Non-Targeted Secondary Metabolite Profile Study for Deciphering the Cosmeceutical Potential of Red Marine Macro Alga Jania rubens—An LCMS-Based Approach
Round 1
Reviewer 1 Report
This paper proposal represents a deep analysis of Jania rubens mineral and secondary metabolites profile, rendering relevant information to support its utilization into cosmeceutical formulations as well as providing some additional data for cosmeceutical anti-oxidant and mineral related claims.
The data is clean and simply presented and the discussion is sufficiently detailed.
The English in the paper shall be revised by a native or fluent English speaker as some colloquial expressions are used and some grammatical and sentence construction issues are present through out the text.
Introduction
line 47 - "..infections caused due to ultraviolet radiation..." change to "..infections caused by to ultraviolet radiation..."
line 53 - Skin ageing is a complex biological process so this sentence is reductionist and shall be reformulated.
line 55 - "... as these agents are mostly safe, non-toxic..." change to "... as these agents are mostly perceived by customers as safe, non-toxic..."
line 61 - "... Till now..." very colloquial style. Change to "until now". this sentence needs reviewing.
line 63 - substitute "contender" for "candidate"
line 89-90 - "Unveiling the secondary metabolite profile... would be useful". Why? Authors should expand a bit more here
Methods
Sample collection - were all the samples collected at a single time point? same season? are they all the same species? this shall be clarified
line 127 - "10 g of dry sample... milled and then extracted thrice..." Typo on thrice? Shall it be twice?
Results
line 195 - how many sample species were analysed? what distinguished them?
Table 1 formatting can be improved (join cells in line number 2 and 8) and in last line add units please.
Figure 1 - legend shall be more complete.
Figure 2 - shouldn't the standard curves of the control reach nearly 100% of total anti-oxidation? can the authors explain why in the FRAP and total antioxidant such is not the case?
line 226, draw a simple conclusion like you did for total phenol content.
lines 274-277 - what known roles have these aromatic compounds? authors should include it here.
lines 279-281 - same as above
Discussion
Line 313 - "Sodium (Na), apart from... " Rephrase this sentence please.
line 364 - further details for the authors hypothesis on DPPH results are needed.
Conclusions
This part could be more explored for example for what this data can contribute to support cosmeceutical claims manufactures adding J.rubens extract can do. It could also include some suggestions of next steps and analysis the authors believe this work could lead to.
Author Response
Introduction
Q: line 47 - ".infections caused due to ultraviolet radiation..." change to "infections caused by to ultraviolet radiation..."
A: The change as per the reviewer’s suggestion has been made.
Q: line 53 - Skin aging is a complex biological process so this sentence is reductionist and shall be reformulated.
A: The sentence has been reformed and rewritten.
Q: line 55 - "... as these agents are mostly safe, non-toxic..." change to "... as these agents are mostly perceived by customers as safe, non-toxic..."
A: The suggested change has been made in the manuscript.
Q: line 61 - "... Till now..." very colloquial style. Change to "until now". This sentence needs reviewing.
A: The sentence has been reformed.
Q: line 63 - substitute "contender" for "candidate"
A: The suggested substitution has been done.
Q: line 89-90 - "Unveiling the secondary metabolite profile... would be useful". Why? Authors should expand a bit more here
A: A Jania rubens extract is being proposed for formulating slimming cosmetics as it apparently promotes the elimination of fats and the synthesis of collagen for smoothing out cellulite. This claim, however, is not substantiated by the scientific literature. In addition to this, J.rubens extract is also being proposed for skin conditioning formulations. But to the best of our knowledge, this is the first comprehensive scientific study to assess its cosmeceutical virtues. Therefore, unveiling the secondary metabolite profile of such a resourceful marine algae would be quite interesting as it may open new avenues for designing algal biorefinery for obtaining high value-added products useful for future cosmetic applications. This has been added to the manuscript.
Method
Q: Sample collection - were all the samples collected at a single time point? same season? are they all the same species? this shall be clarified
A: The sample collection was done in the same season (December) and at the same point of time. The samples were then identified and confirmed as same species by the experts at CSIR-CSMCRI, India and then proceeded for further study.
Q: line 127 - "10 g of dry sample... milled and then extracted thrice..." Typo on thrice? Shall it be twice?
A: Yes. It was a typographical error. The correction has been incorporated in the manuscript. Please note that after formatting, the line number has changed from 127 to 131 in the manuscript.
Results
Q: line 195 - how many sample species were analyzed? what distinguished them?
A: The analysis was carried out in triplicates. The sample species were confirmed to be the same. Please note that the line number has changed from 195 to 201.
Q: Table 1 formatting can be improved (join cells in line number 2 and 8) and in the last line add units, please.
A: The formatting in Table 1 has been changed and the cells in line number 2 and 8 have been merged. The unit has been added to the last line.
Q: Figure 1 - legend shall be more complete.
A: Figure-1 legend has been made more complete and self- explanatory.
Q: Figure 2 - shouldn't the standard curves of the control reach nearly 100% of total anti-oxidation? can the authors explain why in the FRAP and total antioxidant such is not the case?
A: The reducing power of the reference compound (Ascorbic acid) and the scavenging capacity of (Trolox) in the total antioxidant assay were found to be higher than all the tested extracts. However, they did not reach a value of 100% when tested within the range of 1000 µg/mL to 3000 µg/mL during our study. The increase in the value was linear and a higher concentration than the tested range may have led both the standard values to reach 100% as the increment was concentration dependent.
Q: line 226, draw a simple conclusion like you did for total phenol content.
A: A simple conclusion has been drawn and included. Please note that the line number has got changed to 241.
Q: lines 274-277 - what known roles have these aromatic compounds? authors should include it here.
A: The known roles of the aromatic compounds mentioned in lines 274-277 have been included in the manuscript. But after formatting, the line numbers have got changed to 307-311.
Q: lines 279-281 - same as above
A: The known roles of the compounds mentioned in lines 279-281 have been included in the manuscript. The line numbers have been changed post formatting. The new line numbers are 313-316.
Discussion
Q: Line 313 - "Sodium (Na), apart from... "Rephrase this sentence, please.
A: The sentence has been rephrased. The line number has now been changed to 353.
Q: line 364 - further details for the author’s hypothesis on DPPH results are needed.
A: This has been included in the manuscript. The line number has changed to 405.
Conclusions
Q: This part could be more explored for example for what this data can contribute to support cosmeceutical claims manufactures adding J.rubens extract can do. It could also include some suggestions of next steps and analysis the authors believe this work could lead to
A: These features collectively make this coralline alga a promising candidate for its inclusion as an active ingredient in the modern-day cosmeceutical as well as pharmaceutical formulations/products utilized for skin conditioning, skin polishing, anti-aging and skin whitening. J.rubens can, therefore, be considered as a potential natural candidate for improving the quality of modern day cosmetics. However, further studies undertaking the purification as well as clinical trials of the purified components is required for its inclusion as an integral ingredient. This study provides the base for the future perspective of considering J.rubens in the designing of algal biorefinery and other eco-designs to obtain high value-added products. This addition has been done in the manuscript under ‘Conclusion’ section.
Author Response File: 
Author Response.doc
Reviewer 2 Report
The manuscript submitted is apt to the current readers in this field. The J. rubens availability and harvesting the same for various bioactive applications is quite an interesting study. However, the authors need to consider below points before its publication in cosmetics journal.
1. Considering this species availability in India, the authors need to confirm the species identification and its repository in CSIR-NIO or any concerned agencies while they are studying the organism in depth. This need to include in the methods section under sampling.
2. Major research activities in this paper are related to antioxidative properties of the extracted metabolites, however, any small application of the selected leads in the form of a formulation or cream to study in animals would be more challenging to address the case. If any such data available, please include.
3. Moreover, the novelty lies only in the LCMS approach but several other studies in different species have used extensively these antioxidative methods for claiming the cosmetic applications.
4. The values of antioxidative activities are almost on par with the reference standard, Ascorbic acid (at least for few activities), however, the authors need to understand the economic production of the Ascorbic acid when compared with the claimed metabolites, where the industrial development of final product (cream or formulation etc.) will be highly expensive I guess.
5. Last but not least, several of the sentences framed in the manuscript are not in accordance to the scientific writing. Request the authors to re-check the English presentation one more time before its final publication.
Author Response
Q: 1. Considering this species availability in India, the authors need to confirm the species identification and its repository in CSIR-NIO or any concerned agencies while they are studying the organism in depth. This need to include in the methods section under sampling.
A: The authors have identified and confirmed the species with the experts at CSIR-CSMCRI before studying this species in detail. This has been incorporated in the method section.
Q: 2. Major research activities in this paper are related to antioxidative properties of the extracted metabolites, however, any small application of the selected leads in the form of a formulation or cream to study in animals would be more challenging to address the case. If any such data available, please include.
A: No such study related to the application of the identified metabolites in the form of a formulation or a cream to check its effect on animals has been done by us. Therefore, no such data is available with us.
Q: 3. Moreover, the novelty lies only in the LCMS approach but several other studies in different species have used extensively these antioxidative methods for claiming the cosmetic applications.
A: To the best of our knowledge, this is the first detailed study on the secondary metabolite profiling of this species highlighting its cosmeceutical virtues. The study encompasses not only the antioxidative potential but also its mineral content along with its secondary metabolite profile which also contains pharmaceutically important metabolites. The novelty further lies in the fact that this is a baseline study that shall pave way for considering this species as a potential candidate in cosmetic formulations.
Q: 4. The values of antioxidative activities are almost on par with the reference standard, Ascorbic acid (at least for few activities), however, the authors need to understand the economic production of the Ascorbic acid when compared with the claimed metabolites, where the industrial development of final product (cream or formulation etc.) will be highly expensive I guess.
A: Algal metabolites represent an interesting source of active ingredients for the cosmetic market, although in terms of costs they still remain expensive due to the research and biotechnological processes needed to generate and maintain them. Therefore, in order to overcome this limitation, studies related to alternative biotechnological tools and techniques for making the process economically viable are under progress.
Q: 5. Last but not least, several of the sentences framed in the manuscript are not in accordance to the scientific writing. Request the authors to re-check the English presentation one more time before its final publication.
A: As per the reviewer’s suggestion, we have tried to re-frame the language of the manuscript according to the requirements of the Journal.
Author Response File: Author Response.doc
Reviewer 3 Report
This manuscript describes a nice study aiming to evaluate important parameters of Jania rubens such as mineral composition, antioxidant potential and the presence of bioactive molecules using non-targeted metabolite profiling. The work is interesting and carefully performed, however, there are a few points, which need to be addressed by the authors:
Main points:
1) Fig 1: the results of total phenol content and total flavonoid content are expressed as μg/mL. The authors should consider expressing the results as μg per mg of dry weight. In addition, standard deviation is missing from the bars.
2) Fig 2: the results of antioxidant assays are expressed as μg/mL. The authors should consider expressing the results as mg per mg of dry weight. In addition, standard deviation is missing from the experimental points.
3) The authors evaluated the bioactivity parameters using methanol/water as an extraction medium. Why ? Did the authors study another solvent ?
4) The authors evaluated the total antioxidant capacity through the formation of a phosphomolybdenum complex. Why the authors chose this assay? A short explanation/discussion should be added in the manuscript.
5) How do the results of the study (mineral composition, antioxidant potential, total flavonoid, total phenol content) compared with those of other micro and macroalgae. The authors should discuss their findings in light of the available literature.
Minor points:
1) Throughout the manuscript: replace ml with mL.
2) Throughout the manuscript: correct chemical formulas (e.g. CaCO3) with subscripts and ions formulas (e.g. Fe2+) with superscripts.
Author Response
Main points:
Q: 1. Fig 1: the results of total phenol content and total flavonoid content are expressed as μg/mL. The authors should consider expressing the results as μg per mg of dry weight. In addition, standard deviation is missing from the bars.
A: The results of the total phenol content and total flavonoid content have been changed and expressed as mg/mg seaweed (DW). The standard deviation bars have been added.
Q: 2. Fig 2: the results of antioxidant assays are expressed as μg/mL. The authors should consider expressing the results as mg per mg of dry weight. In addition, standard deviation is missing from the experimental points.
A: The results of the antioxidant assays have been calculated in terms of %Scavenging capacity according to the formula mentioned in the manuscript. However, the FRAP assay result has been expressed as mg/g seaweed (DW). Standard deviation values have been added to the graphs.
Q: 3. The authors evaluated the bioactivity parameters using methanol/water as an extraction medium. Why? Did the authors study another solvent?
A: The methanol/water extraction medium was selected as the % yield of the J.rubens extract (from 1 gram dry weight seaweed biomass) was found to be the highest (12.36%) with this combination. In case of other solvents like water (3.97%) ethanol (6.13%), ethyl acetate (2.05%), diethyl ether (2.63%), acetone (0.91%) and isopropanol (0.99%), the % yield values obtained were less. These results led us to the selection of this particular medium as the maximum number of metabolites could be extracted using this combination.
Q: 4. The authors evaluated the total antioxidant capacity through the formation of a phosphomolybdenum complex. Why the authors chose this assay? A short explanation/discussion should be added in the manuscript.
A: The evaluation of the total antioxidant capacity was done using the phosphomolybdenum method as it evaluates both fat-soluble as well as water-soluble antioxidants (total antioxidant capacity). The phosphomolybdenum method is a simple, cheap and good alternative to the other laborious methods used for assessing the total antioxidant capacity. This assay was carried out in order to get a broader understanding of the total antioxidant capacity exhibited by J.rubens rather than determining the antioxidant potential of individual constituents. Moreover, it gives a clear understanding of the changes in the antioxidant activity in relation to the oxidative stress and is widely used in a variety of complex mixtures used in pharmaceutical and cosmetic preparations. This explanation has been added to the manuscript.
Q: 5) How do the results of the study (mineral composition, antioxidant potential, total flavonoid, total phenol content) compared with those of other micro and macroalgae. The authors should discuss their findings in light of the available literature.
A: This part has been added to the manuscript in the ‘Discussion’ section.
Minor points:
Q: 1. Throughout the manuscript: replace ml with mL.
A: ‘ml’ has been replaced with ‘mL’ throughout the manuscript.
Q: 2. Throughout the manuscript: correct chemical formulas (e.g. CaCO3) with subscripts and ions formulas (e.g. Fe2+) with superscripts.
A: The chemical formulas have been corrected throughout the manuscript as per the reviewer’s suggestion.
Author Response File: Author Response.doc
Round 2
Reviewer 2 Report
The responses are quite convincing. However, small advise on the reference citation in text as below:
The names of the Author of the references should be cited in the text (wherever necessary) and then the ref. No. will follow e.g., the authors mentioned that "The total phenolic content in this study was determined following the protocol as per [26]" in the Page 4 (Line No. 140).
This an be reframed as "The total phenolic content in this study was determined following the protocol as per Lim et al. [26]." Please check other published papers in this journal and revise the same in the entire manuscript by following the journals guidelines.
Author Response
Q. The names of the Author of the references should be cited in the text (wherever necessary) and then the ref. No. will follow e.g., the authors mentioned that "The total phenolic content in this study was determined following the protocol as per [26]" in the Page 4 (Line No. 140). This can be reframed as "The total phenolic content in this study was determined following the protocol as per Lim et al. [26]."
A. As per the Reviewer’s suggestion, we have reframed the sentences and indicated the respective author's name followed by the respective reference number.
Author Response File: Author Response.doc
