Skin Antiaging Effects of the Fermented Outer Layers of Leaf Skin of Aloe barbadensis Miller Associated with the Enhancement of Mitochondrial Activities of UV_b-Irradiated Human Skin Fibroblasts

Round 1

Reviewer 1 Report

The authors describe the antiaging effects of fermented outer layers of aloe on human fibroblasts, which may be related to the enhancement of mitochondrial activity after irradiation of cells with UVb radiation. I have the following comments regarding the manuscript that will improve its quality.

1. Authors should not use the colloquial expression "aloe leaf skin" but "outer layers of aloe leaf" or "aloe leaf outer layers". The authors should specify which layers are included in the outer surface of the aloe leaf used.
2. Abstract. “The fermented extract (AF) increased the activities of mitochondrial reductase and the cytochrome complex II system by significantly reducing reactive oxygen species (ROS) production, even under UVb stress conditions, and also increased DPPH free radical scavenging activities compared with the hot water extract of aloe leaf skin (AW) and quercetin itself.”

“complex II” instead of “cytochrome complex II system”

“and significantly reduced reactive oxygen species (ROS) production“ instead of “by significantly reducing reactive oxygen species (ROS) production”

This one sentence indicates that the authors make semantic mistakes.

3. Fig. 1. Parts b) and c) should be of the same scale. Please mark the quercetin peak.
4. The authors measure overall mitochondrial metabolic activity (MTT assay) and complex II activity. To interpret the results correctly, the activity of complexes I and III, which are also important in aging and are mainly responsible for the mitochondrial production of ROS, must also be examined.
5. The authors do not explain whether the 24-hour incubation of fibroblasts with aloe extracts leads to changes in mitochondrial biogenesis (the number of mitochondria). Without it, it is impossible to correctly interpret the results obtained. Therefore, the authors should measure the level of mitochondrial proteins.
6. The authors measure cellular ROS production. It is also very important to measure mitochondrial ROS production to conclude about the influence of the aloe extracts on fibroblasts via the mitochondria.
7. In the discussion, the authors should discuss in more detail the potential impact of aloe extracts on mitochondria (see recent review on flavonoids effect on mitochondria, Kicinska 2020, Molecules).

Author Response

Dear Reviewer 1,

Thank you for your generous reviewing our manuscript. Please find an attached file of 'Responses to reviewers' comments' and a revised manuscript that has been reflected the reviewer's comments.

Truly yours,

H. LEE

Author Response File: Author Response.pdf

Reviewer 2 Report

The study seems well-performed and the topic is innovative.
I raise the following major concerns:

1. The Results and Discussion sections lack interpretation and description of the significance of the findings in light of what was already known about this topic. The findings and their implications should be discussed in the broadest context possible and limitations of the work should be highlighted.
2. Due to long and complex sections of results and discussion, the conclusions should be revised and written in points. It would be clearer to the readers.

3. The bibliography seems to be out of date. Twenty-six out of 49 publications are older than 10 years, and 10 out of those 26 are more than 20 years old. I think there is a lot of research in this area significantly more up to date.

         4. There are small mistakes like: in verse 346 there should be „by-  products”, verse 262 should be rewritten, verse 584 – should be „2015” instead of „215”, in verse 354 a control value should be given.

Author Response

Dear Reviewer 2,

We, all of authors thank you so much for your kind comments on our manuscript. Please find an attached file of 'Responses to reviewers' comments' and a revised manuscript that has been reflected the reviewer's comments.

Truly yours,

H. LEE

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript of Hyeon Woo Lee reports Skin antiaging effects of the fermented outer layers of leaf skin of Aloe barbadensis miller associated with the enhancement of mitochondrial activities of uvb-irradiated human skin fibroblasts. The data obtained show that that fermentation of the skin of aloe leaves increases the activation of mitochondria and inhibits the photo-aging of UVb-irradiated skin fibroblasts.

Although the manuscript is interesting, various changes need to be made to the manuscript for publication.

Revision:

1- Some references in the methods are not appropriate refer to authors who have not really developed the methodologies described. For example, the MTT assay. Please enter the right references;

2- The authors must provide information on the linearity of the calibration obtained for the quantization of quercetin;

3- Graphic c in Fig. 8 not giving statistical information. Was the experiment done only once? Please add more information; the error bar is not visible.

Author Response

Dear Reviewer,

All of authors really thank you for your efforts of reviewing our manuscript. Please find two attached files of a revised manuscript and Responses to reviewers' comment.

Sincerely yours,

H. LEE 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors addressed some of the comments. They explained why they did not see the need to perform the required additional functional tests (measurement of the activity of complexes I and III and measurement of mitochondrial ROS). However, the authors still do not explain whether the 24-hour incubation of fibroblasts with aloe extracts leads to changes in mitochondrial biogenesis (the number of mitochondria). In my opinion, measuring the number of mitochondria by detecting the mitochondrial protein (VDAC or COX) is necessary for the correct interpretation of the results. The authors should perform at least this immunodetection.

Author Response

Dear Reviewer 1,

Again, thank you for your kind comments on our manuscript. Please find attached files of Responses to reviewers' comments and Re-revised manuscript.

Sincerely yours,

H. LEE

Author Response File: Author Response.pdf

Reviewer 3 Report

Dear Authors,
considering the changes made in revised manuscript, the manuscript can be accepted for publication if the Editor agree.