Residues of St. John’s Wort (Hypericum perforatum) Tea Infusions/Water Extracts as a Valuable Source of Tocotrienols: An Extraction Study
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Authors
Your manuscript “Residues from tea infusions/aqueous extracts of St. John’s wort (Hypericum perforatum) as a valuable source of tocotrienols: an extraction study” is about a suitable and interesting integration of tocotrienol extraction as a new and valuable source of these compounds with high health value.
The St. John’s wort processing schemes that could improve its value chain are suitable since they use green chemistry and inexpensive methodology.
This viewpoint, of using residues as raw material for extraction of secondary metabolites, has been recurrent for many years. Pharmacists and chemists use residues from various plants to produce by-products for different purposes. Sometimes, they are the basis for the production of drugs or for the hemisynthesis of other new drugs. My overall assessment is good regarding the preparation of the manuscript and the work plan developed for the extractions and respective analyses of these bioactive compounds.
My only question is:
Since hyperforin is also present in the plant residues of this plant, since it does not go into the water infusion, and is extracted by n-hexane or n-heptane and can also be found in 96% ethanolic extracts,
1- Why do you never report this situation?
2- Why is it not found in your extracts.
I accept the manuscript in the present form but maybe add with the information requested.
Sincerely
Author Response
We sincerely thank you for all the comments, remarks, and suggestions that have contributed to enhancing the manuscript and its scientific quality. The graphical abstract, manuscript, and supplementary materials have been improved accordingly. Provided changes are marked in red font. For the literature we used references manager software, therefore the changes are not highlighted.
Reviewer 1
Comment 1: Your manuscript “Residues from tea infusions/aqueous extracts of St. John’s wort (Hypericum perforatum) as a valuable source of tocotrienols: an extraction study” is about a suitable and interesting integration of tocotrienol extraction as a new and valuable source of these compounds with high health value.
The St. John’s wort processing schemes that could improve its value chain are suitable since they use green chemistry and inexpensive methodology.
This viewpoint, of using residues as raw material for extraction of secondary metabolites, has been recurrent for many years. Pharmacists and chemists use residues from various plants to produce by-products for different purposes. Sometimes, they are the basis for the production of drugs or for the hemisynthesis of other new drugs. My overall assessment is good regarding the preparation of the manuscript and the work plan developed for the extractions and respective analyses of these bioactive compounds.
Response 1: Thank you for the positive overview.
Comment 2: My only question is: Since hyperforin is also present in the plant residues of this plant, since it does not go into the water infusion, and is extracted by n-hexane or n-heptane and can also be found in 96% ethanolic extracts, Why do you never report this situation? Why is it not found in your extracts.
Response 2: Thank you for your comment. Hyperforin was not a subject of the study and the project. We initially assumed that this compound is already well-studied and that further investigation would not yield significantly novel findings. Therefore, a hyperforin standard was not purchased to explore this specific topic. Presently we see this topic differently and see some scientific potential. To acknowledge this topic, a brief discussion was included, and the necessity for future research in this area was emphasized. (Page 10, middle and bottom part)
Comment 3: I accept the manuscript in the present form but maybe add with the information requested. Sincerely
Response 3: Thank you for the positive overview.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript describes a study on the content of tocotrienols (T3) and tocopherols (T) in St. John’s Wort residues using hydroethanolic extraction. While there are plenty of studies on the chemical components of St. John’s Wort, research specifically focusing on tea residues is limited. The findings may contribute to phytochemistry and the usage of herbal by-products. However, the manuscript would benefit from improved clarity and more detailed results.
Main Comments
1. It appears that standards of T3 and T were used for quantification in St. John’s Wort and tea residue extracts. However, the manuscript should provide more details on the bioanalytical assay method, including the linear range, accuracy, and precision. At least a partial validation is necessary.
2. Are the chromatograms in Figure 1 original? They appear to have been processed.
3. The study method included the LC-MS method, but no corresponding results were presented.
4. In Figure 2, the y-axis represents recovery. What reference was used as 100%?
5. In Figure 3, the content appears to reach the plateau at higher ethanol levels. Linear regression may not be the best choice, while nonlinear regression should be considered.
Minor Concerns
1. The English should be carefully edited to improve clarity.
2. Is there a specific reason for using 96.2% ethanol?
Comments on the Quality of English LanguageProofreading is needed.
Author Response
We sincerely thank you for all the comments, remarks, and suggestions that have contributed to enhancing the manuscript and its scientific quality. The graphical abstract, manuscript, and supplementary materials have been improved accordingly. Provided changes are marked in red font. For the literature we used references manager software, therefore the changes are not highlighted.
Reviewer 2
Comment 1: The manuscript describes a study on the content of tocotrienols (T3) and tocopherols (T) in St. John’s Wort residues using hydroethanolic extraction. While there are plenty of studies on the chemical components of St. John’s Wort, research specifically focusing on tea residues is limited. The findings may contribute to phytochemistry and the usage of herbal by-products. However, the manuscript would benefit from improved clarity and more detailed results. Main Comments
Response 1: Thank you for the positive overview.
Comment 2: It appears that standards of T3 and T were used for quantification in St. John’s Wort and tea residue extracts. However, the manuscript should provide more details on the bioanalytical assay method, including the linear range, accuracy, and precision. At least a partial validation is necessary.
Response 2: Thank you for your comment. Some details about the used concentration range of tocochromanols and the obtained LOD and LOQ values were provided in the manuscript in the material section since the present study did not focus on the RPLC method development. (Page 6, top part)
Comment 3: Are the chromatograms in Figure 1 original? They appear to have been processed.
Response 3: Thank you for your comment. The obtained chromatograms are original. They have been improved to enhance the line thickness and description. The chromatograms are obtained by LabSolutions (version 5.124), Shimadzu Corporation Software, using the postrun option and data comparison. The data comparison feature allows for the analyst to apply a ‘base shift’ option, between the chromatograms and using different colors. Subsequently, the figure was transferred to a graphics program to thicken the lines and add descriptions. The primary goal was to ensure the chromatograms are clearly visible and well-described. The full original chromatograms of the saponified sample and extracted samples with different percentages of ethanol are provided in the Supplementary Materials.
Comment 4: The study method included the LC-MS method, but no corresponding results were presented.
Response 4: Thank you for your comment. Yes, mass spectrometry (MS) was employed only to confirm the identification of tocotrienols and tocopherols according to a previously established method [26], this is mentioned in the manuscript. The MS study was employed in the present study to avoid further concerns about the identification of tocotrienols in St. John's wort inflorescences. (Page 6, middle part)
Comment 5: In Figure 2, the y-axis represents recovery. What reference was used as 100%?
Response 5: Thank you for your comment. The saponification protocol was provided below the figure and the description was improved for clarity. (Page 8, middle part)
Comment 6: In Figure 3, the content appears to reach the plateau at higher ethanol levels. Linear regression may not be the best choice, while nonlinear regression should be considered.
Response 6: Thank you for your comment. We agree. The logarithmic trend was chosen and it seems that it fits well. (Page 9, middle part)
Comment 7: Minor Concerns. The English should be carefully edited to improve clarity.
Response 7: Thank you for your comment. The manuscript was improved by the English native speaker Arianne Soliven, PhD.
Comment 8: Is there a specific reason for using 96.2% ethanol?
Response 8: Thank you for your comment. The following explanation was added: “The highest purity ethanol concentration from SIA Kalsnavas Elevators (Jaunkalsnava, Latvia) (96.2 %) was used and its hydroethanolic solutions for the recovery of tocochromanols from residues of St. John's wort tea infusions/water extracts due to lower costs of solvent.” (Page 3, bottom part)
Author Response File: Author Response.pdf
