Essential Oil from Arnica Montana L. Achenes: Chemical Characteristics and Anticancer Activity
, Joanna Jakubowicz-Gil 3, Krystyna Winiarczyk 4 and Radosław Kowalski 5Round 1
Reviewer 1 Report
Dear Editor,
I have now completed reviewing the manuscript titled as «Essential oil of Arnica montana L. achenes: chemical characteristics and anticancer activity» with ID: molecules-633446.
In present study authors reported for the first time the composition and anticancer activities of essential oil from A. achenes. Obviously, this work stands on two pillars, the phytochemical analytical and the biological. The biological part seems to offer interesting knowledge concerning the application of natural products as probable anticancer agents. On the other hand concerning the analytical part, there are some issues that should be discussed/improved.
In more detailed, achenes of the plant Arnica montana were distilled for the first time and the obtained essential oil analyzed also for the first time. Moreover, the authors compared their findings in correlation to plants’ different ages. When this kind of experimentation is taking place, researchers should be very careful in providing all the necessary information. In this case, none information is given in subsection 4.3.1. concerning the mass of the raw material used for distillation, the yield of the essential oil derived or the number of replicates that authors should contact. Analytically, the latter is of great importance not only because data provided could be considered as random but also because, as mentioned above, a comparison between achenes from plants of different ages is one of manuscript’s topic.
Also, according to the authors in subsection 4.3.3.
“The qualitative analysis was carried out on the basis of MS spectra, which were compared with the spectra of the NIST library (50) and with data available in the literature (51-52). The identity of the compounds was confirmed by their retention indices (53) taken from the literature (51-52) and our own data for standards described previously (10, 12)”.
The authors should add a column indicating the method that has been used for the identification of essential oils’ ingredients as well as a column providing the theoretical RI values. According to my opinion the only positive method to identify the ingredients of an essential oil, is to compare retention time and mass spectroscopic data of oil’s components with those of purchased authentic compounds, at least the major ones. The above is of great importance especially in the case of a novel essential oil, such as herein in which the biological activity is been associated and thoroughly discussed with the major component 2, 5-dimethoxy-p-cymene presence. Although, authors referred in two of their former works only in the case of article (10) authentic standards have been used. Specifically, only decanal, E-caryophyllene and caryophyllene oxide in Table 1 could be positively identified based on comparison to authentic standards. According to my opinion, at least the identity of the major component (not only as a percentage but also as biological importance) should be proved either by comparison with a purchased sample or by isolation and subsequently thorough spectroscopic study, otherwise it is questionable.
Moreover,
According to authors Lines 108-109:
“The results of a systematic study on the morphology of glandular trichomes and anatomy studies will be presented in a separate paper”
If that is the case, I really do not understand the necessity to provide present data in this manuscript (subsections 2.1., Lines 194-210, subsection 4.2., Figure 1) particularly when this topic is not given in the title of the manuscript.
Lines 92-95.
Since the comparison of essential oil's profile and biological activity derived from achenes' of plants of different ages is one of the manuscript’s critical topics, it should be also referred to in the objectives of this study.
Authors should check and correct all over the text for the appropriate nomenclature e.g. 2, 5-Dimethoxy-p-cymene (not capital the first letter, italics the Greek letters or letters suggesting para, alpha, trans, E-caryophyllene etc.).
Line 111. Irrelevant capture.
Line 131. It is not IR retention indices.
Line 118 and all over the text.
change the percentage layout e.g. from (48,09, 53,06%) to (48,09 and 53,06 %)
Lines 155, 262
0.5 μl ml ?
Lines 274-284.
The comparison as well the discussion is not acceptable. The literature data that authors used are concerning to the activity of quercetin in synergism with a synthetic drug and not as an individual compound, as herein.
Lines 275-276.
Since no yield of the derived essential oils are given (according to experimentation), this statement is questionable.
Overall, my final decision is that the present manuscript could be considered for publication only after major revision.
Author Response
Response to reviewers' comments
We would like to thank the anonymous reviewers for their useful comments in the development of this paper. We have thoroughly processed all comments and found that changes had greatly improved the clarity of our manuscript. The paper has been corrected in accordance with the reviewers’ comments. Additionally, the style and grammar changes in the whole text have been made.
Reviewer 1:
Dear Editor,
I have now completed reviewing the manuscript titled as «Essential oil of Arnica montana L. achenes: chemical characteristics and anticancer activity» with ID: molecules-633446.
In present study authors reported for the first time the composition and anticancer activities of essential oil from A. achenes. Obviously, this work stands on two pillars, the phytochemical analytical and the biological. The biological part seems to offer interesting knowledge concerning the application of natural products as probable anticancer agents. On the other hand concerning the analytical part, there are some issues that should be discussed/improved.
In more detailed, achenes of the plant Arnica montana were distilled for the first time and the obtained essential oil analyzed also for the first time. Moreover, the authors compared their findings in correlation to plants’ different ages. When this kind of experimentation is taking place, researchers should be very careful in providing all the necessary information. In this case, none information is given in subsection 4.3.1. concerning the mass of the raw material used for distillation, the yield of the essential oil derived or the number of replicates that authors should contact. Analytically, the latter is of great importance not only because data provided could be considered as random but also because, as mentioned above, a comparison between achenes from plants of different ages is one of manuscript’s topic.
- Corrected in accordance with the recommendations of the Reviewer. In subsection 4.3.1 such information as the mass of the raw material used for distillation, and the number of replicates is given. The yield of the essential oil derived from achenes of 3-year-old and 4-year-old plants is given in Table 1.
Also, according to the authors in subsection 4.3.3.
“The qualitative analysis was carried out on the basis of MS spectra, which were compared with the spectra of the NIST library (50) and with data available in the literature (51-52). The identity of the compounds was confirmed by their retention indices (53) taken from the literature (51-52) andour own data for standards described previously (10, 12)”.
The authors should add a column indicating the method that has been used for the identification of essential oils’ ingredients as well as a column providing the theoretical RI values. According to my opinion the only positive method to identify the ingredients of an essential oil, is to compare retention time and mass spectroscopic data of oil’s components with those of purchased authentic compounds, at least the major ones. The above is of great importance especially in the case of a novel essential oil, such as herein in which the biological activity is been associated and thoroughly discussed with the major component 2, 5-dimethoxy-p-cymene presence. Although, authors referred in two of their former works only in the case of article (10) authentic standards have been used. Specifically, only decanal, E-caryophyllene and caryophyllene oxide in Table 1 could be positively identified based on comparison to authentic standards. According to my opinion, at least the identity of the major component (not only as a percentage but also as biological importance) should be proved either by comparison with a purchased sample or by isolation and subsequently thorough spectroscopic study, otherwise it is questionable.
- Corrected in accordance with the recommendations of the Reviewer. In subsection 4.3.3 a column indicating the method that has been used for the identification of essential oils’ ingredients as well as a column providing the RI values have been add. The literature under table was added.
When MS detection is used, standards are not necessary in every case. One of the advantages of mass spectrometry is the possibility of qualitative analysis based on available MS libraries or literature. Of course, standards are needed when using gas chromatography with classic detectors. In addition, in qualitative analysis we use the retention index calculated on the basis of the separation of the alkanes mixture, this is the second parameter that allows identification and is comparable with the data contained in relevant libraries and literature. The main components of arnica oil in the presented work are not substances that were identified for the first time in the studied species and are well described in the literature, which is why in this case we did not need standards. Identification of the essential oil ingredients was made in accordance with the applicable rules by persons who have experience in this field (Radosław Kowalski). In the presented work, we conducted an assessment of the biological activity of arnica oil, while we prepare paper on determining the activity of the main components of essential oil and in the next paper we will work with pure standard substances.
Moreover,
According to authors Lines 108-109:
“The results of a systematic study on the morphology of glandular trichomes and anatomy studies will be presented in a separate paper”
If that is the case, I really do not understand the necessity to provide present data in this manuscript (subsections 2.1., Lines 194-210, subsection 4.2., Figure 1) particularly when this topic is not given in the title of the manuscript.
- Corrected in accordance with the recommendations of the Reviewer. Subsections 2.1., Lines 194-210, subsection 4.2., Figure have been removed. New numbering of sections and figures is given.
Lines 92-95.
Since the comparison of essential oil's profile and biological activity derived from achenes' of plants of different ages is one of the manuscript’s critical topics, it should be also referred to in the objectives of this study.
- Corrected in accordance with the recommendations of the Reviewer. Information about different age of plants is given in the objectives of this study.
Authors should check and correct all over the text for the appropriate nomenclature e.g. 2, 5-Dimethoxy-p-cymene (not capital the first letter, italics the Greek letters or letters suggesting para, alpha, trans, E-caryophyllene etc.).
- Corrected in accordance with the recommendations of the Reviewer. The nomenclature was check and correct in table 1 and in the text.
Line 111. Irrelevant capture.
- Corrected in accordance with the recommendations of the Reviewer. Subsection 4.2., Figure 1, and part of discussion has been removed. New numbering of sections and figures is given.
Line 131. It is not IR retention indices.
- Corrected in accordance with the recommendations of the Reviewer.
Line 118 and all over the text.
change the percentage layout e.g. from (48,09, 53,06%) to (48,09 and 53,06 %)
- Corrected in accordance with the recommendations of the Reviewer.
Lines 155, 262
0.5 μl ml ?
- Corrected in accordance with the recommendations of the Reviewer. The units were corrected in the manuscript – µl/ml.
Lines 274-284.
The comparison as well the discussion is not acceptable. The literature data that authors used are concerning to the activity of quercetin in synergism with a synthetic drug and not as an individual compound, as herein.
- Thank you for critical remark. Indeed, sorafenib and temozolomide are synthetic compounds and the correlation between them shouldn’t be discussed in the article. Therefore, such information has been removed from the text.
Lines 275-276.
Since no yield of the derived essential oils are given (according to experimentation), this statement is questionable.
- The yield of the essential oil derived from achenes of 3-year-old and 4-year-old plants is given in table 1.
Overall, my final decision is that the present manuscript could be considered for publication only after major revision.
Reviewer 2 Report
The article entitled “Essential oil of Arnica montana L. achenes: chemical characteristics and anticancer activity” performed the characterization of the chemical composition of essential oils (EO) derived from Arnica montana L. achenes and also investigated the effect of the analyzed EO on induction of apoptosis and autophagy in human anaplastic astrocytomca MOGGCCM and glioblastoma multiforme T98G cell lines.
The manuscript is suitable to be accepted in Molecules, after major revisions. First of all, the authors should consult the author guidelines, since Fig. should be Figure, Tab should be Table, and the references should be [10,12] and not (10,12). Moreover, in my opinion the authors should revise the references, since 87 references are too much references for this study.
All abbreviations should be described for the first time, such as GC/MS in the abstract, and GC/MS should be GC-MS.
Lines 28 to 30: this information also appears in lines 119 to 122, and 217 to 219. Please remove the information related to retention time, and add this information in Table 1. The authors should pay attention to 2,6-diisopropylanisol, according to the authors the retention time is 8.55 min, but this is not in agreement with the information in Table 1, the retention index. Moreover, the authors say that is thymol (8.63 %), but in the Table 1, I see thymol, methyl ether (O-Methylthymol). In Table 1, the authors use RI, but in footnotes wrote IR. The compounds name should be uniform.
Lines 42 to 43: “….metabolite class (sesquiterpene……and essential oils)…” essential oils is metabolite class?
The units should be the same through the manuscript, sometime appears µL others µl.
The concentration was expressed as µl ml, please clarify this, µl/ml??.
Line 326: In all manuscript appears GC-MS, but then in this line appears GC-MS/MS, this is correct?
Line 330: “…40-870 Da”, why the authors used this mass range, when were looking for volatile metabolites?
Author Response
Response to reviewers' comments
We would like to thank the anonymous reviewers for their useful comments in the development of this paper. We have thoroughly processed all comments and found that changes had greatly improved the clarity of our manuscript. The paper has been corrected in accordance with the reviewers’ comments. Additionally, the style and grammar changes in the whole text have been made.
Reviewer 2:
The article entitled “Essential oil of Arnica montana L. achenes: chemical characteristics and anticancer activity” performed the characterization of the chemical composition of essential oils (EO) derived from Arnica montana L. achenes and also investigated the effect of the analyzed EO on induction of apoptosis and autophagy in human anaplastic astrocytomca MOGGCCM and glioblastoma multiforme T98G cell lines.
The manuscript is suitable to be accepted in Molecules, after major revisions. First of all, the authors should consult the author guidelines, since Fig. should be Figure, Tab should be Table, and the references should be [10,12] and not (10,12). Moreover, in my opinion the authors should revise the references, since 87 references are too much references for this study.
All abbreviations should be described for the first time, such as GC/MS in the abstract, and GC/MS should be GC-MS.
- Corrected in accordance with the recommendations of the Reviewer – abbreviation was described. The references have been revised and renumbered: 15 titles were remove, ad 2 new articles were added.
Lines 28 to 30: this information also appears in lines 119 to 122, and 217 to 219. Please remove the information related to retention time, and add this information in Table 1. The authors should pay attention to 2,6-diisopropylanisol, according to the authors the retention time is 8.55 min, but this is not in agreement with the information in Table 1, the retention index. Moreover, the authors say that is thymol (8.63 %), but in the Table 1, I see thymol, methyl ether (O-Methylthymol). In Table 1, the authors use RI, but in footnotes wrote IR. The compounds name should be uniform.
- Thank you for critical remark. The nomenclature was check and correct in table 1 and in the text. Table 1 was supplemented – retention indices taken from literature eidentified based on comparison to standards were given.In lines 28-30 the percent share of the main components in the EO from the arnica achenes are presented. The nomenclature was checked and correct in table 1 and in the text. In lines 119-122 the percent share of the major class of compounds, and in lines 217-219 the number of constituents in relation to literature data are presented.
Lines 42 to 43: “….metabolite class (sesquiterpene……and essential oils)…” essential oils is metabolite class?
- Corrected in accordance with the recommendations of the Reviewer.
The units should be the same through the manuscript, sometime appears µL others µl.
The concentration was expressed as µl ml, please clarify this, µl/ml??.
- Corrected in accordance with the recommendations of the Reviewer. The units were corrected in the manuscript – µl/ml.
Line 326: In all manuscript appears GC-MS, but then in this line appears GC-MS/MS, this is correct?
- Corrected in accordance with the recommendations of the Reviewer.
Line 330: “…40-870 Da”, why the authors used this mass range, when were looking for volatile metabolites?
- The used range of 40-870 Da results from setting parameters for the simultaneous analysis of extracts containing heavier fractions in terms of the analyzed chemical composition. In addition, this mass range is not incorrect when analyzing essential oils, which is confirmed by the literature.
Reviewer 3 Report
The manuscript entitled "Essential oil of Arnica montana L. achenes: chemical characteristics and anticancer activity" describes the effects of the essential oil of Arnica montana on two cancer cell lines.
The work is very interesting, since this plant exert notable effects on two cancer cell lines of very aggressive and widespread tumours.
My suggestions are listed in the following points:
The results are clearly presented in figures 2-5, and the main effect is apoptosis, therefore it should be useful to have the IC50 values relative to the different tested plants (3- or 4-year old) and the different cell lines (MOGGCCM and T98G) summarized in a table, to easily compare the obtained results Another important missing point is the cytotoxicity of the EO in healthy cells, this assay should be important to assess that Arnica montana essential oil induces apoptosis only in cancer cell lines It is not clear the meaning of the unit of measurement “µl ml”. The authors may better explain this aspect and also specify the unit of measurement on the X axis of figures 2-5.Author Response
Response to reviewers' comments
We would like to thank the anonymous reviewers for their useful comments in the development of this paper. We have thoroughly processed all comments and found that changes had greatly improved the clarity of our manuscript. The paper has been corrected in accordance with the reviewers’ comments. Additionally, the style and grammar changes in the whole text have been made.
Reviewer 3:
The manuscript entitled "Essential oil of Arnica montana L. achenes: chemical characteristics and anticancer activity" describes the effects of the essential oil of Arnica montana on two cancer cell lines.
The work is very interesting, since this plant exert notable effects on two cancer cell lines of very aggressive and widespread tumours.
My suggestions are listed in the following points:
The results are clearly presented in figures 2-5, and the main effect is apoptosis, therefore it should be useful to have the IC50 values relative to the different tested plants (3- or 4-year old) and the different cell lines (MOGGCCM and T98G) summarized in a table, to easily compare the obtained results Another important missing point is the cytotoxicity of the EO in healthy cells, this assay should be important to assess that Arnica montana essential oil induces apoptosis only in cancer cell lines
- Corrected in accordance with the recommendations of the Reviewer. Table 2 presenting IC50 values obtained after MOGGCCM and T98G cell treatment with essential oils was included according to Reviewer suggestion. Experiments on healthy cells were conducted (as a standard procedure) but no cytotoxic effect was observed. Such information was included in the Materials and methods as well as in the Results.
It is not clear the meaning of the unit of measurement “µl ml”. The authors may better explain this aspect and also specify the unit of measurement on the X axis of figures 2-5.
- Corrected in accordance with the recommendations of the Reviewer. The units were corrected in the manuscript – µl/ml, and X axis was described in the titles of all figures.
Round 2
Reviewer 1 Report
Dear authors,
Indeed, a great effort has been done concerning the revised manuscript meaning that in the present form it is suitable for publication. As a minor recommendation, I will insist (as a chemist) on correcting the nomenclature as I have already pointed out according to my earlier revision
"Authors should check and correct all over the text for the appropriate nomenclature e.g. 2, 5-Dimethoxy-p-cymene (italics the Greek letters or letters suggesting para, α, β, trans, E-caryophyllene, etc.)."
Also, in line 122 please correct the word "Table 1" with a capital T.
Reviewer 2 Report
The manuscript entitled "Essentialoil from Arnica montana L. achenes: chemical chaacterisica and anticancer acitivity" shoudl be accepted in current form.
Reviewer 3 Report
The manuscript can now be accepted
