Anxiolytic and Antidepressant-Like Effects of Conyza canadensis Aqueous Extract in the Scopolamine Rat Model

Round 1

Reviewer 1 Report

this is an interesting paper on antioxidant activity and rats anxiolitic model of conyza cnADIENSIS extracts such as the elevated plus-maze and forced swimming tests were performed. comments: good work, on a typical plant widely used in traditional medicine in Morocco for the treatment of varied diseases.  weakness: need to perform quantitation of compounds using ich standard guidelines, adding recoveries etc of at least one compounds. table 1 do not have standard deviation for example. need to add ethical comitee on animals permission.how many animals were used? please check english language accordingly.

Author Response

Thank you for your suggestions. We took into consideration all aspects indicated by you as follows:

1. The quantification of compounds was included in the material and methods in 3.2. - Chemical assessment of the extract, more clarifications and specifications were also included. LOD and LOQ were included too.
2. The standard deviation was included in the table (Table 1).
3. The ethical aspects were included in the initial manuscript, however, we have now indicated exactly the number of the accepted protocol. We made sure the number of animals is specified correctly.

Reviewer 2 Report

Opinion related to paper entitled: Anxiolytic and antidepressant-like effect of Conyza canadensis aqueous extract in the scopolamine model.

 

            The work is interesting. The most interesting part of work is the high anxiolytic and antidepressant activity of extracts. Authors attribute these activities to the presence of phenolic fraction especially flavonoids. I have serious doubts about this. I would attribute the effect on the central nervous system, for example, to the presence of volatile fraction (essential oils). I suggest deeper literature analysis of the phytochemical composition in order to identify fraction acting as an anti-anxiety and antidepressant.

Typical activities that can be attributed to flavonoids are diuretic, spasmolytic, anti-inflammatory, tannins (catechins) antihemorrhagic, etc.

Authors demonstrated strong antioxidant, chelating iron ions and inhibiting LOX activity (potential anti-inflammatory activity) of extracts.

Figure 2. Form figures a, b, c we can see a strong dependence of the reaction rate on the extract concentration at lower extract concentrations. At the higher concentration of extract the plateau is established and the reaction rate does not increase despite increasing the concentration of extract. Why Authors calculate straight lines for all concentration range. It is better calculate these lines for low substance concentrations. For example, from Figure 2a, for lower concentration of substance, it is clear that quercetin is more active than the CC extract. The description of the results concerning antioxidant properties is very unclear, it should be corrected. The work contains mistakes that should be removed. Description of axes (fig. 2) should be mg/mL not mg. Check the result “IC50 was 126.03 μg/mL”. If data in table 1 are averages, the information on error should be given.

Author Response

Thank you for your suggestions. We took into consideration all aspects indicated by you as follows:

1. Although the anxiolytic and antidepressant activities could be induced also by volatiles, there is plenty of data that indicates flavonoids such as luteolin, apigenin, quercetin or polyphenolic acids (rosmarinic acid, chlorogenic acid, ellagic acid etc) can modulate neural activity. E.q. apigenin is an established GABA-ergic modulator. We investigated this in relation to the extract that we included in our study. Water extracts usually do not isolate volatile which are lipophilic compounds. Our research is unique by the fact that the material and the type of extract is different from all other studies on Conyza published up to date. We specified these aspects and we included more comparable data to prove our results. 
2. Corrections were made to figure 2 a, b, c, and the entire text referring to antioxidant activity was improved.

Reviewer 3 Report

 

The paper by Jamila El-Akhal and colleagues is a translational work that includes analytical aspects, in vitro experiments and in vivo data. The experimental design is clear and the amount of data sufficient, but the paper does not meet the standard for publication at this stage. In particular, some improvements should be made, especially concerning the data presentation quality. I report some observations below.

 

• In the abstract, avoid using too many abbreviations.
• The first part of Introduction (lines 32-42) is quite long and too vague. The Introduction should me more closely focused on the topic.
• The use of CC abbreviation should be explained in the text.
• Quality of Figure 1 should be improved: text is not readable and the figure overall is not very clear. The authors could consider resubmitting a higher quality version as supplementary file. The same holds true for graphics in Figure 2. Even if resolution is sufficient, providing graphics as “default” Excel objects is not sufficient for meeting the standards required by a Journal of this level. Most importantly, according to the reported data, a linear fit of data points does not make sense.
• Spacing between paragraphs is missing in some cases.
• Methods description could be improved and be more specific.
• Conclusion section is too brief and do not provide enough comments on the observed results.

Author Response

Thank you for your suggestions. We took into consideration all aspects indicated by you as follows:

1. The abstract was modified and the abbreviations were eliminated.
2. Lines 32-42 were improved, they are included as a justification for our plant choice which is a traditional Maroccan remedy.
3. CC explanation was included in the text.
4. All figures were improved and enlarged writing was included in them, some corrections were made to Figure 2. The linear data fit was eliminated. More correlation was made in the text referring to antioxidant activity. 
5. Methods were improved and were made more specific.
6. The conclusion paragraph was improved also. 

Reviewer 4 Report

The authors evaluated the phytochemical composition (phenolic compounds) and  anxiolytic and antidepressant-like effects of the Conyza canadensis aqueous extract. The data presented is interesting, however improvements are needed for the identification/quantification of the phenolic compounds.

Please answer to the following comments and make the improvement in the manuscript:

1. How did you identify the compounds? Please add the information of the lamda max of absorption of each compound identified. If standards were used, please add as supplementary materials the chromatograms of the chemical standards and how they compare with the retention time and spectra characteristics of the data presented in the manuscript.
2. Please add the standard deviation of the compounds quantified.
3. Please better describe in materials and methods how the compounds were identified. 

 

Table 1 include the standard deviation. 

Author Response

Thank you for your suggestions. We took into consideration all aspects indicated by you as follows:

1. Regarding the identification of the compounds, we included more specifications into material and methods section 3.2. - The chemical analysis. Initially, we were concerned about plagiarism and we wrote the method with fewer data, sorry about that.
2. We have a UV spectrum library (which we cannot include as supplementary material) that is registered in the Chromelon software and the Cobra wizard processing methods matches the time retention and UV spectra to each peak from the chromatogram. We consider that a correct match is only for the compounds which have a percentage of at least 95% identity.
3. We included several data such as LOD and LOQ for two of our standards. Calibration curves were also obtained for the standards and we included a phrase referring to it.

Round 2

Reviewer 3 Report

The authors improved the former version of the manuscript and provided a new version in which they addressed my comments. The paper was improved, also from the chemical point of view. The author meets the standard for publication. Nevertheless, some minor improvements are still required. For example:

• check the use of capital letters (iron/Iron)
• formatting should still be improved (e.g. Figure 2)
• revision mode should be removed

Author Response

Thank you. We removed the revision mode and we spell checked all word and changed where needed. We uploaded al figures with higher resolution. 

Reviewer 4 Report

The authors attended the comments raised by this reviewer. 

Author Response

Thank you