Fractions and Compounds Obtained from Transformed Plant Cell Cultures of Lopezia racemosa Show Anti-Inflammatory and Cytotoxic Activities

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General Comments:
This manuscript presents interesting findings with potential relevance to the field; however, several aspects of the study need to be clarified and improved before it can be considered for publication. In particular, the manuscript would benefit from substantial re-organization to enhance clarity and scientific rigor.

Please consider re-arranging the Results section to begin with a detailed description of your fractionation steps, and move Scheme 1 to the beginning of this section for better context. You may also consider assigning a clearer or more concise name to your combined fractions to aid readability (eg replace numders with letters: 1-12=A, 12-15=B etc).

The experimental design should be reconsidered or more thoroughly justified. Typically, studies either test a crude extract and then isolate and test multiple compounds, or follow a bio-guided approach where all fractions are tested and the most active ones are subjected to phytochemical analysis. In this context, the following issues should be addressed:

• Why are the fractions C2F12–15 and their subfractions excluded from Table 1 and Table 3?
• Why was only C2F12–15 further fractionated, when C2F21–23 and C2F28–29 also showed activity?
• For C2F21–23 and C2F28–29, please consider including a phytochemical description. If further fractionation is not feasible, an LC-MS analysis would still provide useful information—active fractions without any phytochemical characterization weaken the overall conclusions.

Additional specific points that should be addressed include:

• Please revise the abstract to clearly state that C3F33 is a mixture of two triterpenes.
• Include the positive control in Table 3 and again indicate that C3F33 is a mixture.
• Remove all result-related content from the Introduction; this section should focus solely on background and the study’s aims.
• For known/previously described compounds no extended NMR description is needed. The authors should combine a paragraph regarding fractionation, with short hplc and nmr analysis (eg According to NMR analysis (1 and 2D), fraction C3F33 showed the presence of compounds 1 and 2 in mixture, which were found to be 3-O-[(E)-feruloyl]-maslinic acid (1) and 3-O-[(E)-fer- 199 uloyl]-corosolic acid (2), according to the literature). Table 4 and Figure 6 should be moved to SI.
• Carefully proofread the manuscript for typographic and grammatical errors throughout.

In the Materials and Methods section, please include the following missing information:

• Ethical committee approval number for the animal studies
• References supporting each of the experimental protocols used
• The number of cells used in the cytotoxicity assay
• Solvent calibration values for NMR analysis
• General information about consumables (e.g., origin of solvents, cell lines, and positive controls)

 

Minor corrections:

l.2: Please remove the botanical authority from the plant’s name in the title and add it in the first time mentioned in the abstract (l.20 and remove from l.25).

l.56: Please induce more details about the traditional use of this plant.

l.59: Please include references regarding the previously reported biological activities.

l.144: Please add citation for the NCI.

Author Response

Comments 1 

This manuscript presents interesting findings with potential relevance to the field; however, several aspects of the study need to be clarified and improved before it can be considered for publication. In particular, the manuscript would benefit from substantial re-organization to enhance clarity and scientific rigor.

Please consider re-arranging the Results section to begin with a detailed description of your fractionation steps, and move Scheme 1 to the beginning of this section for better context. You may also consider assigning a clearer or more concise name to your combined fractions to aid readability (eg replace numders with letters: 1-12=A, 12-15=B etc).

R1. : We have reorganized the Results section to begin with a detailed description of the fractionation procedure in order to provide better context for the reader. Scheme 1 has also been moved to the beginning of this section to improve the overall understanding of the process. In addition, the names of the combined fractions have been modified, replacing numerical ranges with letters: FA (12–15), FB (19–20), FC (21–23), FD (28–29) to enhance clarity and facilitate readability. 

Comments 2. The experimental design should be reconsidered or more thoroughly justified. Typically, studies either test a crude extract and then isolate and test multiple compounds, or follow a bio-guided approach where all fractions are tested and the most active ones are subjected to phytochemical analysis. In this context, the following issues should be addressed:

• Why are the fractions C2F12–15 and their subfractions excluded from Table 1 and Table 3?
• Why was only C2F12–15 further fractionated, when C2F21–23 and C2F28–29 also showed activity?

R2: In our study, the experimental approach was not bio-guided. From the outset, our intention was to identify and isolate triterpene- and phytosterol-type compounds that had been previously reported in the LRT7.31 cell line, which is related to LRTC3.1. For this reason, the design focused on fraction C2F12–15, which exhibited chemical characteristics similar to those in which such compounds had been detected.

Comments 3: Fractions C2F21–23 and C2F28–29 were not selected for further fractionation because their preliminary phytochemical profiles did not match the type of metabolites we were targeting.

R3: Regarding the tables, the subfractions derived from C2F12–15 were not included in Tables 1 and 3 because these tables were specifically dedicated to presenting the activity data of the original fractions obtained from the extract, prior to any further fractionation. The only exception is fraction C3F33, which turned out to be a compound not previously reported.

Comments 4: For C2F21–23 and C2F28–29, please consider including a phytochemical description. If further fractionation is not feasible, an LC-MS analysis would still provide useful information—active fractions without any phytochemical characterization weaken the overall conclusions.

R4: The phytochemical characterization of fractions C2F21–23 and C2F28–29 has not yet been completed due to time constraints. However, we acknowledge the importance of this analysis and plan to conduct further studies, including LC-MS analysis, to complement this information in future reports.

Comments 5: Additional specific points that should be addressed include:

Please revise the abstract to clearly state that C3F33 is a mixture of two triterpenes. Include the positive control in Table 3 and again indicate that C3F33 is a mixture. Remove all result-related content from the Introduction; this section should focus solely on background and the study’s aims.

R5: The requested changes have been made. The abstract was revised to clearly indicate that fraction C3F33 is a mixture of two triterpenes. In addition, all result-related content was removed from the Introduction section, which now focuses exclusively on the background and objectives of the study. 

Comments 6: For known/previously described compounds no extended NMR description is needed. The authors should combine a paragraph regarding fractionation, with short hplc and nmr analysis (eg According to NMR analysis (1 and 2D), fraction C3F33 showed the presence of compounds 1 and 2 in mixture, which were found to be 3-O-[(E)-feruloyl]-maslinic acid (1) and 3-O-[(E)-fer- 199 uloyl]-corosolic acid (2), according to the literature). Table 4 and Figure 6 should be moved to SI.

R6: The NMR data for previously reported compounds have been shortened, and a single paragraph now summarizes the fractionation process along with a brief HPLC and NMR analysis. Fraction C3F33 is described as a mixture containing compounds 1 and 2, identified as 3-O-[(E)-feruloyl]-maslinic acid (1) and 3-O-[(E)-feruloyl]-corosolic acid (2), based on 1D and 2D NMR analyses and in agreement with the literature.
In addition, Table 4 and Figure 6 have been moved to the Supplementary Information (SI), as recommended.

Comments 7: Carefully proofread the manuscript for typographic and grammatical errors throughout.

In the Materials and Methods section, please include the following missing information:

• Ethical committee approval number for the animal studies
• References supporting each of the experimental protocols used
• The number of cells used in the cytotoxicity assay
• Solvent calibration values for NMR analysis
• General information about consumables (e.g., origin of solvents, cell lines, and positive controls)

R7: All requested information has been included: ethics approval number, references for experimental protocols, cell number for the cytotoxicity assay, NMR solvent calibration values, and details on consumables (solvents, cell lines, and positive controls).

Comments 8: Minor corrections:

l.2: Please remove the botanical authority from the plant’s name in the title and add it in the first time mentioned in the abstract (l.20 and remove from l.25).

l.56: Please induce more details about the traditional use of this plant.

l.59: Please include references regarding the previously reported biological activities.

l.144: Please add citation for the NCI

R8: The botanical authority was removed from the title and adjusted throughout the manuscript, retaining it only in the first mention in the abstract (line 20), as requested. Additional information on the traditional use of the plant was included based on the Atlas de las plantas de la medicina tradicional mexicana, and references to previously reported biological activities were added. Lastly, the NCI citation was included at line 212.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is in total a solid piece of work with isolation and elucidation leading to bioactivity tests. There are just a few minor things i would have liked to be added to the manuscript:

 

1.) Please add the parameters used for NMR analysis in the materials and methods

2.) Please add the parameters of the MS Experiments in the materials and methods

3.) If possible perform some HRMS experiments to further give conficidence to your elucidation. (only if available)

 

In total i could wholeheartly recommend a minor revision

Author Response

Comments 1:The manuscript is in total a solid piece of work with isolation and elucidation leading to bioactivity tests. There are just a few minor things I would have liked to be added to the manuscript:

1.) Please add the parameters used for NMR analysis in the materials and methods
2.) Please add the parameters of the MS Experiments in the materials and methods
3.) If possible perform some HRMS experiments to further give conficidence to your elucidation. (only if available) 
In total i could wholeheartly recommend a minor revision

R1: The parameters used for NMR and mass spectrometry analyses have been added to the Materials and Methods section. Currently, only the reported results are available, so additional HRMS experiments could not be performed; however, we will consider this suggestion for future studies.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The study addresses a timely and relevant topic with potential scientific value, but few issues need to be addressed for it to meet publication standards. A detailed review is provided below.

• The primary focus of this study is anti-inflammatory activity. However, the Introduction section centers too much on cancer, which is not the main objective. The authors should realign the Introduction to clearly reflect the study’s focus, significance, and objectives. Clearly state the aim of the study and highlight the gap it addresses in current research.
• The paper mentions hairy root induction by Agrobacterium rhizogenes, but provides no detailed results on the development of the callus. These results should be included to complete the experimental narrative.
• PCR analysis is included, but a quantitative Q-PCR is necessary to properly validate gene expression. Please include this or explain why it was not performed.
• In all figures that include statistical analysis, indicate what the asterisks represent. Also, specify which groups were compared in the post hoc analysis.
• There are several typos and formatting issues throughout the manuscript. For example, full scientific names are used repeatedly. Use the full name only on first mention, then abbreviate appropriately.
• Why was cytotoxicity not tested in a mouse model? Justify this decision or consider including it. Also, provide a dose-response curve for the cell-line cytotoxicity assay.
• The Conclusion should be more focused on the key findings of the study and also touch on future directions.
• The Discussion needs more comparative analysis with relevant previous studies to strengthen the interpretation of results.

Author Response

The study addresses a timely and relevant topic with potential scientific value, but few issues need to be addressed for it to meet publication standards. A detailed review is provided below.

• The primary focus of this study is anti-inflammatory activity. However, the Introduction section centers too much on cancer, which is not the main objective. The authors should realign the Introduction to clearly reflect the study’s focus, significance, and objectives. Clearly state the aim of the study and highlight the gap it addresses in current research.

R1. The focus of our research were both cancer and inflammation activities, since they are related.

Commentes 2: The paper mentions hairy root induction by Agrobacterium rhizogenes, but provides no detailed results on the development of the callus. These results should be included to complete the experimental narrative.

R2: The cell line used in this study was previously generated through hairy root induction by Agrobacterium rhizogenes, from which the callus was derived and results were previously published. Since the present work focused exclusively on the maintenance and experimental use of this callus cell line, and not on its generation or development, no results related to callus induction or growth are included.

Comments 3: PCR analysis is included, but a quantitative Q-PCR is necessary to properly validate gene expression. Please include this or explain why it was not performed.

R3: In this study, conventional PCR was used to confirm the presence of the transgene in the cell line. qPCR was not performed, as the primary objective was the isolation and identification of bioactive compounds rather than gene expression quantification. However, in future studies, if we plan to evaluate gene expression levels using qPCR in order to compare different cell lines and explore potential correlations with their respective phytochemical profiles, we will do it.

Commentes 4: In all figures that include statistical analysis, indicate what the asterisks represent. Also, specify which groups were compared in the post hoc analysis.

There are several typos and formatting issues throughout the manuscript. For example, full scientific names are used repeatedly. Use the full name only on first mention, then abbreviate appropriately.

R4: Clarifications have been added to all figures that include statistical analysis, indicating the meaning of the asterisks and specifying the groups compared in the post hoc analysis. In addition, typos and formatting issues throughout the manuscript have been corrected. Scientific names are now written in full only at first mention and appropriately abbreviated thereafter.

Comments 5: Why was cytotoxicity not tested in a mouse model? Justify this decision or consider including it. Also, provide a dose-response curve for the cell-line cytotoxicity assay.

R5: This study focused on a preliminary evaluation of cytotoxicity using an in vitro system, aiming to characterize the effects of the treatment on specific cell lines. The use of a murine model was not considered at this stage due to budget constraints and because the action mechanisms could be determined after the exploratory nature of this research.

Comments 6: The Conclusion should be more focused on the key findings of the study and also touch on future directions.

R6: We appreciate the reviewer’s comment. The Conclusion section has been revised to focus on the key findings of the study, including the isolation of a previously unreported compound mixture in L. racemosa, the significant anti-inflammatory activity of the crude extract, and the selective cytotoxicity of specific fractions. Clear future directions have also been added, such as optimizing metabolite production using elicitors, fully characterizing active fractions, and validating the observed in vitro effects through in vivo studies.

Comments 7: The Discussion needs more comparative analysis with relevant previous studies to strengthen the interpretation of results.

R7: The discussion was primarily focused on the results obtained, as there is currently limited information available on the isolated compounds and few previous studies on Lopezia racemosa. Nevertheless, relevant comparisons were included where possible, and the novelty of the findings was emphasized within the phytochemical context of the species. We believe this work contributes valuable data that may serve as a reference for future comparative studies.

 

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors addressed all my concerns. Therefore, I am recommending publishing this article in its current format.