Evolution of Phytochemical Variation in Myrtle (Myrtus communis L.) Organs during Different Phenological Stages

Round 1

Reviewer 1 Report

Accept without amendments

Author Response

No amendments received

Reviewer 2 Report

All comments are given in the manuscript attached below.

Comments for author File: Comments.pdf

Author Response

-Have you used any standard compound? Please, explain in more details how you calculate %w/w in Table 1 (line 127).

Thank you for your precise comment. In essential oil analysis with GC and GC-MS devices, the percentage of compounds is presented in general and the sign “%w/w” was removed from table 1. (Added in now lines 124-125 and changed in now line 238)

 

-Antioxidant activity of what, EOs or extracts. or both of them? (line 151)

Thank you for your comment. In the present study, we evaluated the antioxidant properties of the extract, which were revised in the manuscript. Now in sections 2.7, line 151, and 3.3, line 312

 

-Is this correct? “The high antioxidant properties in this plant occur due to the existence of the phenolic and flavonoid compounds in the EO.”

Thank you for your comment. Essential oil was changed to extract. Now in line 372.

Reviewer 3 Report

The manuscript “Evolution of Phytochemical Variation in Myrtle (Myrtus communis L.) Organs During Different Phenological Stages” gives a detailed insight into the essential oil composition (and content) of various plant organs (leaves, flowers, fruits) of Myrtus communis collected from Iran during five different phenological stages as well as their total phenolic and total flavonoid content and DPPH radical scavenging activity. Although the study shows a lot of potential, the paper should be thoroughly revised before publication considering it has some drawbacks such as missing or incorrect data. Also, interpretation of obtained data should be improved.

 

Major remarks:

In the Introduction part, the authors state that the objective of this study was to, among other things, examine the antioxidant activity and correlations with phytochemical constituents and to “identify possible constituents responsible for this effect”.  However, the last is not visible from their results since they have, if understood correctly (Table 2 states “different myrtle organs”), examined only the activity of extracts (not of the essential oils) and have studied the phytochemical composition of only the essential oils (and not of the extracts). Other examples of unsupported or incorrect claims: “we found in this study that biosynthesis and accumulation of phenol and flavonoids occur independently in each plant organ” (they were different but were they independent); “The high antioxidant properties in this plant occur due to the existence of the phenolic and flavonoid compounds in the EO” (phenolic compounds and flavonoids were determined in the extracts).

 

The rows 428-435 seem to be redundant. It is not clear how they contribute to the authors’ own findings. Please consider what conclusions could be drawn from your results. The conclusion “…collecting the myrtle at the vegetative and flowering stages, especially the leaves organs, would be of considerable importance.” is a bit vague. It is not clear how and for whom? The authors should elaborate more on that.

 

Some questions considering methodology: Was the extraction also performed in triplicate and at what temperature was it performed? For how long were the filtrates evaporated under the fume hood? The Folin-Ciocalteu method cited (Reference 44) is a personal communication. Please check if all data are (correctly) given or if the original method was somewhat modified (e.g., Folin-Ciocalteu reagent and sodium carbonate concentration, wavelength). Manufacturer names are not given for the used reagents. Correlation analysis methodology description is missing?

 

Please compare the data given in tables (including Table S1) to those in the text (including the abstract) because there are many inconsistencies. For example, ɑ-pinene content in the leaves, limonene in the flowers (rows 25,26), rows 385-388 etc. Why are the data in rows 382-383 not shown?

 

Minor remarks:

In the abstract, name the plant part in which the given values of total phenol and flavonoid content were obtained.

Consider omitting or changing some (unnecessary) sentences to make the text more concise. For example, “The positive correlation between phytochemical compounds means that the amount and production of each component are correlated, that there is a direct relationship between two variables”; or “but there is still not complete information about the influence of the different organs at different phenological stages on the secondary metabolites and the antioxidant properties” (rows 79-80) and “no research has been done on the amounts of phytochemical compounds at different phenological stages and in various organs of the plant” (rows 85-86), which are quite similar.

row 51 - a word is missing: “…and to improve oral”

 

row 75 - consider replacing “purify” with “remove”, “neutralize” or “scavenge”

 

row 105 - kg

 

row 114 – were subjected to hydro-distillation?

 

row 140 – UV-Vis spectrophotometer (PG Instruments)

 

row 256 – Linalyl acetate?

 

Titles of tables: Table 1 and 2 – why is the title a part of the table? Table 3 – the term “in different phenological stages” requires larger font size. Consider a shorter term for the last phenological stage such as “Post-dispersal stage” and/or providing the Table 1 in horizontal position or expanding the first column so that the entry numbers etc. would be in the same row. Compound names are not all given in capital letters (while in the text capital letters should be replaced by small letters, e.g., rows 382-384).

 

For easier following, please add compound numbers (x1, x2 etc.) in parentheses in Table S1.

 

Latin names of species including plants (varieties and subspecies), bacteria and fungi should be given in italics (e.g., see References 3,12,13,15,19,25,49,71) and in the text plant names should be given in full at first mention (author abbreviations included).

 

Reference list should be re-checked considering the use of punctuation (e.g., journal abbreviations), italics (e.g., References 53,55), capital letters (Reference 11), typing errors and/or missing spaces between words (e.g., rows 503-Herpes, 541, 588, 657-American).

 

Throughout the text, the authors frequently use the definite article “the”, especially in combination with “myrtle”, which is not necessary in most of the cases.

Author Response

-Comments and Suggestions for Authors

All issues that need to be corrected is listed as follows:

The manuscript “Evolution of Phytochemical Variation in Myrtle (Myrtus communis L.) Organs During Different Phenological Stages” gives a detailed insight into the essential oil composition (and content) of various plant organs (leaves, flowers, fruits) of Myrtus communis collected from Iran during five different phenological stages as well as their total phenolic and total flavonoid content and DPPH radical scavenging activity. Although the study shows a lot of potential, the paper should be thoroughly revised before publication considering it has some drawbacks such as missing or incorrect data. Also, interpretation of obtained data should be improved.

Thank you for your comments. Based on your valuable comments, we improved the results and discussion sections.

 

Major remarks:

-In the Introduction part, the authors state that the objective of this study was to, among other things, examine the antioxidant activity and correlations with phytochemical constituents and to “identify possible constituents responsible for this effect”.  However, the last is not visible from their results since they have, if understood correctly (Table 2 states “different myrtle organs”), examined only the activity of extracts (not of the essential oils) and have studied the phytochemical composition of only the essential oils (and not of the extracts). Other examples of unsupported or incorrect claims: “we found in this study that biosynthesis and accumulation of phenol and flavonoids occur independently in each plant organ” (they were different but were they independent); “The high antioxidant properties in this plant occur due to the existence of the phenolic and flavonoid compounds in the EO” (phenolic compounds and flavonoids were determined in the extracts).

Thank you for your comment. In the present study, we evaluated the antioxidant properties of the extract, which were revised in the manuscript, since the extract contains more complete compounds than essential oils and has also more effects than essential oils. It has been corrected in the whole of manuscript. Now in sections 2.7 and 3.3 and lines 82-88.

 

-The rows 428-435 seem to be redundant. It is not clear how they contribute to the authors’ own findings. Please consider what conclusions could be drawn from your results. The conclusion “…collecting the myrtle at the vegetative and flowering stages, especially the leaves organs, would be of considerable importance.” is a bit vague. It is not clear how and for whom? The authors should elaborate more on that.

Thank you for your comment. We removed the redundant sentences (rows 428-435).

We improved the conclusions “The results of this study could serve as a guideline for growers to produce desirable metabolites with undeniable economic benefits to both the food and pharmaceutical industries. Collecting leaves of myrtle at the vegetative and flowering stages is imperative because, in general, the amount of essential oil content and phytochemical compounds in these two stages and in the leaf organ is higher.” Now in lines 462-466.

 

-Some questions considering methodology: Was the extraction also performed in triplicate and at what temperature was it performed?

Thank you for your comment. We improved the methodology section. Also, the extraction was done in three replications. Now in line 127.

 

-For how long were the filtrates evaporated under the fume hood?

For 48 hours. Now in line 131.

 

-The Folin-Ciocalteu method cited (Reference 44) is a personal communication. Please check if all data are (correctly) given or if the original method was somewhat modified (e.g., Folin-Ciocalteu reagent and sodium carbonate concentration, wavelength).

Thank you for positive comment. We cited the original reference. Now in lines, 135, 592 and 593.

Singleton, V. L.; Orthofer, R., Lamuela-Raventós, R.M. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol. 1999, 299, 152-178. Academic press.

 

-Manufacturer names are not given for the used reagents.

We added the manufacturer names. Now in lines 136, 137, 140, 146, 147, 152.

 

-Correlation analysis methodology description is missing?

It was added. Now in lines 162-163.

 

-Please compare the data given in tables (including Table S1) to those in the text (including the abstract) because there are many inconsistencies. For example, ɑ-pinene content in the leaves, limonene in the flowers (rows 25,26), rows 385-388 etc. Why are the data in rows 382-383 not shown?

Thank you for your comment. We compared all the data in the text with table and corrected the inconsistencies.

Minor remarks:

-In the abstract, name the plant part in which the given values of total phenol and flavonoid content were obtained.

Thank you for your comment. We corrected the sentences “the greatest amounts of total phenol and flavonoid content were obtained for the extract made of the before flowering stage in leaves”. Now in line 30.

-Consider omitting or changing some (unnecessary) sentences to make the text more concise. For example, “The positive correlation between phytochemical compounds means that the amount and production of each component are correlated, that there is a direct relationship between two variables”; or “but there is still not complete information about the influence of the different organs at different phenological stages on the secondary metabolites and the antioxidant properties” (rows 79-80) and “no research has been done on the amounts of phytochemical compounds at different phenological stages and in various organs of the plant” (rows 85-86), which are quite similar.

Thank you for your comment. We removed the sentences “The positive correlation between phytochemical compounds means that the amount and production of each component are correlated, that there is a direct relationship between two variables”.

We removed the sentences “but there is still not complete information about the influence of the different organs at different phenological stages on the secondary metabolites and the antioxidant properties” (rows 79-80).

We removed the sentences “no research has been done on the amounts of phytochemical compounds at different phenological stages and in various organs of the plant” (rows 85-86)

-row 51 - a word is missing: “…and to improve oral”

Thank you for your comment. We corrected the missing word “and to improve oral wound” Now in line 52.

 

-row 75 - consider replacing “purify” with “remove”, “neutralize” or “scavenge”

Thank you for your comment. We replaced the “purify” with “scavenge” Now in line 75.

 

-row 105 – kg

Thank you for your comment. We replaced “Kg” with “kg”. Now in line 100.

 

-row 114 – were subjected to hydro-distillation?

Thank you for your comment. We corrected the sentences “Every sample of the organs was chopped using a blender at low speed and 100 g of the dried sample of different organs included leaves, fruits and flowers were subjected to hydro-distillation for 3 hours”. Now in lines 109-110.

 

-row 140 – UV-Vis spectrophotometer (PG Instruments)

We corrected. Now in lines 138-139.

 

-row 256 – Linalyl acetate?

Thank you for your comment. NO. Methyl citronellate and geranyl acetate had the highest amount among the studied organs.

 

-Titles of tables: Table 1 and 2 – why is the title a part of the table? Table 3 – the term “in different phenological stages” requires larger font size. Consider a shorter term for the last phenological stage such as “Post-dispersal stage” and/or providing the Table 1 in horizontal position or expanding the first column so that the entry numbers etc. would be in the same row. Compound names are not all given in capital letters (while in the text capital letters should be replaced by small letters, e.g., rows 382-384).

Thank you for your comment. We removed the extra row in the table 1 and 2 and placed the title outside the table.

In table 3, we corrected the font size for “in different phenological stages”. Now in line 417.

We changed Table 1 in horizontal position

We replaced the capital letters with small letters in rows 382-384. Now in lines 390-395.

 

-For easier following, please add compound numbers (x1, x2 etc.) in parentheses in Table S1.

Thank you for your comment. We added add compound numbers in Table S1.

 

-Latin names of species including plants (varieties and subspecies), bacteria and fungi should be given in italics (e.g., see References 3,12,13,15,19,25,49,71) and in the text plant names should be given in full at first mention (author abbreviations included).

Thank you for your comment. We corrected the references 3,12,13,15,19,25,49,71

 

-Reference list should be re-checked considering the use of punctuation (e.g., journal abbreviations), italics (e.g., References 53,55), capital letters (Reference 11), typing errors and/or missing spaces between words (e.g., rows 503-Herpes, 541, 588, 657-American).

Thank you for your comment. Corrected

 

-Throughout the text, the authors frequently use the definite article “the”, especially in combination with “myrtle”, which is not necessary in most of the cases.

Thank you for your comment. We removed definite article “the” in combination with “myrtle”.

Round 2

Reviewer 3 Report

Major remarks:

rows 84-85 – “…no research has yet been presented about the variations in phytochemical compounds and their effect on the levels of antioxidant properties”, please be more precise which variations you mean since there are research that considered different variations in M. communis (e.g., among genotypes, fruit parts, fruit harvesting times) such as those from Yangui et al., 2021 (South Afr J Bot 137, 35-45), Wannes and Marzouk, 2012 (J Food Biochem 37, 585-594), reference 26

rows 464-466 – “Collecting leaves of myrtle at the vegetative and flowering stages is imperative because, in general, the amount of essential oil content and phytochemical compounds in these two stages and in the leaf organ is higher.”, please rephrase since according to presented results, the EO content was highest for leaves in flowering and after full-mature fruit dispersion stage (Figure 2; “According to the results, the highest EO content was achieved in leaves, at the flowering stage of the plant (1.25%). The same result was obtained in the leaves after the full-mature fruit dispersion stage”)

In Table 1, linalyl acetate is marked in bold (not methyl citronellate), but the former was not mentioned in the text even though its content was generally higher in different stages than that of methyl citronellate; consider adding this compound to discussion (e.g., before rows 260-263) and also putting methyl citronellate in bold in the Table; remove “(Fatty esters)” and “(phenylpropene)” from the same table or add missing classes for other compounds

Minor remarks:

row 52 – improve oral wound “healing”

row 66 – unripe “fruits”…ripe “fruits”

Table 1, row 14 and Table S1 – Linalyl acetate (in capital)

rows 517, 601, 658-659 – Culex; typicus; Vaccinium macrocarpon (not American cranberry) should be in italic

Figures 3 and 4 – unripe/ripe “fruit” (not friut)

Author Response

Reviewer 3

-rows 84-85 – “…no research has yet been presented about the variations in phytochemical compounds and their effect on the levels of antioxidant properties”, please be more precise which variations you mean since there are research that considered different variations in M. communis (e.g., among genotypes, fruit parts, fruit harvesting times) such as those from Yangui et al., 2021 (South Afr J Bot 137, 35-45), Wannes and Marzouk, 2012 (J Food Biochem 37, 585-594), reference 26

Thank you for your comments. Based on your valuable comments, we improved the manuscript. Now in lines 85- 87.

rows 464-466 – “Collecting leaves of myrtle at the vegetative and flowering stages is imperative because, in general, the amount of essential oil content and phytochemical compounds in these two stages and in the leaf organ is higher.”, please rephrase since according to presented results, the EO content was highest for leaves in flowering and after full-mature fruit dispersion stage (Figure 2; “According to the results, the highest EO content was achieved in leaves, at the flowering stage of the plant (1.25%). The same result was obtained in the leaves after the full-mature fruit dispersion stage”)

Thank you for your comment. We improved the conclusion section. Now in lines 466 and 467.

 

- In Table 1, linalyl acetate is marked in bold (not methyl citronellate), but the former was not mentioned in the text even though its content was generally higher in different stages than that of methyl citronellate; consider adding this compound to discussion (e.g., before rows 260-263) and also putting methyl citronellate in bold in the Table; remove “(Fatty esters)” and “(phenylpropene)” from the same table or add missing classes for other compounds

Thank you for your comment. We corrected table 1, based on your valuable comments

 

 

Minor remarks:

row 52 – improve oral wound “healing”

We added the healing. Now in line 52.

row 66 – unripe “fruits”…ripe “fruits”

Thank you for your comment. We corrected the fruit. Now in line 66.

Table 1, row 14 and Table S1 – Linalyl acetate (in capital)

Thank you for your comment. Corrected. Please see Table 1 and Table S1.

rows 517, 601, 658-659 – Culex; typicus; Vaccinium macrocarpon (not American cranberry) should be in italic

Thank you for your comment. Corrected. Now in lines 532, 616, 673-674.

Figures 3 and 4 – unripe/ripe “fruit” (not friut)

Thank you for your comment. Corrected, please see figures 3 and 4.

Author Response File: Author Response.docx