Effect of Supercritical Fluid Extraction Process on Chemical Composition of Polianthes tuberosa Flower Extracts

Round  1

Reviewer 1 Report

Experiment was conducted rather straight-forward way, and it is presented clearly.
Results shown could be useful for small-scale industrial fractionation, but not facilitating it as such. Only the effect of pressure and temperature, which are shown as surface response graphs in Figure 2 might be too little to choose conditions. I am not personally able to comment does the presented graphs best illustrate the potential of different end-uses of the extracts by changing extraction conditions, but I hope this was the intention of the authors. In addition, probably CO2 density vs. temperature would show the differences more clearly.Lines 139-144 authors conclusion of the results however is confusing. As the results are given only as area-% of the extract, the portion of individual compound (mass-%) and the end-use potential remains ambiguous.

Author Response

Authors are agree with the comments about that only the effect of pressure and temperature were the only studied factors. But the aim of this research was to understand how chemical composition of extracts was affected by the extraction process, pressure and temperature in supercritical extraction are the factors that affects carbon dioxide solvation. Meanwhile, factors as time, particle size, carbon dioxide flow, etc. are factors that affects recovery efficiency or yield extraction.

The results are shown in % area because the effects of extraction conditions over chemical profile can be described in a good way and with good accuracy, and the effect of changes in chemical profile of the extracts can be observed with % area. 

Author Response File: Author Response.pdf

Reviewer 2 Report

The paper presents the supercritical fluid extraction of Polianthes tuberosa flowers, and the effect of the extraction conditions on the chemical composition of the extracts. Although the subject is of scientific interest, the main drawback is the poor discussion of the results. The authors show the results obtained from 12 experiments but they are not commented in deep.  Moreover I have found several disagreements between the results presented and the comments made by the authors, thus I am unable to recommend this manuscript for publication.

·         Lines 55-56.  “None of these researches studied the process conditions as particle size, humidity content, carbon dioxide flow rate..” . But the authors neither studied these parameters at the present work. They just studied the effect of pressure and temperature.

·         A description of the supercritical fluid extractor should be included in section 2.2

·         How was the recovery evaluated? Weighing the vessel after the extraction? There is a high variability between the experiments conducted in the same conditions (for example between experiments 5 and 11)

·         The extraction was always performed in dynamic mode? The possibility of working in static mode for some minutes was not taken into account?

·         Lines 110-112: “Higher yields obtained in this work could be due to different factors as carefully postharvest handling of tuberose flowers, drying conditions and particle size used for extraction”. In my opinion these results could be due to the higher pressures assayed. The authors should comment the effect of pressure on the recovery.

·         How was the extraction time selected?

·         The results should be commented in deep.

·         I can see from table 2, that authors had the standards for most of the compounds identified in the extracts. In my opinion the quantification should be done using standard calibration curves.  Quantification based on area percentage is not accurate because not all the compounds identified had the same response factor.

·         Lines 139-144: How were the compounds, which extraction is affected by pressure and temperature, determined? . The variation of methyl antranilate  percentage, in the different extracts, is similar to that of farnesol. Moreover, in my opinion ethyl limolenate is also affected by the extraction conditions (0.5 %  at 38 MPa/333.15 K, and 1% at 18 MPa/313.15 K)

Authors should discuss which conditions are the best to obtain extracts with value in the fragrance and cosmetic industry, and which conditions should be used to obtain extracts with antimicrobial activity.

Author Response

Lines 55-56.  “None of these researches studied the process conditions as particle size, humidity content, carbon dioxide flow rate..” . But the authors neither studied these parameters at the present work. They just studied the effect of pressure and temperature.

Authors are agree with the comments about that process conditions as particle size, humidity content, carbon dioxide flow rate… were not evaluated, but the aim of the research was to study, how the main parameters that affects carbon dioxide solvation capability in supercritical fluid process, impacts the chemical composition profile of Polianthes tuberosa extracts. It is known that factors as time, particle size, carbon dioxide flow, etc. are factors that affects recovery efficiency or yield extraction but not solvation of carbon dioxide. Besides, and for a better understanding the lines 55-56 were rewrote.

A description of the supercritical fluid extractor should be included in section 2.2

Attending this suggention, a schematic diagram of the process have been added into section 2.2

How was the recovery evaluated? Weighing the vessel after the extraction? There is a high variability between the experiments conducted in the same conditions (for example between experiments 5 and 11).

Authors are agree about the high variability between experiments 5 and 11. Experiments were carried out carefully and under the same conditions, but variability could be at the sample recovery, because at the time of depressurizing the CO2 an expansion occurs which causes a drop in temperature and therefore a freeze in the sampling nozzle that we cannot control and could affect the sample. Unfortunately we only had limited among of flowers and we were not able to repeat this experiment, but it is also known that this variability affects only yield of extract and do not affects chemical composition because of the process extraction, which was the aim of this work.   

The extraction was always performed in dynamic mode? The possibility of working in static mode for some minutes was not taken into account?

Yes, it was performed in dynamic mode because the objective of this work was to evaluated factors that affect solubility of compounds. The static mode was not evaluated, since this mode will affects efficiency of extraction process, not solubility.

Lines 110-112: “Higher yields obtained in this work could be due to different factors as carefully postharvest handling of tuberose flowers, drying conditions and particle size used for extraction”. In my opinion these results could be due to the higher pressures assayed. The authors should comment the effect of pressure on the recovery.

Authors are agree that high pressure it is also important besides factors as carefully postharvest handling of tuberose flowers, drying conditions and particle size, so discussion of this factor was added to manuscript in lines 110-112.

How was the extraction time selected?

Extraction time was selected by previous studies reported by Gosh et al. and also by Reverchon et al., but as we mentioned, extraction time does not affect solubility.

The results should be commented in deep.

An improvement of discussion was made to manuscript.

I can see from table 2, that authors had the standards for most of the compounds identified in the extracts. In my opinion the quantification should be done using standard calibration curves.  Quantification based on area percentage is not accurate because not all the compounds identified had the same response factor.

Authors are agree with the comments about, but the results are shown in % area because the effects of extraction conditions over chemical profile can be described in a good way and with good accuracy, and the effect of changes in chemical profile of the extracts can be observed with % area.

It should be noted that in order to optimize the recovery of a particular compound from tuberose extracts, then it will be necessary to quantify with the calibration curves.

Lines 139-144: How were the compounds, which extraction is affected by pressure and temperature, determined? . The variation of methyl antranilate  percentage, in the different extracts, is similar to that of farnesol. Moreover, in my opinion ethyl limolenate is also affected by the extraction conditions (0.5 %  at 38 MPa/333.15 K, and 1% at 18 MPa/313.15 K).

The analysis of the variation in the chemical composition of the extracts at different pressures and temperatures was carried out by means of a statistical analysis, using the same experimental design, but evaluating the effect of factors process over the percentage of relative area of each metabolite identified. The analysis could have been done for all the compounds but it was only made to the important compounds and that give characteristic scent of tuberose: methyl isoeugenol, heptacosene, bezyl benzoate and methyl anthranilate as reported by Sheela et al. and by Reverchon et al.

Authors should discuss which conditions are the best to obtain extracts with value in the fragrance and cosmetic industry, and which conditions should be used to obtain extracts with antimicrobial activity.

The manuscript was modified, adding the pressure conditions to which the compounds with value in the different industries are obtained

Author Response File: Author Response.pdf

Reviewer 3 Report

This document reports the effect of temperature and pressure in the chemical composition of extracts from Polianthes Tuberosa flowers.   

This study is presented in a comprehensive form, characterization of extracts has been correctly performed and may be of interest for the scientific community interested in extraction of natural products by supercritical CO₂. In my opinion this study can be published in Processes after some comments are addressed:

1)     Some English misspells should be corrected, as example, in page 1, line 31, given should be giving. Other errors can be found in the text.

2)     An overall good reproducibility can be observed among experiments performed at the same conditions, however, some of them present some differences in yield (Table 1) as in experiments 5 and 11 that are reflected as well in the analysis of the extracts (Table 2, for n-Hexatriacontano for example, but as well for other compounds): authors should explain these differences in yield (Table 1) and in compositions. Other case are experiments 1 and 7.

Author Response

This document reports the effect of temperature and pressure in the chemical composition of extracts from Polianthes Tuberosa flowers.   This study is presented in a comprehensive form, characterization of extracts has been correctly performed and may be of interest for the scientific community interested in extraction of natural products by supercritical CO2. In my opinion this study can be published in Processes after some comments are addressed:

1)          Some English misspells should be corrected, as example, in page 1, line 31, given should be giving. Other errors can be found in the text.

English misspells were corrected and a new english grammar review was done.

2)          An overall good reproducibility can be observed among experiments performed at the same conditions, however, some of them present some differences in yield (Table 1) as in experiments 5 and 11 that are reflected as well in the analysis of the extracts (Table 2, for n-Hexatriacontano for example, but as well for other compounds): authors should explain these differences in yield (Table 1) and in compositions. Other case are experiments 1 and 7).

Authors are agree about the differences in yield between experiments. Experiments were carried out carefully and under the same conditions, but variability could be at the sample recovery moment, because at the time of depressurizing the CO₂ an expansion occurs which causes a drop in temperature and therefore a freeze in the sampling nozzle that we cannot control and could affect the sample. Unfortunately we only had limited among of flowers and we were not able to repeat this experiment, but it is also known that this variability affects only yield of extract and do not affects chemical composition because of the process extraction, which was the aim of this work.  

Author Response File: Author Response.pdf

Reviewer 4 Report

The manuscript by Fragoso-Jimenez investigates the effect of SFE parameters on a natural products extraction. This in particular is not novel. The novelty belongs in both the specific plant being extracted and in the comparison of SFE to other extraction methods. Thus, the paper, while it merits publication, is merely average. To be a more impactful paper, the focus should shift to the comparison of the various extraction methods employed. Other than that, my only concerns of significance with the paper are (a) the temperature range used was rather narrow, thus the effect of temperature is negligible in most cases and (b) the are some minor issues with proper use of the English language, so editorial review is needed.

Author Response

The manuscript by Fragoso-Jimenez investigates the effect of SFE parameters on a natural products extraction. This in particular is not novel. The novelty belongs in both the specific plant being extracted and in the comparison of SFE to other extraction methods. Thus, the paper, while it merits publication, is merely average. To be a more impactful paper, the focus should shift to the comparison of the various extraction methods employed. Other than that, my only concerns of significance with the paper are:

(a) The temperature range used was rather narrow, thus the effect of temperature is negligible in most cases.

The temperature ranges were proposed based on previous studies and where they recommend not working at higher temperatures to avoid losses of the volatile components, which would affect the chemical profile of the extract. These studies are cited in section 2.2 of the manuscript

(b) The are some minor issues with proper use of the English language, so editorial review is needed.

English misspells were corrected and a new english grammar review was done.

Author Response File: Author Response.pdf

Reviewer 5 Report

Please, see the attached file 

Comments for author File: Comments.docx

Author Response

The scientific level is adequate, and the results obtained in this study will be useful for the extraction of volatile compounds from this and similar plants. However, there is a list of issues that requires revision prior publication. In this sense, I recommend the publication of the manuscript after minor revision. Please, see the following specific comments:

*Please, revise the punctuation marks of the entire manuscript: there are a lot of commas that should be dots.

English misspells were corrected and a new english grammar review was done.

*Lines 19 and 21: Please, change “were obtained” to “were identified”.

Changed and besides english misspells were corrected and a new english grammar review was done

*Line 44: Please, change “have been” to “has been”.

Changed and besides english misspells were corrected and a new english grammar review was done

*The SFE abbreviation was introduced at the beginning of the manuscript but then it is not used. Please, change all “supercritical fluid extraction” by “SFE”.

Correction done.

*Lines 50-51: Please, explain the benefits of SFE compared to enfleurage.

Benefits are now mentioned in lines 36-39 in the modified manuscript.

Please, compare SFE with other green extraction methods such as ultrasound-assisted extraction or ionic liquid-based extraction.

Discussion of these technics were done in the modified manuscript

*Please, explain in the results section why the rest of parameters that influence the extraction (i.e. the amount of plant or the extraction time, drying conditions of the plant, particle size…) were not optimized.

Explanation was added in the modified manuscript as:

Higher yields obtained in this work could be due to different factors as higher pressure process which affects the capability of solvation of non-polar compounds, carefully postharvest handling of tuberose flowers, drying conditions and particle size used for extraction, because these factors were reported as important factors for jasmine and lavender extraction [19, 22]. These factors were not evaluated in this work because the aim of the research was to study, how the main parameters that affects carbon dioxide solvation capability in supercritical fluid process impacts the chemical composition profile of Polianthes tuberosa extracts. It is known that factors as time, particle size, carbon dioxide flow, etc. are factors that affects recovery efficiency or yield extraction but not solvation of carbon dioxide.

*Section 2.3: Please, include the following conditions during the GC-MS analysis: transfer line temperature, type of mass analyzer, source temperature, and quadrupole temperature (if this mass analyzer was employed).

The chromatographic system used for analysis of extracts was coupled to a MSD 5972A detector, this model does not allow to program independently the temperatures of quadrupole, ionization source and transfer line as mass detectors in current models.

*Section 3.1: Please, include the yield reported in reference [2], even if it is not comparable with the reported value in the manuscript. I also suggest comparing the yield of the first extraction of ref [2] with the obtained yield, if the value was reported.

Yields are already reported in section 3.1

*Lines 110-112: If the drying conditions and particle size are important factors that influence the yield, I do not understand why they were not optimized…

Authors are agree with the comments about that only the effect of pressure and temperature were the only studied factors. But the aim of this research was to understand how chemical composition of extracts was affected by the extraction process, pressure and temperature in supercritical extraction are the factors that affects carbon dioxide solvation. Meanwhile, factor as particle size will affects recovery efficiency or yield extraction. In the other hand, dehydration process should be interesting to evaluate because it might be a loss of volatile compounds during process.

*Section 3.1: Is there interaction between the two studied SFE factors?

Yes, there is an interaction between the two studied SFE factors, however this interaction is not statistically significate that’s why it is not mentioned.

*Lines 145-148: How many analytes were identified in ref. [4,6,11]? Please, include the information in the text.

Discussion of these compounds were done in manuscript.

Author Response File: Author Response.pdf

Round  2

Reviewer 2 Report

In my opinion results have not been commented in deep; in fact taking into account the data shown there is not any conditions where the aroma compounds of the tuberose flowers were obtained in a higher proportion with respect to the other compounds such as fatty acids or terpenes. Moreover the chemical composition of the different extracts, are very similar and any data about standard deviation are given.

I am sorry, but in my opinion there is not enough new findings that justify its publication.