Effect of Plant Growth Regulators on Different Explants of Artemisia ludoviciana under Photoperiod and Darkness Conditions and Their Influence on Achillin Production

Round 1

Reviewer 1 Report

The manuscript processes-1809924 established callus and plantlets from different explants of Artemisia ludoviciana under light or dark conditions. Besides, achilin from the donor plant and established callus was extracted and quantified. The work appears to have been performed in an acceptable manner. However, it will be helpful to the readers if the following points are considered and addressed.

 

Major concerns:

Results and discussion probably need improvement or be more organized to increase the accessibility and readability of the manuscript. For example, why do the authors need the information in Section 3.1 since some were discussed in the subsequent sections? Also, "discussion" is lacking in this section. It would be good to cite Table 1 or 2 when mentioning the results (instead of at the end of the paragraph).

L46-47: "The production of achillin may be limited if it is obtained from wild plants. In this regard, in vitro culture protocols are of great interest for future applications...". It would be good if the authors could further explain why achillin production may be limited if it is from wild plants. This is because the amount of achillin in wild plants is higher than in vitro culture unless scaled up to commercial settings.

 

Minor concerns:

Abstract: Please provide the full name for the first time mentioned.

L43: "Recent studies...". Please provide at least two references to support such claims.

L51: "this type of culture". Please be more specific. In vitro culture in general or callus culture?

Section 2.1: Could this information be integrated into each experiment? It probably would be easier for readers to follow how the experiments were conducted.

L164-165: Is this necessary since this information was repeated in Section 3.1?

Tables 2 and 3: Are these results generated from the experiment for Table 1? Or they were separate experiments?

Figures 1-4: Please insert a scale bar for each image. Also, please specify the days of capturing these images in the figure caption, e.g., after 20 days of culture.

L367: "(1)" refers to?

Author Response

Thank you very much for your valuable comments, the file of the answers is attached

Author Response File: Author Response.docx

Reviewer 2 Report

 

The authors of this article stated in the Abstract that the aim of the study was to isolate the sesquiterpene lactone achillin from the wild plant of Artemisia ludoviciana (Asteraceae) and to identify a callus culture from different explants capable of producing achillin. Actually, some of the authors of the present manuscript have already published an article in 2019 on Pharmaceutics (reference 15) where they reported an almost identical isolation procedure for achillin from the leaves of Artemisia ludoviciana; therefore, this article cannot be aimed at the isolation of achillin, unless the authors have come up with a totally new strategy. The authors should just report that, in the present study, they isolated achillin with a yield of 257 mg from 12.34 g of dried leaves, according to an isolation procedure already described [ref. 15]. Moreover, there is an anomaly between the two extraction solvents used: in the present article, the leaves were extracted using a mixture of dichloromethane and methanol (95:05, v/v), whereas, in ref. 15, the solvent was a mixture of methanol and water (95.05, v/v). The polarity difference between these solvents is clearly very big, so I think that one of the two solvent mixtures is wrong (see J. Nat. Prod., 1988, ref. 22).

In my opinion, the article deserves publication on Processes only after some major revisions.

With regard to the GC/MS analysis, the article lacks with some important details on achillin such as the chemical formula, the molecular weight, the optical rotation (it is a chiral natural product). Furthermore, the authors should report the m/z values chosen in the SIM mode (they simply reported that “target ions” were used for quantitation). But what is should be improved for sure is the GC/MS chromatogram showed in Fig. 5, for the following reasons:

1) why the authors cut the chromatogram at 20.60 minutes and did not show the full baseline? I searched for other chromatograms in the SM, since the authors reported, at lines 337-338, that “Chromatograms, as well as data about the quantification method, are reported in Supplementary Material (Figures S1-S3)”, but I did not find anything; a full chromatogram should be given in the Figure.

2) what are the peaks preceding that of achillin (retention time: 21.60 min)? Since the method uses mass spectrometry as the detector, their identification should be easy.

3) what is eluting from the column in the run time between 22.60 and 23.20 minutes?? The column should be better equilibrated before consecutive injections!

4) what is the concentration of achillin (expressed in micrograms per gram of dry biomass) in the chromatogram showed? Although the value is given in Table 4, it should be reported also in the caption to Figure 5 to give an idea of the soundness of the quantitative method.

 

 

 

 

Author Response

Thank you very much for your valuable comments, the file of the answers is attached

Author Response File: Author Response.docx

Reviewer 3 Report

In the study - Effect of plant growth regulators on different explants of Artemisia ludoviciana under photoperiod and darkness conditions and their influence on achillin production-  Mariana Sánchez-Ramos et al. made a study about few species of the genus Artemisia mainly biosynthesize sesquiterpene lactones. The study conduct to ideea that the achillin is a guaianolide-type sesquiterpene lactone isolated from Artemisia ludoviciana had antibacterial and

anti-inflammatory activities. Some comments which greatly enhance the understanding of the paper and its value are presented below. 

Specific issues that require further consideration are:

1. The title of the manuscript is matched to its content.

2. The structure of the manuscript is rather proper.

3. The Introduction  covers the cases, but should be better focused in order for the reader to understand why this work is performed.

4. In my opinion, the current state of knowledge relating to the manuscript topic has not been covered and clearly presented.

5. An analysis of the manuscript content and the References shows that the manuscript under review constitutes a summary of the Author(s) achievements in the field.

6. In my opinion, some new additional experiments needed, research not conducted correctly.

7. The bibliography, comprising 46 references, look to be not representative and exhaustive.

8. I suggest expanding the conclusions.

9. The presentation of the results must be revised to highlight the scientific findings of the Authors clearly.

The research work is very interesting and deserves to be published but only after a major revision.

Best regards, 

Author Response

Thank you very much for your valuable comments, the file of the answers is attached

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The authors have answered to the reviewer suggestions, but there are still some open items:

In Section 2.1, the sentence “Detection was performed in selective ion-monitoring (SIM) mode and peaks were identified and quantitated using target ions” has not been changed and the m/z values chosen in the SIM mode were not given.

The chromatogram depicted in Figure 5c still presents a cut baseline (from 20.60 to 23 minutes) with a cluster of unknown and coeluting peaks in the run time between 22.60 and 23.20 minutes. The authors are strongly encouraged to replace the chromatogram in Fig. 5c with the one provided in the Supplementary Material (Fig. S4). A proposed picture could be the one attached below.

Comments for author File: Comments.pdf

Author Response

appreciable reviewer,
thank you very much for your comments, the indicated changes have been made, the attached file describes the changes

Author Response File: Author Response.docx

Reviewer 3 Report

After the changes made, from my point of view, the article can be published as it is.

Author Response

Thank you very much for your comments dear reviewer

Author Response File: Author Response.docx