Elastase/Collagenase Inhibition Compositions of Citrus unshiu and Its Association with Phenolic Content and Anti-Oxidant Activity

Round 1

Reviewer 1 Report

This work shows Elastase/collagenase inhibition compositions of Citrus unshiu and association with phenolic content and antioxidant activity to identify compounds that may have an application as anti-aging products. This is interesting.

Authors may make some corrections to improve work.

The introduction should show a broader description of the skin aging process. When and why do elastase and collagenase work in the aging process? How important are these on tops in the aging process?

In 2.3 Sample preparation

Is methanol added to a volume of 1.5ml? 

Where you read:

The supernatant was transferred the supernatant to a 1.5 mL tuve and the residue was washed with 0.5 mL 80% (v/v) MeOH.

It should be read:

The supernatant was transferred to a tuve and the residue was washed with 0.5 mL 80% (v/v) MeOH.

Figure 2 shows inhibition of elastase activity and collagenase. Inhibition values greater than 100 are not understood(The most collagenase inhibition showed in the EJF stage (106%)). It would be good to show the inhibition values of elastase activity and collagenase related to control. Give the control a value of 100% elastase activity or collagenase and represent the other measures relating to that value of the control:

Inhibition % = (value of activity del treatment / value of activitydel control) x 100

Finally, I suggest a statistical analysis type: Mann-Whitney U test.

In addition, a statistical analysis could be included to see the differences with the control.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript composed by Kim and co-workers reports investigations on anti-aging potential and antioxidant activity of several developmental stages of Citrus unshiu fruit and tissues. Methanolic citrus extracts have been studied for what concerns their  anti-aging effect, evaluating by in vitro elastase and collagenase inhibition assay, as well as their total phenolic or flavonoid content (TPC and TFC, respectively) by means of DPPH and FRAP assay.  Experiments are well-planned, sufficiently described and making use of consolidated published methods (Refs 13 and 19).

In my opinion the discussion is scarcely detailed. The authors did not give a clear explanation of the results they obtained, but they rather listed a series of other previous works to support or simply argue for the relevant aspects of the studies they described. A more active contribution of the authors would be welcomed, for example, on a potential relationship between the chemical structures of the four candidates used in the collagenase inhibitory assay and their anti-aging effect. Concluding, I agree to publish this manuscript in Applied Sciences journal, but after reviewing of the Discussion section.      

Author Response

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Reviewer 3 Report

This study describes the anti-aging potential of different C. unshiu samples, evaluated in vitro by the anti-elastase and anti-collagenase activity assays, and their composition in candidate compounds analyzed by GC-MS, as well as the total phenolic and flavonoid contents and antioxidant activity. The subject of this study is worthy of investigation, but a major revision is suggested to improve the presented manuscript. Some comments and suggestions are listed below:

- Line 21. Indicate the meaning of HMF.

- The abstract does not discuss the total phenolic analysis. It should be improved accordingly.

- Lines 56-59. The analysis of total phenolics and flavonoids was not mentioned.

- Line 56. Consider rewriting the sentence.

- Subsections 2.6., 2.7. and 2.8. Present the calibration curves obtained for gallic acid and catechin.

- Subsections 2.8. and 2.9. Indicate how the results of the antioxidant activity and cytotoxicity assays were expressed.

- Lines 178-186. The first two sentences should be reviewed. The meaning of the sentence “After elimination of the same compounds from the two extracts…” is not clear; it must be explained. Explain how the four compounds were selected. In addition, it is referred that “Which of these candidate substances has a anti-aging effect was evaluated…”; therefore, the Material and Methods section should be improved according to all tests performed.

- Indicate which control was used in the cytotoxicity assay.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Changes made by the authors satisfy my requirements. The paper can be published after an accurate English editing, since something is still inexact in the form as well as in verbs and terms.

Author Response

Thank you for your comments,

We did check again our manuscript and changed inexact form as well as verbs a,d terms

Kind regards. 

Reviewer 3 Report

The reviewers' suggestions were considered in the revised version of the manuscript.

Author Response

Thank you for your comment