Antioxidant Activity and Phenolic Compound of Rosemary Under Artificial LED Lights

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

in this manuscript, the effects of different light type on plant growth (length only), the free radical scavenging activity and content of total flavonoid, total phenlic and several individual compounds were evaluated. I have few questions for the authors as follows.

in section 2.1 the number of plant pots tested should be addressed. picture of plants before light treatment should be given for reference. final extract was prepared to a final concentration of 10mg/ml and used for further examination, the volume of each group and biomass used should be addressed, because this is very important.
in section 2.2 the solvent of DPPH solution should be addressed. four concentrations of extract was diluted, why use four concentrations?
the inhibition rate calculation fomula should be given.
in 2.6, the sample was dissolved in ……, what is the sample? herb powder or extract?
in 3.1 only total length was measured in different groups, which might be not very reflective. why don't the authors measure the total biomass increase of each group at last week? that should be more convencing. the herb is kind of flexible and is not as strong as a tree plant. in figure 2, why the length of each group is so different at week 0? should they be the same or similar before light treatment?
in 3.2, the free radical scavenging activity was shown for different concentrations and treated samples. I suggest you use the IC50 valve to illustrate the differences between groups. in figure 3a and 4b there is a text box in Korean which should be deleted.
in section 3.4, the contents of 6 compounds were determined using HPLC at the last week, which in my opinion is not a good design. why don't the authors sample the treated plants at different treat time and used for the dymatic analysis? the contents of different compound or total compounds are expressed as ug/ml，I suggest authors change the unit to mg/g biomass of treated plants. or it would be confusing to conclude the effects of different light on the accumulation of compounds of interest.

Author Response

Response to reviewer 1

 

All revised texts are marked in red, except for the figures.

 

in this manuscript, the effects of different light type on plant growth (length only), the free radical scavenging activity and content of total flavonoid, total phenlic and several individual compounds were evaluated. I have few questions for the authors as follows.

 

Q1: in section 2.1 the number of plant pots tested should be addressed. picture of plants before light treatment should be given for reference. final extract was prepared to a final concentration of 10mg/ml and used for further examination, the volume of each group and biomass used should be addressed, because this is very important.

R1: It has been revised as requested by the reviewer, but there are no photos of the plants for reference before lighting treatment.

Q2: in section 2.2 the solvent of DPPH solution should be addressed. four concentrations of extract was diluted, why use four concentrations?

the inhibition rate calculation fomula should be given.

R2:

1. Solvent of the DPPH Solution: Thank you for pointing out the need to specify the solvent used in the DPPH solution in Section 2.2. In this study, the DPPH solution was prepared using methanol as the solvent. This solvent was chosen because DPPH radicals remain stable in it and it is widely used for antioxidant activity evaluation. We revised the manuscript to clearly include this information.
2. Reason for Using Four Concentrations: The four different concentrations of the extract were used to determine whether the antioxidant activity is concentration-dependent. By testing multiple concentrations, we were able to compare the antioxidant activity across different levels and accurately calculate the IC₅₀ value. This approach allows for a more quantitative assessment of the extract’s antioxidant potential.

 

Q3: in 2.6, the sample was dissolved in ……, what is the sample? herb powder or extract?

R3: The sample mentioned in section 2.6 refers to extract. Thus, in section 2.6, I revised to “ The extract was diluted~”.

 

Q4: in 3.1 only total length was measured in different groups, which might be not very reflective. why don't the authors measure the total biomass increase of each group at last week? that should be more convencing. the herb is kind of flexible and is not as strong as a tree plant. in figure 2, why the length of each group is so different at week 0? should they be the same or similar before light treatment?

R4:

1. Justification for Measuring Only Total Length: Thank you for your insightful comment. In this study, we chose to measure total length rather than total biomass increase because the herb we studied has a relatively flexible structure, making it difficult to obtain accurate biomass measurements without causing potential damage. Additionally, total length is a widely used indicator in studies of similar plant species and provides a practical way to assess growth differences under different conditions. However, we acknowledge that measuring biomass could provide additional insights, and we will consider this approach in future studies.
2. Explanation for Initial Length Differences in Figure 2: We appreciate your observation regarding the differences in initial length at week 0. Ideally, all groups should start with similar lengths before the light treatment. However, slight variations occurred due to natural biological differences in the herb samples, even though we selected plants within a specific size range. These variations were taken into account during data analysis, and we ensured that they did not significantly affect the overall trends observed.

 

Q5: in 3.2, the free radical scavenging activity was shown for different concentrations and treated samples. I suggest you use the IC50 valve to illustrate the differences between groups. in figure 3a and 4b there is a text box in Korean which should be deleted.

R5:

Thank you for your suggestion regarding the presentation of free radical scavenging activity in Section 3.2. While IC₅₀ is commonly used to compare antioxidant activities, we chose to present the results as percentage values for the following reasons:

1. Direct Representation of Activity – Percentage values provide a straightforward and intuitive way to compare the scavenging activity across different concentrations and treated samples without requiring additional calculations.
2. Concentration-Dependent Trends – Presenting the data as percentage inhibition allows for a more detailed visualization of how the activity changes with different concentrations. This is particularly useful when the relationship between concentration and activity does not follow a strictly linear pattern.
3. Avoiding Potential Misinterpretation – In some cases, the IC₅₀ value might not be accurately determined, especially if the highest tested concentration does not reach 50% inhibition. By showing percentage values, we ensure that all data points are included, providing a more comprehensive representation of the results.

 

And, figure 3a and 4b were revised clearly.

 

Q6: in section 3.4, the contents of 6 compounds were determined using HPLC at the last week, which in my opinion is not a good design. why don't the authors sample the treated plants at different treat time and used for the dymatic analysis? the contents of different compound or total compounds are expressed as ug/ml，I suggest authors change the unit to mg/g biomass of treated plants. or it would be confusing to conclude the effects of different light on the accumulation of compounds of interest.

R6:

1. Justification for Sampling Only at the Last Week

"Thank you for your insightful suggestion regarding dynamic analysis over different treatment periods. In this study, we focused on evaluating the final accumulation of phenolic compounds at the end of the experimental period rather than conducting multiple time-point analyses. This approach was chosen for the following reasons:

Final Accumulation Assessment: Our primary objective was to determine how different light treatments influence the ultimate accumulation of phenolic compounds rather than their fluctuation over time. Since phenolic accumulation is a cumulative process, the final content reflects the long-term effects of the light treatments.

Practical Considerations: Sampling at multiple time points would require destructive harvesting, which could introduce variability due to differences in plant growth rates and biomass allocation over time. To ensure consistency, we chose to analyze the samples at a single, standardized endpoint.

Comparable Methodology: Similar studies evaluating the impact of light conditions on phenolic content often analyze samples at a single time point to ensure comparability and minimize experimental variability. However, we acknowledge that dynamic analysis could provide additional insights and will consider this approach in future research."

2. Justification for Expressing Phenolic Compound Content in μg/mL

"Regarding the unit of measurement, we expressed the phenolic compound content in μg/mL because:

Extract-Based Comparison: Our study focused on comparing the phenolic content in the extracted solutions rather than on a per-biomass basis. This allows for direct comparison of the bioactive compound concentrations in the extracts used for biological activity assays.

Consistency with Antioxidant Activity Data: Since antioxidant activity assays were performed using the same extracts, maintaining consistency in concentration units (μg/mL) helps to correlate the compound content with radical scavenging activity.

Avoiding Variability Due to Biomass Differences: Expressing the results as mg/g biomass may introduce additional variability due to differences in moisture content and extraction efficiency among samples. Using μg/mL allows for a more controlled and reproducible comparison.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The effects of different LED lighting conditions on the biological activity of Rosmarinus officinalis have been reported. The experimental results showed that rosemary grown under red light had the highest growth rate and antioxidant capacity, and contained higher total phenolics and total flavonoids. I agree that these are exciting research topics. However, the manuscript must be crucible-revised before it meets precise research requirements

1.          In the abstract, the author only describes the experimental data, and does not put forward effective opinions on the purpose and significance of the experiment and the future agricultural application and development.

2.          In 2.1, please describe the cultivation and growth conditions of rosemary before the treatment of different light sources, including planting methods, growth time, light, temperature and humidity conditions for plant growth. Please also explain why these four different light sources have not been used from the time of planting?

3.          Korean logo appears in Fig.3 and 4, please reprocess the picture.

4.          The discussion section is mixed with the description of the results and is not deep enough. As an agronomy journal, I advise authors to expand the discussion section more on potential agronomic implications of the results, including future perspective for agriculture in the light of their preliminary findings.

Author Response

Response to reviewer 2

 

P.S.: The revised sentences are highlighted in red.

 

The effects of different LED lighting conditions on the biological activity of Rosmarinus officinalis have been reported. The experimental results showed that rosemary grown under red light had the highest growth rate and antioxidant capacity, and contained higher total phenolics and total flavonoids. I agree that these are exciting research topics. However, the manuscript must be crucible-revised before it meets precise research requirements

 

Q1: In the abstract, the author only describes the experimental data, and does not put forward effective opinions on the purpose and significance of the experiment and the future agricultural application and development.

R1: It has been revised as requested by the reviewer.

 

Q2: In 2.1, please describe the cultivation and growth conditions of rosemary before the treatment of different light sources, including planting methods, growth time, light, temperature and humidity conditions for plant growth. Please also explain why these four different light sources have not been used from the time of planting?

R2: It has been revised including planting methods, growth time, light, temperature and humidity conditions as requested by the reviewer.

• why these four different light sources have not been used from the time of planting?: The initial growth phase under uniform white fluorescent light ensured that all plants reached a similar developmental stage before LED treatment, minimizing variations due to early growth differences. This approach was adopted to standardize initial plant conditions and allow for a more accurate comparison of the effects of different LED treatments.
•  

Q3: Korean logo appears in Fig.3 and 4, please reprocess the picture.

R3: It has been revised as requested by the reviewer.

 

Q4: The discussion section is mixed with the description of the results and is not deep enough. As an agronomy journal, I advise authors to expand the discussion section more on potential agronomic implications of the results, including future perspective for agriculture in the light of their preliminary findings.

R4: It has been revised as requested by the reviewer.

 

 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This paper examines the relationship between the antioxidant activity and phenolic compounds of rosemary grown under different wavelengths of LED light. It was shown that red light increases the antioxidant activity and the amount of secondary metabolites in rosemary extracts. Here are a few minor revision points. Please refer to these for making corrections.

 

Minor comments

1.       P.3. The numbers in the title of the vertical axis in Figure 1(a)  should be written in superscript.

2.       L.127. TPC is an abbreviation. The full name should be written in parentheses.

3.       L.139, L.240 and Table 1. p of “p-coumaric acid” should be written in italics.

4.       L.144 and L.151. The size of the column should be written.

5.       L.176. “in” should be written between “than” and “the”.

6.       Figs.2-4 and Table 1. Explanation of what this character (W, R, G and B) represents is required in the figures and the table.

7.       Figs.2-4. Explanation of the lowercase letters above the bar graph and the statistical analysis methods is required in figure legends.

8.       L. 210 and Fig.4. An explanation of “GAE” should be added.

9.       L. 2104and Fig.4. An explanation of “QE” should be added.

10.   Table 1. Explanation of the lowercase letters above the bar graph and the statistical analysis methods is required.

11.   Table 1. The unit for representing the amount of compounds is currently in ug/ml, but it would be clearer if expressed ug/g of plant material.

Comments on the Quality of English Language

The English in this manuscript is understandable.

Author Response

Response to reviewer 3

 

P.S.: The revised sentences are highlighted in red.

 

Comments and Suggestions for Authors

This paper examines the relationship between the antioxidant activity and phenolic compounds of rosemary grown under different wavelengths of LED light. It was shown that red light increases the antioxidant activity and the amount of secondary metabolites in rosemary extracts. Here are a few minor revision points. Please refer to these for making corrections.

 

Minor comments

 

Q1: P.3. The numbers in the title of the vertical axis in Figure 1(a) should be written in superscript.

R1: It has been revised according to the reviewer's opinion.

 

Q2: L.127. TPC is an abbreviation. The full name should be written in parentheses.

R2: It has been revised according to the reviewer's opinion.

 

Q3: L.139, L.240 and Table 1. p of “p-coumaric acid” should be written in italics.

R3: It has been revised according to the reviewer's opinion.

 

Q4: L.144 and L.151. The size of the column should be written.

R4: It has been revised according to the reviewer's opinion.

 

Q5: L.176. “in” should be written between “than” and “the”.

R5: It has been revised according to the reviewer's opinion.

 

Q6: Figs.2-4 and Table 1. Explanation of what this character (W, R, G and B) represents is required in the figures and the table.

R6: It has been revised according to the reviewer's opinion.

 

Q7: Figs.2-4. Explanation of the lowercase letters above the bar graph and the statistical analysis methods is required in figure legends.

R7: It has been revised according to the reviewer's opinion.

 

Q8: L. 210 and Fig.4. An explanation of “GAE” should be added.

R8: It has been revised according to the reviewer's opinion.

Q9: L. 2104and Fig.4. An explanation of “QE” should be added.

R9: It has been revised according to the reviewer's opinion.

 

Q10: Table 1. Explanation of the lowercase letters above the bar graph and the statistical analysis methods is required.

R10: It has been revised according to the reviewer's opinion.

 

Q11: Table 1. The unit for representing the amount of compounds is currently in ug/ml, but it would be clearer if expressed ug/g of plant material.

R11: Thank you for your valuable suggestion regarding the unit representation in Table 1. We understand the importance of clarity in data presentation and appreciate your feedback. However, we chose to express the compound concentrations in µg/mL rather than µg/g of plant material for the following reasons:

 

Standard Practice in HPLC Analysis

 

The concentration of phenolic compounds was measured in the liquid extract obtained from rosemary samples using HPLC analysis, where the standard practice is to report values in µg/mL.

This allows for direct comparison with similar phytochemical studies and ensures consistency with existing literature.

Avoiding Extraction Efficiency Variability

 

Converting the data to µg/g of plant material would require additional normalization based on extraction efficiency, which can introduce variability.

Factors such as solvent choice, extraction conditions, and plant tissue properties can influence the yield, making µg/mL a more direct and reliable unit for quantifying compound concentrations in the extract.

Relevance to Practical Applications

 

In many industrial applications, such as herbal medicine, cosmetics, and functional foods, rosemary extracts are used in liquid form.

Reporting concentrations in µg/mL aligns with how these compounds are typically measured and utilized in real-world scenarios.

Potential Inclusion of Both Units for Clarity

 

If necessary, we can provide an additional conversion of the values in µg/g of plant material as supplementary information or within the table to enhance clarity while retaining µg/mL for consistency with analytical methodologies.

 

 

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

ADDITIONAL COMMENTS

“Antioxidant activity and phenolic compound of rosemary under artificial LED lights” 

I. Article Overview

The article investigates the effects of different LED lights on growth, antioxidant activity and accumulation of phenolic compounds in rosemary (Rosmarinus officinalis). The authors analyse the influence of white, red, blue and green light on the biologically active constituents in the plant using DPPH and ABTS assays, as well as HPLC analysis for phenolic compounds.

The study is well structured and includes an in-depth analysis of rosemary growth and antioxidant accumulation.

II. Methods used:

DPPH and ABTS tests to assess antioxidant activity. Folin-Chikalteau method to measure total phenolic content. HPLC analysis for identification and quantification of phenolic acids (rosmarinic, gallic, caffeic, etc.).

Assessment: The methods are well selected, but information on internal controls and reproducibility of measurements is lacking.

III. Discussion and interpretation of the results

The authors correctly interpret the results by comparing them with previous studies.

The role of the light spectrum in plant metabolism is examined.

It is concluded that red light favors the accumulation of bioactive compounds.

It should be mentioned that while the article has some useful aspects, I also noticed some weaknesses.

The useful aspects are related to the relevance, application in life, scientific justification, as well as the methodology and adequacy of the results to support the objectives of the study and future application:

ü  Relevance of the study – the topic is important for agriculture, pharmacy and the food industry.

ü  Well-described methodology – includes spectrophotometric tests and HPLC analysis.

ü  Clear and well-presented results – include graphs, tables and statistical analysis.

 

In addition, I found it lacking in-depth research:

v  Lack of in-depth analysis of the mechanisms – it is not explained why red light leads to increased phenol content.

v  Lack of internal controls – it would be useful to have additional control groups (e.g. natural lighting conditions). This should be taken into account as a recommendation.

v  Limited discussion of the application of the results – it would be useful to consider the potential industrial applications of the study. I give it as a recommendation.

IV. Conclusion

The article provides an important contribution to the understanding of the effect of light on the antioxidant properties of rosemary. Despite some methodological weaknesses, the study is well structured and substantiated.

 

The manuscript is suitable for publication in your journal.

Comments for author File: Comments.pdf

Author Response

Response to reviewer 4

 

All of the shortcomings in the manuscript have been revised to reflect the reviewers' comments.

 

P.S.: The revised sentences are highlighted in red.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

the authors have made major improvement of this manuscript. I suggest the acceptance of this revise manuscript.

Author Response

Dear reviewer 1

 

All have been revised to reflect reviewer's opinions.

Thank you so much.

 

Best regards,

Prof. Eun Soo Seong

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript Number:

Titel：Antioxidant activity and phenolic compound of rosemary under artificial LED lights

The revised manuscript has been greatly altered and improved. Some of the latest reports were quoted, the writing was standardized, and some errors have been corrected. Based on the current modification content, I recommend it can be accepted after minor revisions.

Liquan Guo

Author Response

Dear reviewer 2

 

All have been revised to reflect reviewer's opinions.

Thank you so much.

 

Best regards,

Prof. Eun Soo Seong