Effect of Harvesting Time Variations on Essential Oil Yield and Composition of Sage (Salvia officinalis)

Round 1

Reviewer 1 Report

All issues that need to be corrected is listed as follows: 1. Lines 19-20, “Harvesting between … (0.599%)” this statement is a contradiction. 2. Line 23, “trans” should be in italic. Please check the whole manuscript and correct them. 3. Table 1, please add a full name of “EC”. 4. Figure 1, temperature in y axis is a typo. 5. Figure 1, please add a full name of “PPFD”. 6. Line 158, “…retention time (RI) and indices (KI)…”. I suggest that it should be modified to “…retention indices (RI)…”. 7. Line 162, “9 – 25oC” this in not correct. 8. Line 166, dimension of HP-5 column is not correct. 9. Line 173, film thickness of DB-5 column is not correct. 10. Line 199, “The highest yield of EO was observed from 10 am to 6 pm”, I suggest it should be modified to “The higher yield of EO was observed from 10 am to 6 pm”. 11. Figure 3, the authors did not mention that how to measure the “EO content (%)”. Please add this information in the methods section. 12. Line 245, cis-thujone (36.93-46.18%), this data did not match with those in Table 2. Please check all data. 13. Table 2, what does the title “T” in the first column means? Please add the information in a proper place. 14. Table 2, “ρ-Cymene” it should be “p-Cymene”. 15. Table 2, “Β-Gurjunene” it should be “β-Gurjunene”. 16. Table 2, There are two “α-Humulene” in a Table. This is not correct. Please correct the compound name. 17. Line 271, “caryophyllen”, a typo. 18. Line 288, the aim of the “correlation analysis” is not clear. Please add more information. 19. Line 309, data in Table 3 were not described in this section? Please check. 20. Figure 4, what the dash line means? And, how the authors determine the value of the dash line? Please add the information. 21. Line 354, “Oliveria decumbens” should be in italic. 22. Table 4, “β--Pinene”, a typo. 23. Figure 5, if possible, the authors can add the loading plot in this figure. This would help readers understand the PCA results. 24. Line 411, “Cis-Thujon”, typos. 25. In conclusion section, I would suggest the authors focus on the optimal harvesting time for the sage EO. In most part of this paragraph, the authors shed light on the effect of temperature and light intensity on the EO. However, it should be better to move to the discussion section. Because the authors did not intensively evaluate the effect of these two parameters.

Author Response

Reviewer 1

We are grateful to this reviewer for a very thorough review and for posing many important questions. Modifications are highlighted in red in the text.

 Comments and Suggestions for Authors

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

1. Lines 19-20, “Harvesting between … (0.599%)” this statement is a contradiction.

Thank you for your comment. Corrected. Now in lines 19-20 it reads “Harvesting between 4:00 and 6:00 pm revealed the highest EO percentage (1.14%), whereas between 04:00 and 06:00 am indicated the minimum EO percentage (0.599 %).”

2. Line 23, “trans” should be in italic. Please check the whole manuscript and correct them.

Thank you for your comment. It has been corrected in the whole manuscript.

3. Table 1, please add a full name of “EC”.

Thank you for your comment. We spelled out the name in Table 1.

4. Figure 1, temperature in y axis is a typo.

Thank you for your comment. We corrected the typo in Figure 1.

5. Figure 1, please add a full name of “PPFD”.

Thank you for your comment. We spelled out the name in Figure 2.

6. Line 158, “…retention time (RI) and indices (KI)…”. I suggest that it should be modified to “…retention indices (RI)…”.

Thank you for your comment. We modified to retention indices and Kovats indeces (now in line 162).

7. Line 162, “9 – 25oC” this in not correct.

Corrected. Now in line 166. “9-25 C”.

8. Line 166, dimension of HP-5 column is not correct.

Thank you for your comment. Corrected. Now in line 171.

9. Line 173, film thickness of DB-5 column is not correct.

Thank you for your comment. Corrected. Now in line 177.

10. Line 199, “The highest yield of EO was observed from 10 am to 6 pm”, I suggest it should be modified to “The higher yield of EO was observed from 10 am to 6 pm”.

Thank you for your comment. Corrected. Now in line 203.

11. Figure 3, the authors did not mention that how to measure the “EO content (%)”. Please add this information in the methods section.

Thank you for your comment. We changed in: The obtained EO was then dried by anhydrous sodium sulphate (British Pharmacopoeia, 1988) and EO content (w/w %) was determined on an EO weight to dry weight basis (Method section). Now in lines 145-147.

12. Line 245, cis-thujone (36.93-46.18%), this data did not match with those in Table 2. Please check all data.

Thank you for your comment. We changed in the text as (34.38-46.18%). Now in line 254.

13. Table 2, what does the title “T” in the first column means? Please add the information in a proper place.

Thank you for your comment. It was a compounds numbering. It has been changed to "No".

14. Table 2, “ρ-Cymene” it should be “p-Cymene”.

We changed ρ-Cymene to p-Cymene in the table 2.

15. Table 2, “Β-Gurjunene” it should be “β-Gurjunene”.

We changed Β-Gurjunene to β-Gurjunene in the table 2

16. Table 2, There are two “α-Humulene” in a Table. This is not correct. Please correct the compound name.

Corrected. There were two lines with the same compound.

17. Line 271, “caryophyllen”, a typo.

Corrected. Now in line 280.

18. Line 288, the aim of the “correlation analysis” is not clear. Please add more information.

We improved the correlation analysis section. Now in lines 312-314 and 334-339.

19. Line 309, data in Table 3 were not described in this section? Please check.

Thank you for your comment. Table 3 described in lines 354-358.  This table shows the comparison between the mean values obtained from the cluster analysis, which confirms the cluster grouping and demonstrates a significant difference between the groups in essential oil composition.  Therefore, this table (Table S2) presented in supplementary file.

20. Figure 4, what the dash line means? And, how the authors determine the value of the dash line? Please add the information.

The dashed lines on the dendrogram resulting from the cluster analysis indicate a "cut off line", which indicates that at this point, the best grouping is obtained based on variance criteria, and the obtained groups have significant differences. In cluster analysis grouping, researchers' decisions always have a high priority. The cutting point is determined mathematically. For this experiment, we used XLSTAT software, which has the capability of automatically determining the cutting line. It has been added in the Figure 4 caption.

21. Line 354, “Oliveria decumbens” should be in italic.

Corrected. Now in line 389.

22. Table 4, “β--Pinene”, a typo.

It has been corrected in table 3.

23. Figure 5, if possible, the authors can add the loading plot in this figure. This would help readers understand the PCA results.

Thank you for your comment. We added the loading plot and screen plot in Fig 5.

24. Line 411, “Cis-Thujon”, typos.

Corrected. Now in line 434.

25. In conclusion section, I would suggest the authors focus on the optimal harvesting time for the sage EO. In most part of this paragraph, the authors shed light on the effect of temperature and light intensity on the EO. However, it should be better to move to the discussion section. Because the authors did not intensively evaluate the effect of these two parameters.

Thank you for your valuable suggestion. We improved the conclusion section. Now in lines 423-442.

Reviewer 2 Report

Title: is suitable

Abstract:

-- P1/L18, the authors claimed that they made isolated of essential oils by hydro-distillation, they should say that they made an extract of EO by hydro-distillation.

-- P1/L19-20 the sentence: “The harvest between 4:00 and 6:00 p.m. m. presented the highest percentage of OE (1.14%), while between 04:00 and 06:00 p.m. m. presented the minimum percentage of EO (0.599%)”, it is confusing, it seems the same hours.

Introduction:

-- P2/L 77-80. The phrase: “In order to better predict the best harvest time for EO from medicinal plants, it is important to know how these phytochemical compounds change over time. Therefore, the main objective of the current study was to investigate the variety of changes in salvia EOs during day and night to determine the optimal harvest time to achieve maximum EO yield.” It should go after, mix in with the last paragraph of the introduction.

Materials and methods:

--P3/L113-127 In the field study, it is not mentioned if there was fertilization, or control of pests and diseases and how they were.

-- P3/L130 There is confusion in the different collection times, where it says 8:00-8:00 pm, it should say 6:00-8:00.

-- P3/L136 It is recommended to put the fresh samples for hydrodistillation, instead of drying them for 10 days, since these can be contaminated or lose very volatile compounds.

-- P3/L142-143, the sentence: “The EO obtained was then dried with sodium sulfate (British Pharmaco-142 poeia, 1988) to calculate the percentage of EO” is wrong (sodium-sulfur is not used to calculate the EO% ).

-- P3/L142, sodium sulfate, is anhydrous?

-- P3/L144, use “coupled” instead of “attached”.

-- P4/L158 Kovats Index (KI).

-- P5/L185, should say analyzes “were” performed.

Results and Discussion:

-P5/L199-200, the authors mention high yields and report content in percentage, the yield should refer to the extraction (mL of extract/g of sample), please correct this.

-P5/L 207-208, the sentence: “The results showed that the percentage of EO increased from 6 am to 4 pm and then showed a slight decrease. Although our results showed that the EO levels increased at 00:00 to 02:00 am”, it is somewhat confusing, it can be said: The results showed that the percentage of EO increased from 6 am to 4 pm and then showed a slight decrease, but returning to increased slightly at 00:00 to 02:00 am.

-P6/L243, the authors mention that they identified 32 different compounds, however, table 2 shows 33 compounds.

-P6/L 245, the authors mention that the best EO compound was cis-thujone with 36.93 to 46.18%, however, in table 2 values ​​from 34.93 to 46.18% are shown.

Conclusions:

-They should be more specific.

References:

-They must be cited according to the author guide: Abbreviated Journal Name

Author Response

Reviewer 2

We are grateful to this reviewer for a very thorough review and for posing many important questions.  Modifications are highlighted in red in the text.

 Comments and Suggestions for Authors

Title: is suitable

Abstract:

-- P1/L18, the authors claimed that they made isolated of essential oils by hydro-distillation, they should say that they made an extract of EO by hydro-distillation.

Thank you for your comment. Corrected. Now in line 18.

-- P1/L19-20 the sentence: “The harvest between 4:00 and 6:00 p.m. m. presented the highest percentage of OE (1.14%), while between 04:00 and 06:00 p.m. m. presented the minimum percentage of EO (0.599%)”, it is confusing, it seems the same hours.

Thank you for your comment. Now in lines 19-20.

Introduction:

-- P2/L 77-80. The phrase: “In order to better predict the best harvest time for EO from medicinal plants, it is important to know how these phytochemical compounds change over time. Therefore, the main objective of the current study was to investigate the variety of changes in salvia EOs during day and night to determine the optimal harvest time to achieve maximum EO yield.” It should go after, mix in with the last paragraph of the introduction.

Thank you for your comment. Corrected. Now in lines 107-112. it reads “Hence, to better predict the best time to harvest sage plants according to the EO content and composition, several statistical analyses such as cluster and principal component analysis were carried out in order to identify the changes which occur during the day and night in the EO content and composition of sage plants.  Thus, the main objective of this study was to determine the optimal harvest time for sage EO production by observing various changes that occur during the day and night”.

Materials and methods:

--P3/L113-127 In the field study, it is not mentioned if there was fertilization, or control of pests and diseases and how they were.

Thank you for your comment. Weeding was done by hand. There were no pests or diseases observed during the plant growing. This information has been added. Now in lines 128-129.

-- P3/L130 There is confusion in the different collection times, where it says 8:00-8:00 pm, it should say 6:00-8:00.

Thank you for your comment. Corrected. Now in lines 132.

-- P3/L136 It is recommended to put the fresh samples for hydrodistillation, instead of drying them for 10 days, since these can be contaminated or lose very volatile compounds.

Thank you for your recommendation. Sage plants are dried by farmers after harvest, so farmers want to use dried plants for industrial production. This is commercial standard practice. In fact, extensive literature and commercial processors suggest to extract the EO from dried material. This is because the fresh material is not reliable in terms of constant water content and wilt degree after harvest. We are aware that the drying process can favor volatilization of some compounds, but this is the case if the drying process occur at high temperature in short time. Lastly, the purpose of this study was to determine the percentage of essential oil over the dry matter, a parameter largely used. As a result, dry matter was used to extract essential oils. We hope that these explanations are acceptable for the reviewers, as we do not have the chance to go back and change completely the study done.

-- P3/L142-143, the sentence: “The EO obtained was then dried with sodium sulfate (British Pharmaco-142 poeia, 1988) to calculate the percentage of EO” is wrong (sodium-sulfur is not used to calculate the EO %).

Thank you for your comment. Corrected. Now in line 145.

-- P3/L142, sodium sulfate, is anhydrous?

Yes. In the laboratory, anhydrous sodium sulfate is widely used as an inert drying agent, for removing traces of water from organic solutions.

-- P3/L144, use “coupled” instead of “attached”.

Corrected. Now in line 148.

-- P4/L158 Kovats Index (KI).

Corrected. Now in line 162.

-- P5/L185, should say analyzes “were” performed.

Corrected. Now in line 189.

Results and Discussion:

-P5/L199-200, the authors mention high yields and report content in percentage, the yield should refer to the extraction (mL of extract/g of sample), please correct this.

Thank you for your valuable comment. A percentage of essential oils was used to calculate the results, the weight of the essential oil obtained from the amount of dry matter used for essential oil content calculation.  Our aim was to calculate the percentage of essential oil over the dry matter, and therefore the weight of the essential oil was important for our study. We hope that these explanations are acceptable for the reviewers, as we cannot change the unit of essential oils, because the essential oils has been used in the analysis of compounds.

 

-P5/L 207-208, the sentence: “The results showed that the percentage of EO increased from 6 am to 4 pm and then showed a slight decrease. Although our results showed that the EO levels increased at 00:00 to 02:00 am”, it is somewhat confusing, it can be said: The results showed that the percentage of EO increased from 6 am to 4 pm and then showed a slight decrease, but returning to increased slightly at 00:00 to 02:00 am.

Thank you for your comment. Corrected. Now in lines 216-217.

-P6/L243, the authors mention that they identified 32 different compounds, however, table 2 shows 33 compounds.

There were two lines with the same name “α-Humulene” in a Tables, that we corrected it.

-P6/L 245, the authors mention that the best EO compound was cis-thujone with 36.93 to 46.18%, however, in table 2 values ​​from 34.93 to 46.18% are shown.

Thank you for your comment. We mistakenly reported the wrong number. Now in line 254.

Conclusions:

-They should be more specific.

Thank you for your comment. We improved the conclusion section.

References:

-They must be cited according to the author guide: Abbreviated Journal Name.

References rearranged according to Journal format.

 

Reviewer 3 Report

The current work presents insights into sage essential oils variation and composition after harvesting at different times (day/night). The study is interesting and may contribute to improve sage harvest and EO yield.  However, authors should clarify the methodology (see points described below) and highlight the findings and the novelty of their investigation for the area.

 

Material and methods section

Page 3, line 130 - Harvesting time appears misspelled (8:00-8:00 pm). How many plants were collected to make up each replication?

Page 3 – The same harvesting collection times were performed at three diferent days/weeks or an only time? Authors should clarify this.

Page 3, lines 135-137 – Why material had to be dried for 10 days, instead considering its distillation immediately after harvesting. It is well known that the drying process causes substancial EO losses.

 

Page 3, line 139 – The date in “by Binello et al.,” is not indicated.

Page 4, line 157 – Authors should mention the volume injected in Gas-chromatograph coupled with a Mass Spectrometer equipment.

 

Results and discussion

Authors should improve discussion emphasizing the findings and novelty of the work, not only mention that the results are similar to other studies.

 

It would be interesting to evidenciate negative and/or positive correlations between major OE compounds.

I missed authors recomendations, based on their results, about the one, two or even three best period for sage harvesting, considering OE quality and quantity.

Author Response

Reviewer 3

We are grateful to this reviewer for a very thorough review and for posing many important questions. Modifications are highlighted in red in the text.

Comments and Suggestions for Authors

The current work presents insights into sage essential oils variation and composition after harvesting at different times (day/night). The study is interesting and may contribute to improve sage harvest and EO yield.  However, authors should clarify the methodology (see points described below) and highlight the findings and the novelty of their investigation for the area.

Thank you very much for your positive comments.

 Material and methods section

Page 3, line 130 - Harvesting time appears misspelled (8:00-8:00 pm). How many plants were collected to make up each replication?

We corrected and added the number of plants per replication. Now in line 132.

Page 3 – The same harvesting collection times were performed at three different days/weeks or an only time? Authors should clarify this.

Thank you for your comment.  "At three different days" we replied to this request in the material and method section. Now in line 131.

Page 3, lines 135-137 – Why material had to be dried for 10 days, instead considering its distillation immediately after harvesting. It is well known that the drying process causes substancial EO losses.

Thank you for your recommendation. Sage plants are dried by farmers after harvest, so farmers want to use dried plants for industrial production. This is commercial standard practice. In fact, extensive literature and commercial processors suggest to extract the EO from dried material. This is because the fresh material is not reliable in terms of constant water content and wilt degree after harvest. We are aware that the drying process can favor volatilization of some compounds, but this is the case if the drying process occur at high temperature in short time. Lastly, the purpose of this study was to determine the percentage of essential oil over the dry matter, a parameter largely used. As a result, dry matter was used to extract essential oils. We hope that these explanations are acceptable for the reviewers, as we do not have the chance to go back and change completely the study done.

Page 3, line 139 – The date in “by Binello et al.,” is not indicated.

Corrected. Now in line 142.

 

Page 4, line 157 – Authors should mention the volume injected in Gas-chromatograph coupled with a Mass Spectrometer equipment.

Thank you for your comment. It has been added in the material and method section 2.3.  Now in line 160.

Results and discussion

Authors should improve discussion emphasizing the findings and novelty of the work, not only mention that the results are similar to other studies.

 Thank you for your comment, we improved the discussion section. Now in lines 209-213, 282-287, 301-309, 334-339,

It would be interesting to evidenciate negative and/or positive correlations between major OE compounds.

Thank you for your comment. A positive and negative correlation has been identified between all compounds, which is provided in the supplementary file.

I missed authors recomendations, based on their results, about the one, two or even three best period for sage harvesting, considering OE quality and quantity.

 Thank you for your comment, we improved the result and conclusion section. Now in lines 423-429, and 434-442.

 

Reviewer 4 Report

line 128. Is the duration of 140 days from planting (10.05.2018) until flowering correct? Seems too long ...

 

Writing errors:

line 20: expression ”whereas between 04:00 and 06:00 pm”  seems to be wrong… a little earlier something else is stated…

1. 177: ”40-340amu.”
2. 265: ” (Figures 1 2).”
3. 354: ”Oliveria decumbens” with italics.
4. 392: ”(Salvia officinalis.”

Author Response

Reviewer 4

We are grateful to this reviewer for a very thorough review and for posing many important questions. Modifications are highlighted in red in the text.

 Comments and Suggestions for Authors

line 128. Is the duration of 140 days from planting (10.05.2018) until flowering correct? Seems too long ...

 Yes, it is correct because the flowering period of sage is usually long, especially in cold regions.

Writing errors:

line 20: expression ”whereas between 04:00 and 06:00 pm”  seems to be wrong… a little earlier something else is stated…

Corrected. Now in lines 19-20.

1. 177: ”40-340amu.”

Now in line 181.

2. 265: ” (Figures 1 2).”

Corrected. Now in line 273.

3. 354: ”Oliveria decumbens” with italics.

Corrected. Now in line 389.

4. 392: ”(Salvia officinalis.”

Corrected. Now in line 424.