Tocotrienol Dominance in Celastraceae Family Species’ Seeds: Phylogenetic Patterns

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript titled Tocotrienol Dominance Celastraceae Family Species’ Seeds: Phylogenetic Patterns presents research with high quality of methodology presented. It is a collection of data, which can be helpful for developing further studies according extraction of bioactive chemicals from selected seeds. After minor revision, the manuscript can be accepted for publication in Applied Sciences.

The Authors could consider the following suggestions:

1. Keywords: The Authors should consider adding additional keyword, concerning extraction technique.

2. Line 29: According to journal guidance, the cited webpage should be included in references, thus it should be presented in the manuscript as [1].

3. Introduction: A graph presenting the plants, active chemicals and their possible application and activities would improve the overall quality of the introduction. Also, the general chemical structure of tocochromanol should be provided.

4. Methods: The equation, used for calculating the recovery of extracted chemicals, should be given.

5. Table 1: The table needs to be edited, so the standard deviation values would be in the same lines, as the main value.

6. The article quality would be enhanced, if the efficiency of presented ultrasound-assisted extraction would be compared to conventional extraction method, e.g., in Soxhlet apparatus.

7. The used seeds are originated from different botanical gardens, thus the cultivation methods can vary. The Authors mentioned in the Methods section that to avoid the influence of the origin of the materials, the diversification was prioritized. However, it should be mentioned in the Results section also, to emphasize that the content of tocochromanol in different species is not dependent on cultivation conditions. Otherwise, please discuss the impact of environment on their content.

8. The conclusions should be elaborated more. The most significant results should be provided here, with values and numbers.

Author Response

The manuscript titled Tocotrienol Dominance Celastraceae Family Species’ Seeds: Phylogenetic Patterns presents research with high quality of methodology presented. It is a collection of data, which can be helpful for developing further studies according extraction of bioactive chemicals from selected seeds. After minor revision, the manuscript can be accepted for publication in Applied Sciences.

We thank you for the positive assessment of the manuscript.

The Authors could consider the following suggestions:

Comment 1: Keywords: The Authors should consider adding additional keyword, concerning extraction technique.

Response 1: Thank you for the comment and suggestion. The keyword: “ultrasound-assisted extraction in ethanol” was added.

 

Comment 2: Line 29: According to journal guidance, the cited webpage should be included in references, thus it should be presented in the manuscript as [1].

Response 2: Thank you for the comment. The references were updated.

 

Comment 3: Introduction: A graph presenting the plants, active chemicals and their possible application and activities would improve the overall quality of the introduction. Also, the general chemical structure of tocochromanol should be provided.

Response 3: Thank you for the comment and suggestion. The structure of tocochromanols was added. The representative plant and phytochemicals are included in the graphic abstract. The application aim is to use UAEE as a green tool for searching new potential sources rich in tocotrienols. Additional discussion on this topic was added to the Introduction section.

 

Comment 4: Methods: The equation, used for calculating the recovery of extracted chemicals, should be given.

Response 4: Thank you for the comment and suggestion. The equation was added.

 

Comment 5: Table 1: The table needs to be edited, so the standard deviation values would be in the same lines, as the main value.

Response 5: Thank you for the comment and suggestion. The table has been rearranged to make the results clear.

 

Comment 6: The article quality would be enhanced, if the efficiency of presented ultrasound-assisted extraction would be compared to conventional extraction method, e.g., in Soxhlet apparatus.

Response 6: Thank you for the comment and suggestion. We did a comparison to the most commonly used protocol for tocochromanols extraction, such as saponification. Soxhlet is used for oil extraction mainly, but our sample resources were even too small to perform such an experiment.

 

Comment 7: The used seeds are originated from different botanical gardens, thus the cultivation methods can vary. The Authors mentioned in the Methods section that to avoid the influence of the origin of the materials, the diversification was prioritized. However, it should be mentioned in the Results section also, to emphasize that the content of tocochromanol in different species is not dependent on cultivation conditions. Otherwise, please discuss the impact of environment on their content.

Response 7: Thank you for the comment and suggestion. The topic of potential factors influencing the content of tocochromanols in the seeds was discussed.

 

Comment 8: The conclusions should be elaborated more. The most significant results should be provided here, with values and numbers.

Response 8: Thank you for the comment. The conclusion section was improved, and some discussion has been added to the end of Results and Discussion.

Reviewer 2 Report

Comments and Suggestions for Authors

I am pleased with the writing style of the authors as they wrote unambiguously. The content is clear to a wider readership. However, mentioning a protocol without enumerating the important procedures used in the research is not encouraging. I will therefore advise the authors to provide the details of their study methodology so that it can be followed for repeatability by other researchers.

Secondly, already established abbreviation standards should be followed, for example, UAE for ultrasound-assisted extraction should be maintained instead of using UAEE.  

Author Response

I am pleased with the writing style of the authors as they wrote unambiguously. The content is clear to a wider readership.

We thank you for the positive assessment of the manuscript.

 

Comment 1: However, mentioning a protocol without enumerating the important procedures used in the research is not encouraging. I will therefore advise the authors to provide the details of their study methodology so that it can be followed for repeatability by other researchers.

Response 1: Thank you for the comment. We are unsure to which protocol this comment pertains. Both the saponification and UAEE extraction procedures are described in detail in the manuscript and are supported by literature citations that provide even more exhaustive methodological information. Furthermore, we have now added explicit information on limits of detection (LOD) and quantification (LOQ), clarified the rationale and application of the chemotaxonomic approach, and included all supplementary procedural details that were absent from the initial submission.

 

Comment 2: Secondly, already established abbreviation standards should be followed, for example, UAE for ultrasound-assisted extraction should be maintained instead of using UAEE. 

Response 2: Thank you for the comment and suggestion. The acronym UAE denotes ultrasonic‑assisted extraction in general, irrespective of solvent, whereas UAEE designates ultrasonic‑assisted ethanol extraction specifically—the double "EE" standing for "ethanol extraction." This convention was adopted to avoid the repetitive phrasing "UAE by ethanol" throughout the manuscript. We consider this abbreviation both concise and unambiguous.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript studies the tocochromanols content in seeds of Celastraceae family species. Authors study 125 samples and   confirm tocotrienol dominance in the family, observed in initial screenings. The study shows that tocotrienols are predominant in the Celastraceae family. A streamlined ethanolic extraction protocol is evaluated and deemed suitable for routine screening. The new method is compared with other standard method for extraction of tocochromanols and high percentage of recovery is shown. Other aim for the authors was to explore how tocochromanol profiles of the samples align with the taxonomic relationships within the family.

The authors work with a large sample of species of the family Celastraceae. The selected methods of analysis are very appropriate and well described. The success of the proposed new method for extraction of tocochromanols has been proven. Suplementary files are informative.

• I would recommend that the text at the beginning of Results and Discussion (lines 176-196) be moved to the introduction, or be referred to the discussion after the presentation of the results.
• Where and how was the MANOVA analysis applied?
• Table 1 – it is not clear which value refers to which row.
• PCA analysis – I don’t think the PCA results are well explained in terms of phylogenetic relationships within the family. The text only repeats the information about what the grouping is due to, but not what these results mean in terms of phylogeny. I think the text in the indicated section could be improved (section “3. Tocochromanol Composition as Shaped by Phylogeny”). Such information also absent from the conclusion.

Author Response

The manuscript studies the tocochromanols content in seeds of Celastraceae family species. Authors study 125 samples and   confirm tocotrienol dominance in the family, observed in initial screenings. The study shows that tocotrienols are predominant in the Celastraceae family. A streamlined ethanolic extraction protocol is evaluated and deemed suitable for routine screening. The new method is compared with other standard method for extraction of tocochromanols and high percentage of recovery is shown. Other aim for the authors was to explore how tocochromanol profiles of the samples align with the taxonomic relationships within the family.

The authors work with a large sample of species of the family Celastraceae. The selected methods of analysis are very appropriate and well described. The success of the proposed new method for extraction of tocochromanols has been proven. Suplementary files are informative.

We thank you for the positive assessment of the manuscript.

 

Comment 1: I would recommend that the text at the beginning of Results and Discussion (lines 176-196) be moved to the introduction, or be referred to the discussion after the presentation of the results.

Response 1: Thank you for the comment and suggestion. Changes have been made in accordance with the guidelines.

 

Comment 2: Where and how was the MANOVA analysis applied?

Response 2: Thank you for the comment and suggestion. The statistical analysis section has been clarified. MANOVA was used on the subset of the dataset with known species, without transformation.

 

Comment 3: Table 1 – it is not clear which value refers to which row.

Response 3: Thank you for the comment and suggestion. The table has been rearranged to make the results clear.

 

Comment 4: PCA analysis – I don’t think the PCA results are well explained in terms of phylogenetic relationships within the family. The text only repeats the information about what the grouping is due to, but not what these results mean in terms of phylogeny. I think the text in the indicated section could be improved (section “3. Tocochromanol Composition as Shaped by Phylogeny”). Such information also absent from the conclusion.

Response 4: Thank you for the comment and suggestion. There is limited overlap with our sample pool and that in other phylogenetic studies, but a short discussion has been added.