Review Reports - Volatiles from Selected Apiaceae Species Cultivated in Poland—Antimicrobial Activities

Round 1

Reviewer 1 Report

General remarks:

The article is interesting, but I have some questions to the Authors which - in my opinion - should be answered within the manuscript:

What's the inclusion criteria for the investigated species? The plants are from different geographical regions. What do all the substances have in common (apart from the family of course, but Apiaceae is a big and diverse one).
Not all the plants have records of a folk-medicine use. It would be good to know why the Authors from some plants picked up herb, from others fruit and from some both.
Did the plant part (e.g. herb, fruit) have any correlation with the results (phytochemical or microbiological)?

Detailed remarks:

Introduction:

end of the second passage: the sentence is too long and too complicated

Author Response

According to Reviewer: 1

What's the inclusion criteria for the investigated species? The plants are from different geographical regions. What do all the substances have in common (apart from the family of course, but Apiaceae is a big and diverse one).

Plants from Apiaceae family are abundant source of different phytochemicals/secondary metabolites, but mostly coumarins and essential oils. According to the presence of these natural compounds, species have interesting properties and are widely used in medicine, industry and as condiments. Both Departments of Pharmacognosy, Medical University of Lublin, Poland as well as Lab of Pharmacognosy and Chemistry of Natural products, Athens, Greece, have long-term experience concerning research of Apiaceae representatives (extraction, isolation, qualitative and quantitative analysis as well as evaluation of numerous biological activity e.g. antibacterial or influencing central nervous systems).

Numerous projects involving multidirectional research are carried out especially at the Medical University of Lublin, covering numerous genera of plants from the Apiaceae family.

Several manuscripts were published in the area of analysis of essential oils and their antibacterial properties [Skalicka-Woźniak et al, 2011; Skalicka-Woźniak et al., 2010; Skalicka-Woźniak et al. 2009, Sieniawska et al, 2016; Sieniawska et al., 2013].

Studies are often of a screening nature and include a dozen or more species in their scope. During one of such projects, the collected plant material, this was used mainly for the isolation of coumarin compounds, due to its quantity also allowed the distillation of an appropriate amount of essential oils. The main reason for the selection of species for this study was the fact that they had not been previously studied in terms of their essential oil composition and its antibacterial properties. Some species like Ferula asa-foetida or Orlaya grandiflora were used in folk medicine [Mustafa et al., 2012}, some were studied very well concerning their biological activity (anti-inflammatory or cytotoxic/anticancer) e.g. Torilis japonica [Merez-Sadowska et al. 2020; Song et al., 2018; Kim et al., 2017]. Several species were studied in field of phytochemistry or activity for the first time in frame of collaboration between the Medical University of Lublin and the National & Kapodistrian University of Athens (e.g. Seseli devenyense or Peucedanum luxurians).

References used

Skalicka-Woźniak, K., Łoś, R., Ulz, Z., Głowniak, K., & Malm, A. (2011). Essential oil from fruits of angelica of ficinalis and its antibacterial activity. Annales Universitatis Mariae Curie-Sklodowska, Sectio DDD: Pharmacia, 24(1), 25-31.
Skalicka-Wozniak, K., Los, R., Glowniak, K., & Malm, A. (2010). Comparison of hydrodistillation and headspace solid-phase microextraction techniques for antibacterial volatile compounds from the fruits of seseli libanotis. Natural Product Communications, 5(9), 1427-1430. doi:10.1177/1934578x1000500916
Skalicka-Wozniak, K., Los, R., Glowniak, K., & Malm, A. (2009). Volatile compounds in fruits of peucedanum cervaria (LAP.) L. Chemistry and Biodiversity, 6(7), 1087-1092. doi:10.1002/cbdv.200800236
Sieniawska, E., Świątek, Ł., Rajtar, B., Kozioł, E., Polz-Dacewicz, M., & Skalicka-Woźniak, K. (2016). Carrot seed essential oil—Source of carotol and cytotoxicity study. Industrial Crops and Products, 92, 109-115. doi:10.1016/j.indcrop.2016.08.001
Sieniawska, E., Los, R., Baj, T., Malm, A., & Glowniak, K. (2013). Antimicrobial efficacy of mutellina purpurea essential oil and α-pinene against staphylococcus epidermidis grown in planktonic and biofilm cultures. Industrial Crops and Products, 51, 152-157. doi:10.1016/j.indcrop.2013.09.001
Mustafa, B., Hajdari, A., Krasniqi, F., Hoxha, E., Ademi, H., Quave, C. L., & Pieroni, A. (2012). Medical ethnobotany of the albanian alps in kosovo. Journal of Ethnobiology and Ethnomedicine, 8 doi:10.1186/1746-4269-8-6
Merecz-Sadowska, A., Sitarek, P., Śliwiński, T., & Zajdel, R. (2020). Anti-inflammatory activity of extracts and pure compounds derived from plants via modulation of signaling pathways, especially pi3k/akt in macrophages. International Journal of Molecular Sciences, 21(24), 1-32. doi:10.3390/ijms21249605
Song, D. H., Jo, Y. H., Ahn, J. H., Kim, S. B., Yun, C., Kim, Y., Lee, M. K. (2018). Sesquiterpenes from fruits of torilis japonica with inhibitory activity on melanin synthesis in B16 cells. Journal of Natural Medicines, 72(1), 155-160. doi:10.1007/s11418-017-1123-4
Kim, G. T., Kim, S., & Kim, Y. M. (2017). Torilis japonica extract fraction compound, EGFR-targeted inhibition of cancer abnormal metastasis in A549 lung cancer cells. Oncology Reports, 38(2), 1206-1212. doi:10.3892/or.2017.5771

Not all the plants have records of a folk-medicine use. It would be good to know why the Authors from some plants picked up herb, from others fruit and from some both.

Mostly the aerial parts have been used in folk medicine, included seeds. In the case of our study the herbal parts most widely used have been included. If the quality of the plant material allowed (in case of most species), the essential oil was extracted from the aerial parts during seed formation (herba cum fructi).

For this reason, only ripe fruits were harvested for two species (Torillis japonica (Houtt.) DC., Orlaya grandiflora (L.) Hoffm). In case of Peucedanum luxurians Tamamsch. authors obtained only aerial part (without fruits).

Did the plant part (e.g. herb, fruit) have any correlation with the results (phytochemical or microbiological)?

According to the existing literature the plant part plays a crucial role on the phytochemical results and furthermore on towards bioactivities. In our case the essential oil was extracted from the aerial parts during seed formation (herba cum fructi). Furthermore, in the Conclusion part it has been also stated that “….differences in their chemical profile (of the ess oils’ composition) probably due to different cultivation, climate and geographic conditions.” This explanation is supported by 3 more references on other species of Apiaceae family.

Detailed remarks:Introduction: end of the second passage: the sentence is too long and too complicated

The sentence has been corrected as follows: “The incidence of microbial infections has increased dramatically together with emergence of antimicrobial resistant strains [8], thus discovering alternative potentially effective treatments for such infectious diseases is a challenge. Furthermore phytoconstituents are widely considered as promising agents for antimicrobial therapy [8]”.

Reviewer 2 Report

Present manuscript deals with hydrodistillation of volatile oils, chemical characterization, and antimicrobial activity of obtained volatile oils. As the authors state in the paper, the paper is a report of an ongoing and partially finished project. Although this manuscript has numerous merits, it still has to be improved, major revision is needed.

First of all, the several parts of the manuscript need some rearrangement and improvement.

Introduction
You give a full list of investigated plant in text form and presented in Table 1. It is more than enough to mention the number of the species. The part on the page #2 “The plant material … Tamamsch.” should be removed and the Table 1. should be moved to Materials and methods part.
Results

Could you confirm once again that 16.45% of Glehnia littoralis volatile oil is palmitic acid?

‘Results’ section is reserved strictly for results. You should put in context your result in discussion part. Please, move parts of the paragraphs to discussion section where you are comparing your results with literature data or you are writing about the plant or medical application in folklore etc.

Discussion

In my opinion you have to improve this section significantly. All parts from previous section should be arranged in this part.

Second, I have some comments and questions regarding the content and results:

Why did you choose these specific plants? What was the original story? It would be nice to read in the Introduction section about your hypothesis even from this perspective.
Materials and Methods

Could you provide the exact geographical location where Torilis japonica was collected?

Could you clarify MIC determination? Which recommendation was followed: EUCAST or CLSI?

In the Introduction section you wrote about increasing need for new antibiotics-like compounds. However, there is nothing discussed about your compounds. Give some evaluation of your compounds and compare their potency with clinical applied antibiotics.
In the Discussion section you are explaining the difference between chemical profile of the same species collected in different harvesting period. Could you support your explanation by similar observations on relative taxa reported in the literature?

Third, the manuscript has numerous typos and language inconsistencies:

page #1, paragraph 3: “thus discovering… diseases is challenging” and “agents to be served for antimicrobial therapy” rephrase.
The previous work done by authors should be written in past tense (e.g. “Seseli devenyense … has been studied exhaustively previously [was studied], from [by] our scientific team…”).
Section 3.2., paragraph 1: Please reconsider following expressions: “Very comparable results” and “Such antibacterial results”.
Discussion, paragraph 1, 3^rd line: “they have been never been”
Unify the symbols/unites/names in the manuscript (e.g. ml or mL, 250°C or 250[space]°C, Gram positive or Gram-positive).
Check the typos regarding temperature! Pay attention on ‘°’ symbol on page #3.
mm, min and other units have to be separated by space from the numbers
Check the use of en-dash in your manuscript: in case of interval 4–88 μg/ml, and minus temperature –20 °C en-dash should be used.

Author Response

According to Reviewer: 2
Present manuscript deals with hydrodistillation of volatile oils, chemical characterization, and antimicrobial activity of obtained volatile oils. As the authors state in the paper, the paper is a report of an ongoing and partially finished project. Although this manuscript has numerous merits, it still has to be improved, major revision is needed.

First of all, the several parts of the manuscript need some rearrangement and improvement.

Introduction
You give a full list of investigated plant in text form and presented in Table 1. It is more than enough to mention the number of the species. The part on the page #2 “The plant material … Tamamsch.” should be removed and the Table 1. should be moved to Materials and methods part.

The part on the page #2 “The plant material … Tamamsch.” has been removed and the Table 1 has been moved to Materials and Methods part.

Results

Could you confirm once again that 16.45% of Glehnia littoralis volatile oil is palmitic acid?

We confirm that the percentage of palmitic acid in Glehnia littoralis volatile oil was 16.45%. It is notable that it is so high but it is also supported by the bibliography as previously 23.6 mg of another fatty acid (9-hydroxystearic acid) has been isolated from 54.2 g of petroleum ether extract of Glehnia littoralis [Shouwen Zhang, Fang Cheng, Li Yang, Jinxiang Zeng, Fengyu Han, Xiuling Yu, Yuye Zhu, Guoyue Zhong & Junwei He (2020) Chemical constituents from Glehnia littoralis and their chemotaxonomic significance, Natural Product Research, 34:19, 2822-2827, DOI: 10.1080/14786419.2019.1586697]

Discussion

In my opinion you have to improve this section significantly. All parts from previous section should be arranged in this part.

According to reviewer suggestion, Results and Discussion parts have been merged, in order to have a complete comparison of our results with the bibliography and no repetitions

Second, I have some comments and questions regarding the content and results:

Why did you choose these specific plants? What was the original story? It would be nice to read in the Introduction section about your hypothesis even from this perspective.

Numerous projects involving multidirectional research are carried out especially at the Medical University of Lublin, covering numerous genera of plants from the Apiaceae family.

References used

Skalicka-Woźniak, K., Łoś, R., Ulz, Z., Głowniak, K., & Malm, A. (2011). Essential oil from fruits of angelica of ficinalis and its antibacterial activity. Annales Universitatis Mariae Curie-Sklodowska, Sectio DDD: Pharmacia, 24(1), 25-31.
Skalicka-Wozniak, K., Los, R., Glowniak, K., & Malm, A. (2010). Comparison of hydrodistillation and headspace solid-phase microextraction techniques for antibacterial volatile compounds from the fruits of seseli libanotis. Natural Product Communications, 5(9), 1427-1430. doi:10.1177/1934578x1000500916
Skalicka-Wozniak, K., Los, R., Glowniak, K., & Malm, A. (2009). Volatile compounds in fruits of peucedanum cervaria (LAP.) L. Chemistry and Biodiversity, 6(7), 1087-1092. doi:10.1002/cbdv.200800236
Sieniawska, E., Świątek, Ł., Rajtar, B., Kozioł, E., Polz-Dacewicz, M., & Skalicka-Woźniak, K. (2016). Carrot seed essential oil—Source of carotol and cytotoxicity study. Industrial Crops and Products, 92, 109-115. doi:10.1016/j.indcrop.2016.08.001
Sieniawska, E., Los, R., Baj, T., Malm, A., & Glowniak, K. (2013). Antimicrobial efficacy of mutellina purpurea essential oil and α-pinene against staphylococcus epidermidis grown in planktonic and biofilm cultures. Industrial Crops and Products, 51, 152-157. doi:10.1016/j.indcrop.2013.09.001
Mustafa, B., Hajdari, A., Krasniqi, F., Hoxha, E., Ademi, H., Quave, C. L., & Pieroni, A. (2012). Medical ethnobotany of the albanian alps in kosovo. Journal of Ethnobiology and Ethnomedicine, 8 doi:10.1186/1746-4269-8-6
Merecz-Sadowska, A., Sitarek, P., Śliwiński, T., & Zajdel, R. (2020). Anti-inflammatory activity of extracts and pure compounds derived from plants via modulation of signaling pathways, especially pi3k/akt in macrophages. International Journal of Molecular Sciences, 21(24), 1-32. doi:10.3390/ijms21249605
Song, D. H., Jo, Y. H., Ahn, J. H., Kim, S. B., Yun, C., Kim, Y., Lee, M. K. (2018). Sesquiterpenes from fruits of torilis japonica with inhibitory activity on melanin synthesis in B16 cells. Journal of Natural Medicines, 72(1), 155-160. doi:10.1007/s11418-017-1123-4
Kim, G. T., Kim, S., & Kim, Y. M. (2017). Torilis japonica extract fraction compound, EGFR-targeted inhibition of cancer abnormal metastasis in A549 lung cancer cells. Oncology Reports, 38(2), 1206-1212. doi:10.3892/or.2017.5771

Materials and Methods

Could you provide the exact geographical location where Torilis japonica was collected?

Torilis japonica fruits were collected near Lublin (village Ciecierzyn, located 15 km from Lublin).

Could you clarify MIC determination? Which recommendation was followed: EUCAST or CLSI?

The recommendation EUCAST has been followed, eventhough in opur case we have assayed against standard strains. Moreover, and as for further thoughts and scientific discussion, despite the importance of antimicrobial susceptibility testing (AST) for clinical management of infection and antimicrobial resistance (AMR) surveillance, the methodologies and breakpoints of the two most commonly used systems worldwide, Clinical and Laboratory Standards Institute (CLSI) and European Committee for Antimicrobial Susceptibility Testing (EUCAST), are far from harmonized. Both systems are recommended in the World Health Organization's Global Antimicrobial Resistance Surveillance System (GLASS), but how discrepancies between the two systems will be addressed is unclear. As it is well accepted worldwide, there are breakpoint discrepancies towards the Impact of CLSI and EUCAST on reporting of antimicrobial susceptibility and AMR surveillance (TP Cusack et al. 2019)

In the Introduction section you wrote about increasing need for new antibiotics-like compounds. However, there is nothing discussed about your compounds. Give some evaluation of your compounds and compare their potency with clinical applied antibiotics.

As it is known in the area of herbals, there are not recently approved either extracts and/or pure secondary metabolites with strong antibiotics-like activities. The outcome of the study (as in many others too) would be a promising result of potential antimicrobial activity, for certain herbal preparations (as of essential oils), according their activities against specific microorganisms (Either gram positive, Gram negative or yeasts), towards further applications in cosmetics (antiseptic activities on skin) or in foods preservatives. The results of the antimicrobial activities, as they can be seen in table 4, and their potency with clinical applied antibiotics is surely much weaker.

In the Discussion section you are explaining the difference between chemical profile of the same species collected in different harvesting period. Could you support your explanation by similar observations on relative taxa reported in the literature?

In the Conclusion part it was explained that “differences in their chemical profile probably due to different cultivation, climate and geographic conditions.” The explanation is supported by 3 references on other species of Apiaceae family.

Arif Şanli, Tahsin Karadoğan 2017. Geographical Impact On Essential Oil Composition Of Endemic Kundmannia Anatolica -Mor. (Apiaceae) Afr J Tradit Complement Altern Med., 14 (1): 131-137
Ivana Generalić Mekinić, Vida Šimat, Ivica Ljubenkov, Franko Burčul, Mia Grga, Marija Mihajlovski, Ružica Lončar, Višnja Katalinić, Danijela Skroza, 2018 Influence of the vegetation period on sea fennel, Crithmum maritimum L. (Apiaceae), phenolic composition, antioxidant and anticholinesterase activities, Industrial Crops and Products, 124, 947-953, https://doi.org/10.1016/j.indcrop.2018.08.080.
Kamel Msaada, Karim Hosni, Mouna Ben Taarit, Thouraya Chahed, Mohamed Elyes Kchouk, Brahim Marzouk, 2007Changes on essential oil composition of coriander (Coriandrum sativum L.) fruits during three stages of maturity, Food Chemistry, 102, 4, 1131-1134, https://doi.org/10.1016/j.foodchem.2006.06.046.

Third, the manuscript has numerous typos and language inconsistencies:

page #1, paragraph 3: “thus discovering… diseases is challenging” and “agents to be served for antimicrobial therapy” rephrase.

The sentence has been rephrased to: ”thus discovering alternative potentially effective treatments for such infectious diseases is a challenge. Furthermore phytoconstituents are widely considered as promising agents for antimicrobial therapy [8].”

The previous work done by authors should be written in past tense (e.g. “Seseli devenyense … has been studied exhaustively previously [was studied], from [by] our scientific team…”).

The past tense has been used as suggested by the reviewer.eg Page4: Seseli devenyense Simonkai (Seseli elatum subsp. osseum (Crantz) P.W.Ball.) was studied exhaustively previously, by our scientific team// Page 5: Finally, herba of Peucedanum luxurians was studied phytochemically before, as a source of rare bioactive furanocourmarins with unique structures by our scientific team [3, 6, 7, 8],

Section 3.2., paragraph 1: Please reconsider following expressions: “Very comparable results” and “Such antibacterial results”.

The proposed sentences have been corrected accordingly: “Corresponding results to that of S. libanotis were revealed from S. devenyense (MIC 1.35, 1.20 mg/mL). These results are in agreement with previously published data,..”

Discussion, paragraph 1, 3^rd line: “they have been never been”

The suggested sentence has been corrected: as they have never been studied before for their volatiles

Unify the symbols/unites/names in the manuscript (e.g. ml or mL, 250°C or 250[space]°C, Gram positive or Gram-positive).
Check the typos regarding temperature! Pay attention on ‘°’ symbol on page #3.
mm, min and other units have to be separated by space from the numbers
Check the use of en-dash in your manuscript: in case of interval 4–88 μg/ml, and minus temperature –20 °C en-dash should be used.

The symbols and the units have been corrected/unified through the whole manuscript.

The symbol for the temperature has been corrected, a space has been inserted between units and numbers and the use of en-dash has been corrected too, in the whole manuscript.

Reviewer 3 Report

This work endeavors to describe the phytochemical inventory of essential oils extracted from understudied plant species. This paper suffers from several errors and this reviewer cannot determine whether they are a byproduct of manuscript preparation or the study itself. Either way, they are outlined below.

1) The materials and methods section is incomplete and the study could not be replicated as described. Here is an incomplete list of things missing, which are not discussed below:

-Collection time of year, storage after collection and before drying, and means of collection (simply stating collection is insufficient)

-Drying time/conditions, powdering conditions, how long was the pentane extract stored and in what?

-How much was injected on the GCs? Was it done by hand or autoinjected? What program was used for analysis? What about the other parameters (e.g., scan speed, mass range, etc.)?

-What was your tolerance cutoff for the NIST database – 60%, 70%, 80% similarity?

-1ul of microbe may have been used but how many cells was that (or even what OD)? You can’t do an MIC with 1ul as your units because 1ul of P. aeruginosa is a different number of cells than 1ul of E. coli and this number varies by growth phase (which we don’t know because it is not mentioned).

The materials and methods needs to be rewritten to include the vast amount of missing information.

2) The authors describe the compound inventory from each of their extracts and name specific compounds and percentages of the extract that these compounds represent. In the materials and methods (discussed below), there is little mention of how these compounds were called other than NIST is invoked and bibliographic information, which is not defined – who’s bibliography, the citation? It so happens that, in this citation, there are many ways to determine plausible identities for the general groups of compounds in extracts. These often rely on comparisons to standards or libraries of standards, which are used to determine retention indices. Without using specific standards that co-elute or doing more thorough structural analysis, the true identity of such compounds cannot be determined. This work lacks any description of how compounds were called with an impressive degree of accuracy, which is entirely unsupported by the materials and methods section (there is no way to determine the difference between E- vs. Z-beta-ocimene using this analysis). The methods lacks an explanation of how this data was analyzed and compound names called other than citing an entire book on analysis (we don’t know how they determine the percent of a compound in the extract). This section should be rewritten to detail the process. If a sufficient level of experimentation was conducted to truly define the compounds named herein, the authors should change all mentioned to “putatively identified” and then greatly soften the claims made of individual compounds as they are not supported by the results. The authors may also need to conduct additional control experiments with various standards to verify their results.

3) The authors then go on to do work to determine the MIC of extracts against several microbial pathogens and state specific mg/ml values of each extract resulting in inhibition. There is no way the authors could know this because there is no drying step in the methods. Once extracted, the pentane would have to be rotovaped off (or otherwise removed), lyophilized to remove excess water (or dried with some anhydrous sulfate before evaporation, for instance), accurate dry masses recorded, and then these used to create extract stocks. None of this is mentioned so there is no way the authors could claim a mg/ml MIC was determined. Additionally, the authors never mention what their carrier solvent was to conduct MIC testing because pentane is not good to add to microbial cultures. Methanol or DMSO are commonly used as solvent carriers and negative controls are included (there is almost no mention of a control anywhere in this manuscript). Continuing on, the use of the term “antimicrobial” is incorrect here as the authors have not tested any cell lines. It is possible that they have extracted a cytotoxin and not antimicrobial compounds, which the reader does not know given the lack of cell lines tested. Though not incorrect necessarily, the choice to only use yeast for fungal testing is limiting to the analysis and adding a filamentous fungus would improve the work (Aspergillus niger is a common choice). Also, some of the microbial names are misspelled.

4) The authors make some claims in the first paragraph of the intro and back them up with no sources. Reviews are helpful to substantiate broad claims. The introduction also provides very little introduction. What has been done on the Apiaceae – essential oil analysis? Antimicrobial activities? What was found by other groups? Where is the current state of knowledge and what has been found? There are only broad statements that indicate that something has been worked on but not enough……maybe. Do more with the intro and use it to frame the study. The last paragraph of the intro is more like a materials and methods section and is out of place.

There are also some errors in the writing that could benefit from additional editing. Comma use is dicey at times.

Author Response

According to Reviewer: 3

1) The materials and methods section is incomplete and the study could not be replicated as described. Here is an incomplete list of things missing, which are not discussed below:

-Collection time of year, storage after collection and before drying, and means of collection (simply stating collection is insufficient)

The time of collection of the used plant material was between second half of August and end of September. Exact collection time depended from the plant growth phase, which is not fully strict, as depends mainly on the annual temperature (climate changes, rain etc).

-Drying time/conditions, powdering conditions, how long was the pentane extract stored and in what?

The plant material was harvested by cutting the plant with pruning shears and immediately transferred carefully (to prevent loss of ripe fruit) to the drying room. After drying in room temperature (to avoid loss of essential oil), what took few days, the plant material was cut in small pieces and pulverised in a laboratory grinder and passed through a sieve with a mesh of 1,4 mm.

-How much was injected on the GCs? Was it done by hand or autoinjected? What program was used for analysis? What about the other parameters (e.g., scan speed, mass range, etc.)?

The GC injection is 2μL by hand. The program is referred in the “Material and Methods” in part 2.3: “The column temperature was programmed from 75 ⁰C to 280 ⁰C at a rate of 2.5⁰C/min.”

-What was your tolerance cutoff for the NIST database – 60%, 70%, 80% similarity?

The cutoff for the NIST database was 80% similarity.

In all cases of the antimicrobial bioassay, the tested organisms have to reach a cell concentration of 10⁷ cell/ml. This has been added in the Experimental part, even though the references used, include this as standard.

The materials and methods needs to be rewritten to include the vast amount of missing information.

With full respect to the Reviewer’s comments, we take all into consideration and try to answer and cover in details; we do not think that there is a vast amount of missing information.

On behalf of the authors, I can confirm that for such bioassays, as described and evaluated in a phytochemical study in this manuscript, the methods are standards and somehow trivially are repeated in the Experimental Part. To my long scientific experience in the area (more that 170 publications) since at least, last 15 years, all Editors and Reviewers request to apply short experimental part, with no details, as they are referring to “well-known” methods. In many previous cases the comments were to shorten rather to extend Experimental part.

In the case of the submitted paper, we proceeded with the development of the submitted manuscript, following information and examples from very recent references of the Format- Experimental details, used in the Journal of Processes (Salamatullah et al. 2021; Elansary et al. 2020; Abo Elgat et al. 2020) . All data given were cited in one or two short paragraphs always with appropriate references. The same way has been followed by us too, so there is not lack of information, but contrary the information given has been shortened to comply with journal’s demands. Of course could be more detailed, if requested in this particular case so.

All components were identified by comparison of their mass spectra with those obtained from authentic samples and/or the mass spectral databases.

It was not used only the NIST library but also the Wiley275 and the Adams07, that is why the names are not written as in NIST library invoked. It is referred only the NIST in the manuscript, as it is the one with official Rights (permission of use) in our Dept, but collaboratively with other Research laboratories (out of the University of Athens) we have access to the other libraries for cross-checking, which is always pointed.

The bibliographic data that it is referred in this part 2.3 is specific: Adams, R.P. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry; Allured Publishing Corporation Carol Stream, IL, 2007, 456

A number of standards are used generally in our lab for the analyses of essential oils and our experience on this field is proved by our publications- of the individuals in the group of authors (more than 20 in scopus in well accepted and high IFs scientific Journals). For the example of Z- and E-beta-ocimene it is checked that they are eluted with this order which is in accordance with Adams, 2007.

The percentage of the compounds was confirmed by GC- FID as it is generally accepted in the literature.

Continuing on, the use of the term “antimicrobial” is incorrect here as the authors have not tested any cell lines. It is possible that they have extracted a cytotoxin and not antimicrobial compounds, which the reader does not know given the lack of cell lines tested. Though not incorrect necessarily, the choice to only use yeast for fungal testing is limiting to the analysis and adding a filamentous fungus would improve the work (Aspergillus niger is a common choice). Also, some of the microbial names are misspelled.

The pentane extract was evaporated under nitrogen (this method is generally used from our team as the pentane boiling pentane is at 36 ⁰C) and it is evaporated in a short period of time (some minutes only). The compounds that remain in the extract are in accordance with that identified by GC, GC-MS, as the GC temperature program starts from 60⁰C.

In the 2.2. Experimental part the sentence:”The collected essential oils were dried over anhydrous sodium sulphate.” has been added

Contrary to the suggestion that “antimicrobial” assays and/or activity is incorrect. We cannot agree as Candida species are well known opportunistic pathogenic yeasts, belonging (classified) to the Kingdom of fungi, The approach in this study (as in many others already published studies) was the assay of the particular essential oils against a panel of human pathogenic microorganisms. So 6 Gram positive and Gram negative bacteria and three Candida species (C. albicans, C. glabrata and C. tropicalis) which can cause candidiasis and are together responsible for 50–90% of all cases of candidiasis in humans, so very common and interesting to test the essential oils against them. We welcome always any misspelling or other comment and suggestion towards the amendment of the manuscript.

There are also some errors in the writing that could benefit from additional editing. Comma use is dicey at times.

The bibliography with the number 1 has been added as source for the first paragraph information.

In the Introduction part the following paragraph has been added in order to give to the readers more info for the family and its biological activity and also to frame our work: “Apiaceae is considered as one of the most important plant family, including 3780 species and 434 genera (Sayed-Ahmad et al., 2017). They are mainly distributed in the Mediterranean basin with high economic importance for the food and cosmetic industry (Ngahang Kamte et al., 2018). Plant secondary products typically found in Apiaceae are essential oils, including terpenoids and phenylpropanoids, coumarins and furanocoumarins, sesquiterpenelactones, polyacetylenes (polyines), and further compounds derived from acetate units, such as alkylphthalides and the toxic piperidine alkaloids [Chizzola 2010]. Plants of the Apiaceae family are used widely in folk medicine for the treatment of several human ailments (Zengin et al., 2020). Several species from this family have shown antimicrobial and antioxidant activity and they are considered as promising sources of bioactive agents (Zengin et al., 2020, Hasheminya & Dehghannya 2021, Pateiro et al., 2021).”

The last paragraph of the Introduction: “The plant material … Tamamsch.” has been removed and the Table 1 has been moved to Materials and Methods part.

Round 2

Reviewer 2 Report

Dear Authors,

Thank you for considering my suggestions. In my opinion, your manuscript has been improved significantly.

Reviewer 3 Report

The authors did a thorough job responding to the comments. One comment struck me as odd - why would anyone purposefully shorten the materials and methods section of a paper published online without word or page limits? There is no reason why specifics of any experiment should be omitted from publication in a format where there is no limit on content. The idea that reviewers would purposefully require less procedural information runs quite counter to reproducibility. Perhaps the details should be moved to the supplemental section to conserve space in certain manuscripts but never removed entirely. Without them, how would the reader without 170 publications and a lifetime of experience doing exactly this work know what was done? Details are added for future readers who may not have dedicated a career to a single line of inquiry. While the idea of well-established protocols was invoked, I think we can agree that there are a LOT of well-established protocols, which vary widely by the lab in which they are conducted. Sure, the compound was visualized on TLC by sulfuric acid charring but was it 10%, 20%, 50% acid and at what temperature over what time? Though these may seem trivial, they matter for compounds that can volatilize away during visualization. While I very much disagree with the idea that less information in a materials and methods section adds to the paper in any positive way (I feel it very much detracts from reproducibility), this is a disagreement routed in scientific theory rather than the soundness of work as judged by the response to reviewers. For that reason, I do not see the need to impair publication of the work but, in the future, consider that less is indeed less when you have unlimited space in a paper.