Behavioral and Biochemical Evaluation of Anti-Depressive and Oxidative Stress-Ameliorating Effects of Amber Extract in Adult Male ICR Mice

Round 1

Reviewer 1 Report

- I missed the botanical name in the title;

- In the methodology, adjust the description of the solvents used, as well as the equipment;

- I missed the number referring to the authorization to carry out research with animals;

- Images of the results with low resolution; excessive use of acronyms;

- Update references, some are very old.

Author Response

 

 

Reviewer 1

 

Reviewer`s Comment:

- I missed the botanical name in the title;

- In the methodology, adjust the description of the solvents used, as well as the equipment;

- I missed the number referring to the authorization to carry out research with animals;

- Images of the results with low resolution; excessive use of acronyms;

- Update references, some are very old.

Authors` Response:

 

We thankful for these suggestions.

• We prefer not to include the botanical name of amber in the title.
• We would be grateful if you could suggest how the description of methodology and equipment should be adjusted.
• In line 105, we stated the institution that granted the authorization.
• All acronyms have been defined.
• All references are relevant to our story.

Reviewer 2 Report

Dear Editor,

The manuscript entitled as "Behavioral and biochemical evaluation of anti-depressive and 2 oxidative stress-ameliorating effects of amber extract in adult 3 male ICR mice" was well designed and clearly defined the hypothesis snd the results. Manuscript can be accepted without any revision.

Kind Regards

 

Author Response

 

Reviewer 2

 

Reviewer`s Comment:

Dear Editor,

The manuscript entitled as "Behavioral and biochemical evaluation of anti-depressive and 2 oxidative stress-ameliorating effects of amber extract in adult 3 male ICR mice" was well designed and clearly defined the hypothesis snd the results. Manuscript can be accepted without any revision.

Kind Regards

Authors` Response:

We are grateful to the reviewer for the immense contributions towards the improvement of our paper.

Reviewer 3 Report

Comments and suggestions for author: 

Please correct the following  typo mistake:

 The space between the temp. value and its symbole (degree sign)  should be omitted : 

in page 2 line 88

in page 3 line  1110, 111, 113, 117, 119.  

Author Response

 

 

Reviewer 3

 

Reviewer`s Comment:

Please correct the following  typo mistake:

 The space between the temp. value and its symbole (degree sign)  should be omitted : 

in page 2 line 88

in page 3 line  1110, 111, 113, 117, 119.  

Authors` Response:

We are grateful to the reviewer for the immense contributions towards the improvement of our paper.

 

Kindly find the typo mistakes corrected.

Reviewer 4 Report

The article entitled “Behavioral and biochemical evaluation of anti-depressive and 2 oxidative stress-ameliorating effects of amber extract in adult 3 male ICR mice” is very interesting. However, I have some concerns in this manuscript. These should be rectified before publication.

What was the solvent to powder ratio during extraction?

In nearly all the methodology, there are no references.

This test was performed twice more as planned, why? Give the rationale.

Statistical analysis of data was performed 146 using one-way analysis of variance (ANOVA) and two-way ANOVA, which software was used? You have not mentioned.

Regenerate the graphs with GraphPad by showing the values of each bar so that the presentation of results may be clear.

Why in TST CT (Non-Stressed mice) was not used?

The supplementary figure is not cited in the text.

The authors have not discussed amber extract as nutraceutical. It should be added in the introduction part.

There is no concluding paragraph in this manuscript.

Author Response

Reviewer 4

 

Reviewer`s Comment:

The article entitled “Behavioral and biochemical evaluation of anti-depressive and 2 oxidative stress-ameliorating effects of amber extract in adult 3 male ICR mice” is very interesting. However, I have some concerns in this manuscript. These should be rectified before publication.

What was the solvent to powder ratio during extraction?

In nearly all the methodology, there are no references.

This test was performed twice more as planned, why? Give the rationale.

Statistical analysis of data was performed 146 using one-way analysis of variance (ANOVA) and two-way ANOVA, which software was used? You have not mentioned.

Regenerate the graphs with GraphPad by showing the values of each bar so that the presentation of results may be clear.

Why in TST CT (Non-Stressed mice) was not used?

The supplementary figure is not cited in the text.

The authors have not discussed amber extract as nutraceutical. It should be added in the introduction part.

There is no concluding paragraph in this manuscript.

Authors` Response:

We thank the reviewer for the valuable concerns and suggestions to improve our paper.

• 50g of Amber resin was dissolved with 400mL of 50% ethanol
• Most of the methodologies are instructions from the manufacturing companies of the kits used.
• Repetition of test is necessary to validate the results obtained.
• Statistics were generated using GraphPad Prism 8.0. Kindly find the corrected statement in line 151. `All graphs and statistics were generated using GraphPad Prism software 8.0...`
• We believe that representing results with individual values on each bar will make presented data crowdy.
• TST is a stress test so all animals that take part must be stressed. However, we have the CT (Non-Stressed mice) that must not undergo any form of stress.
• Kindly find the citation of supplementary data added to the methodology on line 107.
• Amber as a nutraceutical has been extensively described as a nutraceutical in our our previous paper titled ` Stress buffering and longevity effects of amber extract on Caenorhabditis elegans ( elegans)`.
• The conclusion of the paper was merged with the discussion and can be found in the last paragraph of the discussion on line 279 since its optional in nutraceuticals.

Reviewer 5 Report

In the manuscript entitled “Behavioral and biochemical evaluation of anti-depressive and oxidative stress-ameliorating effects of amber extract in adult 3 male ICR mice” Somuah-Asanteet. al., investigated the anti-depressive and oxidative stress-ameliorating effects of amber extract in mice subjected to restraint stress. they performed tail suspension test, and their immobility time, body weight before and after stress, levels of stress-related hormones and neurotransmitters, and oxidative stress parameters were assessed. The idea is novel and should be published. The following are the comments regarding the manuscript:

 

1.     It would be great if authors discuss epidemiology briefly in the introduction introduction section.

2.     The authors did not provide an adequate description about available antidepressants in introduction part.

3.     Please include some current references.

4.     It would be wonderful if authors make hypothetical figure in the last to explain the mechanism of action of amber extract as an anti-depressive agent.

5.     As the authors showed that anti-depressive and oxidative stress-ameliorating effects of amber extract through bar diagram, they should perform at least one or two western blot analysis following amber extract treatment (e.g., SOD, CAT), that would be more appealing for researcher.

6.     Authors may create a distinct section for the conclusion.

 

7.     Although manuscript is well written, but it could be more efficient if author check with professional or native English speaker. 

Comments for author File: Comments.pdf

Author Response

 

Reviewer 5

 

Reviewer`s Comment:

In the manuscript entitled “Behavioral and biochemical evaluation of anti-depressive and oxidative stress-ameliorating effects of amber extract in adult 3 male ICR mice” Somuah-Asanteet. al., investigated the anti-depressive and oxidative stress-ameliorating effects of amber extract in mice subjected to restraint stress. they performed tail suspension test, and their immobility time, body weight before and after stress, levels of stress-related hormones and neurotransmitters, and oxidative stress parameters were assessed. The idea is novel and should be published. The following are the comments regarding the manuscript:

1. It would be great if authors discuss epidemiology briefly in the introduction introduction section.
2. The authors did not provide an adequate description about available antidepressants in introduction part.
3. Please include some current references.
4. It would be wonderful if authors make hypothetical figure in the last to explain the mechanism of action of amber extract as an anti-depressive agent.
5. As the authors showed that anti-depressive and oxidative stress-ameliorating effects of amber extractthrough bar diagram, they should perform at least one or two western blot analysis following amber extract treatment (e.g., SOD, CAT), that would be more appealing for researcher.
6. Authors may create a distinct section for the conclusion.
7. Although manuscript is well written, but it could be more efficient if author check with professional or native English speaker. 

Authors` Response:

We thank the reviewer for the valuable comments given to improve our manuscript.

1. We would like to know what epidemiology the reviewer is suggesting.
2. We want to focus on the amber without throwing so much light on commercial antidepressants which are mostly orthodox medications.
3. We believe that the stated references are quite current in relation to the study and the methodologies employed. Though newer methods are being employed in this area of study.
4. We perfectly agree. However, we are still working to determine at length, the possible mechanism of action of amber as an anti-depressant agent.
5. Thank you for this recommendation. We are still working extensively by employing several methods to confirm our hypothesis.
6. Since the conclusion section is optional, we merged it with the discussion to provide an easy flow of the manuscript.
7. Thank you for the recommendation. The author is a native English speaker. However, we will let a second native speaker proofread the manuscript.

Reviewer 6 Report

Please mentioned the source of amber resin (name of the plant).

In 137, you mentioned mouse, in other lines and paragraph you mentioned mice, it must clear either mice or mouse.

In 162, the BUP full form not mentioned only abbreviation is mentioned.

in 221, the paragraph start with full form

Comments for author File: Comments.pdf

Author Response

Reviewer 6

 

Reviewer`s Comment:

Please mentioned the source of amber resin (name of the plant).

In 137, you mentioned mouse, in other lines and paragraph you mentioned mice, it must clear either mice or mouse.

In 162, the BUP full form not mentioned only abbreviation is mentioned.

in 221, the paragraph start with full form

Authors` Response:

 

We thank the reviewer for the valuable concerns to improve our paper.

 

• Kindly find the source of Amber resin stated in line 72. `Amber, a terpenoid-containing resin from the pine tree, has been reported…`
• Thank you for the observation. We prefer to use mice hence kindly find the correction in line 115, 126, 135, 140 and 233.
• In line 102 and 103, BUP group was defined as representing the positive control group of mice treated with bupropion (10 mg/kg body weight).

Reviewer 7 Report

In the present study, Sandra et al. intended to provide a concise, comprehensive behavioral evaluation of the Amber extract titled " Behavioral and biochemical evaluation of anti-depressive and oxidative stress-ameliorating effects of amber extract in adult male ICR mice. The presentation appears excellent overall. The article is well-written, but there are few suggested experiments for validating the hypothesis. Before further publication consideration, it is advised to conduct the suggested experiments.

 

In the forced swim and tail suspension tests, antidepressants that inhibit serotonin and/or norepinephrine reuptake decrease immobility time and increase rodent swinging activity. The authors conducted studies on tail suspension, but the measurement of immobility, the Forced swim test, is absent. The decrease in period of immobility can be viewed as an antidepressant-effect index. It is frequently used to evaluate the efficacy of antidepressants. It is advised to conduct a forced swim test (FST). MAO-A levels are substantially elevated throughout the brains of depressed patients. Do you believe that Amber extract lowers MAO-A levels in the brain? Use the Monoamine oxidase A assay kit to conduct an experiment.

Author Response

 

Reviewer 7

 

Reviewer`s Comment:

In the forced swim and tail suspension tests, antidepressants that inhibit serotonin and/or norepinephrine reuptake decrease immobility time and increase rodent swinging activity. The authors conducted studies on tail suspension, but the measurement of immobility, the Forced swim test, is absent. The decrease in period of immobility can be viewed as an antidepressant-effect index. It is frequently used to evaluate the efficacy of antidepressants. It is advised to conduct a forced swim test (FST). MAO-A levels are substantially elevated throughout the brains of depressed patients. Do you believe that Amber extract lowers MAO-A levels in the brain? Use the Monoamine oxidase A assay kit to conduct an experiment.

 

Authors` Response:

 

We are grateful to the reviewer for these valuable suggestions.

• We studied the effect of amber using forced swimming test (data not shown). However, no substantial difference was obtained between all groups. This could be as a result of a lot of reasons. Thus, the data for this test was omitted as we keep improving the protocol.
• Yes, it is possible that Amber can lower the levels of MAO-A in the brain as it has shown an ability to decrease dopamine and serotonin levels. At present, we are still working to exploiting several biomarkers and we will keep in mind the use of Monoamine oxidase A assay kit.

Round 2

Reviewer 7 Report

In the present study, Sandra et al. aimed to provide a concise, thorough behavioral evaluation of Amber extract titled: " Behavioral and biochemical evaluation of anti-depressive and oxidative stress-ameliorating effects of amber extract in adult male ICR mice”. In my view, the TST is more trustworthy when it is done in combination with other depression models such as the FST or learned helplessness model, olfactory bulbectomy, and anhedonia models. However, overall, the manuscript looks fine and ready for publication.