary materials.

Author Response

Dear Reviewer 1, 

Thank you so much for your time, accuracy, and constructive comments to improve the quality of the revised manuscript. 

We responded point by point to all comments, as follows:

C1. For reliable identification of essential oil components, comparing the obtained mass spectra with those in the NIST library is insufficient; Kovats retention indices must also be taken into account. The authors should address this point.

R1. Thank you for this accurate comment. The following data, supported by suitable references, were added in lines 542-557.

Moreover, Agilent GC/MS databases (such as retention time locking (RTL) libraries,  https://www.agilent.com/en/product/gas-chromatography-mass-spectrometry-gc-ms/gc-ms-application-solutions/gc-ms-libraries, accessed on 05 February 2026) are enriched with retention index (RI) data for standard alkanes, typically using them to calculate Kovats retention indices [130]. Because they are independent of minor experimental variations, retention indices are used to accurately characterize other compounds. Agilent MassHunter software can use the retention times of these alkanes to interpolate the indices for the EO constituents. Then, Agilent libraries [131] provide reliable identification of EOs compounds based on temperature-programmed retention data [132] and supplement the mass spectral matching procedure.

C2. Figure 1. The FTIR spectra of PJO (A) and UBPJO (B) are recommended to be moved to the supplementary materials.

R2. Thank you for this pertinent comment.

Figure 1 became Figure S3 in the Supplementary Material. 

C3. Figure 3. On the rheological properties plots (A and B), a legend must be added to indicate which color corresponds to Dynamic viscosity and which to Shear stress.

R3.  Thank you for this pertinent comment. Due to the previously requested change, Figure 3 was renamed Figure 2 in the revised version. 

The Legend of Figure 2 was modified accordingly.  Moreover, Figures 2A and 2B were modified to better illustrate shear stress and dynamic viscosity. 

C4. The section 2.9. Data Analysis . The results of the correlation analysis need to be discussed in detail. Simply stating the presence of a statistically significant correlation is not sufficient to establish a reliable cause-and-effect relationship. Furthermore, the manuscript does not specify which variables were compared in the correlation analysis. The attached file only indicates that there were six. This data (e.g., the list of variables) must be added to both the main text and the supplementary materials.

R4. Thank you for the accurate comment. 

We respectfully inform Reviewer 2 that all variables are shown in both graphs included in Figure 3. 

We clarified this aspect in the manuscript and expanded the discussion to support our findings with evidence from the scientific literature as follows: 

Line 449-451:

Pearson's correlation was applied to all quantitative variables, and the correlation coefficient (r) and the coefficient of determination (R²) were calculated and plotted in Figures 3A and 3 B. 

Lines 460-483:

Isomenthone, pulegone, hydroxy-p-menthone, and As show substantial correlation with pH (r > 0.854, p < 0.05, Figure 3A). The relationship between isomenthone and pulegone with respect to pH is not necessarily linear; rather, it reflects stability and chemical degradation [128–131]. As environmental pH increases, menthone undergoes oxidation, forming hydroxyl groups; thus, the positive correlation between hydroxy-p-menthone and pH can be explained [132,133]. Literature data confirm that arsenic concentrations in groundwater and soil are generally positively correlated with pH [134].

Estragole, isomenthone, and Pb show a significant correlation with density (r > 0.813, p < 0.05) and shear stress (r > 0.850, p < 0.05), while  Pb, As, and isomenthone are strongly associated with spreadability (r > 0.825, p < 0.05; Figure 3A). These correlations are also reported in the literature. The density of an oil is a function of its constituents. A higher concentration of oxygenated compounds (such as estragole and isomenthone) increases oil density and affects its rheology [135,136]. Moreover, the presence of lead and arsenic in oils—both in industrial lubricants/paints and in crude oil—directly influences their density and rheological properties by creating internal structures that alter oil flownder stress [137,138].

Oxidative stability is significantly associated with isomenthone (r > 0.836, p < 0.05, Figure 3A) [139].

The physicochemical properties of oil samples are intercorrelated. Density is highly correlated with shear stress and dynamic viscosity (r > 0.878, p < 0.05) [140], while pH is strongly associated with rheological properties (r > 0.839, p < 0.05; Figure 3A) [141]. Oxidative stability is significantly associated with pH (r = 0.944, p < 0.05, Figure 3A). This correlation is not linear; it is often better at intermediate pH levels (5-7) than at extremes (very acidic or very alkaline) [142]; our oil samples had pH values of 6.01 and 6.25.

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript provides a more balanced, clearer, and up to date overview of physicochemical properties of oil extract of Usnea barbata L. F.H.Wigg from Călimani Mountains, Romania . The authors have significantly improved the manuscript through a thorough restructuring of the text, updating and expanding the references, and enhancing the academic rigor and internal coherence of the paper. Furthermore, the manuscript is now technically aligned with the journal’s formatting standards. Based on these revisions and the noticeable improvements in the quality of the manuscript, I believe that the revised version meets the requirements and can be accepted for publication.

Author Response

C1. The revised manuscript provides a more balanced, clearer, and up to date overview of physicochemical properties of oil extract of Usnea barbata L. F.H.Wigg from Călimani Mountains, Romania . The authors have significantly improved the manuscript through a thorough restructuring of the text, updating and expanding the references, and enhancing the academic rigor and internal coherence of the paper. Furthermore, the manuscript is now technically aligned with the journal’s formatting standards. Based on these revisions and the noticeable improvements in the quality of the manuscript, I believe that the revised version meets the requirements and can be accepted for publication.

R1. Dear Reviewer 2,

Thank you so much for your time, attention, and professionalism during the second round of revision. 

We are grateful for your positive feedback on the revised version after the first round of peer review. We did our best on this, and the quality of our manuscript improved substantially after the first revision following your constructive suggestions.

Thank you again for everything. We were honored to collaborate with you. 

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The basis for calculating the correlation and determination coefficients requires clarification. The provided file indicates the calculations were performed using six observations (from scatter plots). The nature of these observations should be specified. Without such an explanation in the manuscript, the origin of this data remains unclear. If the analysis relied exclusively on the PJO and UBPJO points from three replicates, it may not be statistically robust, so it might be better to remove this section from the manuscript.

Author Response

C1. The basis for calculating the correlation and determination coefficients requires clarification. The provided file indicates the calculations were performed using six observations (from scatter plots). The nature of these observations should be specified. Without such an explanation in the manuscript, the origin of this data remains unclear. If the analysis relied exclusively on the PJO and UBPJO points from three replicates, it may not be statistically robust, so it might be better to remove this section from the manuscript.

R1. Thank you so much for this pertinent comment. As suggested, it was removed from the manuscript.