Patchouli Alcohol: A Potent Tyrosinase Inhibitor Derived from Patchouli Essential Oil with Potential in the Development of a Skin-Lightening Agent

Round 1

Reviewer 1 Report

The manuscript "Patchouli Alcohol: A Potent Tyrosinase Inhibitor Derived from Patchouli Essential Oil with Implications for Developing a Skin-Lightening Agent" is well written and presents a solid research. I would recommend it for publication with only a few minor issues to be fixed, as pointed below:

Methods: please add some key information on the methods used in the study (e.g. line 164 "Protein concentrations were determined using the Bio-Rad protein assay reagent" what kind of reagent is that, what is the principle of the method etc. Please add similar information for other methods where appropriate)

Line 331 "we assessed its cytotoxic effects on B16-F10 murine melanoma cells" Why have only melanoma cells been used. Why not keratinocytes as well. Please explain in the text.

Throughout the text pleaswe change melanin inhibition/inhibitory to melanin formation/synthesis inhibitory/inhibition (e.g. line 650 "tyrosinase and melanin inhibitory effects " please change to "tyrosinase- and melanin formation-inhibitory effects "

Avoid use of capital letters for some chemical compounds and techniques (e.g. line 114 "Proton Nuclear Magnetic Resonance" please change to "proton nuclear magnetic resonance" line 360 "Kojic acid" please change to "kojic acid")

English language is fine. Only minor issues detected (e.g. capital letters) as described above.

Author Response

Reviewer’s opinion: The manuscript "Patchouli Alcohol: A Potent Tyrosinase Inhibitor Derived from Patchouli Essential Oil with Implications for Developing a Skin-Lightening Agent" is well written and presents a solid research. I would recommend it for publication with only a few minor issues to be fixed, as pointed below:

Response: We would appreciate reviewer 1, who commented positively on our work. The reviewer’s comments and our responses are as follows.

Comment. 1: Methods: please add some key information on the methods used in the study (e.g. line 164 "Protein concentrations were determined using the Bio-Rad protein assay reagent" what kind of reagent is that, what is the principle of the method etc. Please add similar information for other methods where appropriate)

Response: We appreciate your suggestion to provide additional key information on the methods used. In response to your comment regarding the determination of protein concentrations using the Bio-Rad protein assay reagent, we apologize for the lack of detail. The Bio-Rad protein assay reagent utilizes the principle of the Bradford assay, which is based on the binding of Coomassie Brilliant Blue dye to proteins in an acidic solution. This binding causes a shift in the dye's absorption spectrum, leading to a color change that can be quantified spectrophotometrically. We will ensure to include this explanation briefly (Line 164-165).

Comment. 2: Line 331 "we assessed its cytotoxic effects on B16-F10 murine melanoma cells" Why have only melanoma cells been used. Why not keratinocytes as well. Please explain in the text.

Response: Thank you for your comment. We chose B16-F10 melanoma cells to assess cytotoxic effects due to melanoma relevance. Our study observed no cytotoxicity with PEO or PA up to 100 micrograms/mL. Hence, we believe these drug concentrations are safe for topical use. Thus, we focused solely on melanoma cells, omitting validation in other cell lines like keratinocytes. We will incorporate this rationale into our manuscript for clarity (Line 336-338).

Comment. 3: Throughout the text please change melanin inhibition/inhibitory to melanin formation/synthesis inhibitory/inhibition (e.g. line 650 "tyrosinase and melanin inhibitory effects " please change to "tyrosinase- and melanin formation-inhibitory effects ".

Response: We appreciate your suggestion to change "melanin inhibition/inhibitory" to "melanin formation/synthesis inhibitory/inhibition" for clarification. This update was made throughout the text, including the specific example you provided on line 650.

Comment. 4: Avoid use of capital letters for some chemical compounds and techniques (e.g. line 114 "Proton Nuclear Magnetic Resonance" please change to "proton nuclear magnetic resonance" line 360 "Kojic acid" please change to "kojic acid").

Response: Thank you for your valuable feedback regarding the use of capitalization for chemical compounds and techniques in our manuscript. As per your suggestion, we made the necessary revisions accordingly (Line 114, 364).

Reviewer 2 Report

1. The title is consistent with the content of the manuscript.

2. The research methodology is well described .

3. The findings are appropriately presented and described.

4. The literature is up-to-date and appropriately selected.

5. The findings in the manuscript are interesting.

I accept the manuscritpt in present form.

Author Response

Reviewer’s opinion: 1. The title is consistent with the content of the manuscript. 2. The research methodology is well described. 3. The findings are appropriately presented and described. 4. The literature is up-to-date and appropriately selected. 5. The findings in the manuscript are interesting. I accept the manuscript in its present form.

Response: We would appreciate reviewer 2, who commented positively on our work.

Reviewer 3 Report

The article"Patchouli Alcohol: A Potent Tyrosinase Inhibitor Derived from Patchouli Essential Oil with Implications for Developing a Skin-Lightening Agent" is very complex and well done. Base on your research how you expect PEO to be use?

Best regards!

Author Response

Reviewer’s opinion: The article "Patchouli Alcohol: A Potent Tyrosinase Inhibitor Derived from Patchouli Essential Oil with Implications for Developing a Skin-Lightening Agent" is very complex and well done. Based on your research how do you expect PEO to be used? Best regards!

Response: Thank you for your positive feedback on our article. Regarding your question about the expected use of Patchouli Essential Oil (PEO) based on our research, we anticipate several potential applications. Firstly, due to the significant tyrosinase inhibitory effects of patchouli alcohol, a major component of PEO, we believe it holds promise as a natural ingredient in skin-lightening formulations or cosmetics. Its ability to inhibit melanin formation could make it valuable for addressing hyperpigmentation issues such as melasma or age spots. Additionally, given its anti-inflammatory and antimicrobial properties demonstrated previously, PEO may also find application in skincare products targeting acne or other inflammatory skin conditions. Furthermore, PEO could be explored in dermatological treatments or as an adjuvant therapy alongside conventional treatments for conditions such as vitiligo, where melanin production is disrupted. Overall, our research suggests that PEO, particularly its active component patchouli alcohol, has potential as a versatile ingredient in various skincare and dermatological products aimed at addressing pigmentation disorders, inflammation, and microbial infections. Further studies and clinical trials would be necessary to validate its efficacy and safety for these applications.

Reviewer 4 Report

In the present work, the ability to inhibit melanin production of the essential oil of Pogostemon cablin and of one of its main active compounds, patchouli alcohol, was analyzed in B16-F10 (murine melanoma cell line) cell culture. Both the ability to inhibit the enzyme tyrosinase and the effect on the synthesis of this enzyme, were studied.

The article is interesting, it uses well-known techniques, the bibliography is up-date. The results obtained justify the conclusions. However, I believe that some modifications should be made, which in my opinion, would improve the quality of the article.

In my opinion, the discussion should go a little deeper. The first part of the discussion is more of an introduction than a discussion itself. Nothing is discussed about the results of molecular docking.

The abbreviation KA (Kojic Acid) is not defined until line 346. Please define it the first time it is named.

Graphs and tables have numerous abbreviations that should be defined in the figure/table caption. Tables and figures should be independent and must be understandable by themselves, without the need to refer to the text of the article.

Line 450: 50μM, isn't it 40μM?

Review the bibliography and avoid capitalizing each of the words in the title of references.

It would be interesting to have an analysis of the essential oil that is being used, to analyze its content in patchouli alcohol. Although it is true that, in view of the results, this compound seems to be mainly responsible for the inhibitory activity of tyrosinase activity.

Comments for author File: Comments.pdf

Author Response

Reviewer’s opinion: In the present work, the ability to inhibit melanin production of the essential oil of Pogostemon cablin and of one of its main active compounds, patchouli alcohol, was analyzed in B16-F10 (murine melanoma cell line) cell culture. Both the ability to inhibit the enzyme tyrosinase and the effect on the synthesis of this enzyme, were studied. The article is interesting, it uses well-known techniques, the bibliography is up-date. The results obtained justify the conclusions. However, I believe that some modifications should be made, which in my opinion, would improve the quality of the article.

Response: We would appreciate reviewer 1, who commented positively on our work. The reviewer’s comments and our responses are as follows.

Comment. 1: In my opinion, the discussion should go a little deeper. The first part of the discussion is more of an than a discussion itself. Nothing is discussed about the results of molecular docking.

Response: Thank you for your feedback. We have enhanced the discussion section, particularly focusing on molecular docking results' implications. Our revised discussion will delve into the binding interactions between patchouli alcohol and tyrosinase, their significance, and potential applications in developing skin-lightening agents (Line 652-662; 789-799)

Comment. 2: The abbreviation KA (Kojic Acid) is not defined until line 346. Please define it the first time it is named.

Response: Thank you for your feedback. As per your suggestion, we defined the abbreviation "KA" for Kojic Acid when it was first mentioned in the manuscript (Line 118).

Comment. 3: Graphs and tables have numerous abbreviations that should be defined in the figure/table caption. Tables and figures should be independent and must be understandable by themselves, without the need to refer to the text of the article.

Response: Thank you for your feedback. Abbreviations in figures and tables are now accompanied by acronyms in line with your suggestion. This ensures the clarity and independence of each figure and table, making them understandable without reference to the text (Lines 286, 328, 398, 447, 464, and 508).

Comment. 4: Line 450: 50μM, isn't it 40μM?

Response: Thank you for bringing this to our attention. The typographical error has been rectified (Lines 453).

Comment. 5: Review the bibliography and avoid capitalizing each of the words in the title of references.

Response: Thank you for bringing this to our attention. The typographical error has been rectified in the list of references.

Comment. 6: It would be interesting to have an analysis of the essential oil that is being used, to analyze its content in patchouli alcohol. Although it is true that, in view of the results, this compound seems to be mainly responsible for the inhibitory activity of tyrosinase activity.

Response: Thank you for your insightful suggestion. While analyzing the chemical profile of patchouli oil, particularly its patchouli alcohol content, would indeed be intriguing, it's worth noting that such analyses have been conducted by other authors. Therefore, this study focuses on the specific inhibitory activity of patchouli alcohol on tyrosinase rather than on the comprehensive chemical analysis of patchouli essential oil.

References:

1. Srivastava, S.; Lal, R. K.; Singh, V. R.; Rout, P. K.; Padalia, R. C.; Yadav, A. K.; Bawitlung, L.; Bhatt, D.; Maurya, A. K.; Pal, A.; Bawankule, D. U.; Mishra, A.; Gupta, P.; Chanotiya, C. S., Chemical investigation and biological activities of Patchouli (Pogostemon cablin (Blanco) Benth) essential oil. Indus Crop Prod 2022, 188, 115504.
2. Fensia, A.; Aziz, A.; Nazudin; Souhoka, F., Patchouli Oil Isolation and Identification of Chemical Components Using GC-MS. Indo J Chem Res 2020, 8, 108-113.
3. van Beek, T. A.; Joulain, D., The essential oil of patchouli, Pogostemon cablin: A review. Flavor Fragnance J 2018, 33, 6-51. 12.
4. Swamy, M. K.; Sinniah, U. R., A Comprehensive review on the phytochemical constituents and pharmacological activities of Pogostemon cablin: An aromatic medicinal plant of industrial importance. Molecules 2015, 20, 8521-47.