ES2 as a Novel Verbascoside-Derived Compound in the Treatment of Cutaneous Wound Healing
, Stefano Manfredini 1 and Giuseppe Valacchi 1,2,3,*Round 1
Reviewer 1 Report
The paper reports on in vitro and in vivo investigation of wound healing properties of the natural compound Verbascoside and its derivative ES2.
The manuscript needs to be improved before acceptance for publication.
Comments:
- Figure 1 a. If the percentage of cell number with respect to controls is reported, this express cell proliferation and not only cell viability.
What does C means? Is this the control? But in the legend, it is stated that results are reported as percent of control. Thus, control should be 100% and not 70%.
How do you explain that 50 µM of both tested compounds reduced cell number?This should be discussed.
- Animal study. How do you explain that at days 6-9 the wound closure in ES2 group was delayed with respect to control and even with respect to Verbascoside? This should be discussed in the paper.
Add more data. What about side effects? Did you do some histology? How do wounds look like after two days treatment?
-English should be improved.
- Line 21 „has wound healing properties in human keratinocytes” Improve this sentence. The compound can induce/accelerate keratinocyte proliferation. Wound healing can only take place in the whole skin in vivo.
- Line 40 “that takes years to be properly completed”. This is not true for all type of wounds.
Author Response
- Figure 1 a. If the percentage of cell number with respect to controls is reported, this express cell proliferation and not only cell viability.
We thank the reviewer for the comment we believe that to talk about "proliferation" would have been more appropriate a BrdU assay since not always the increased cell viability in % means more cells.
What does C means? Is this the control? But in the legend, it is stated that results are reported as percent of control. Thus, control should be 100% and not 70%.
We agree with the reviewer and we have corrected accordingly Fig.1A
How do you explain that 50 µM of both tested compounds reduced cell number?This should be discussed.
Our data shoed that a dose over 50uM is toxic to the cell. it is possible that high doses of the tested substance will affect the redox homeostasis of the cells and consequently induce cell death. This part has been added to the discussion in the revised manuscript.
- Animal study. How do you explain that at days 6-9 the wound closure in ES2 group was delayed with respect to control and even with respect to Verbascoside? This should be discussed in the paper.
We agree with the reviewer that from day 6 to 9 there is a trend of wound closure delay by ES2 but in our study this is only a trend and it not statistically significant. We have mentioned this issue in the revised manuscript.
Add more data. What about side effects? Did you do some histology? How do wounds look like after two days treatment?
Unfortunately in this preliminary study we were not able to collect samples for histology. We plan to perform a more large study aimed to analyse not only the would morphology but also key players in wound healing such as inflammatory and proliferative markers.
-English should be improved.
The manuscript has been corrected by english medical writer
- Line 21 „has wound healing properties in human keratinocytes” Improve this sentence. The compound can induce/accelerate keratinocyte proliferation. Wound healing can only take place in the whole skin in vivo.
we have corrected the text as suggested.
- Line 40 “that takes years to be properly completed”. This is not true for all type of wounds.
thanks for the comments
Reviewer 2 Report
The paper describes a limited number of experiments carried out to find out if the wound healing efficacy of verbascoside (already described in the literature) could be improved for topical application by adding an acyl group to the molecular structure, increasing its lipophilicity.
My problem with this paper is two-fold:
One: I don't see what this has to do with the field of cosmetics. Wound healing is a medical process, no cosmetic product can claim to "improve wound healing". The distinctions between cosmetics and drugs are quite clear from the regulatory point of view. Furthermore, tests on animals involving cosmetic products or ingredients are clearly forbidden in the EU and an increasing number of countries and regions. This aspect also makes the paper unsuitable for a "cosmetic" journal such as Cosmetics.
Two: the paper finally contains very little useful information. The differences in "efficacy" between the verbascoside and the ES2 variant are minute; in each graph one finds at best one or two data points with a * (p<0.05), but all other data points are not significantly different. For ex: Figure 2 c: only at 24h at 10µM do we see a difference. Same for Figure 3, where only on day 2 there is a significant difference noted. By the way, the "wound healing ratio" term is not explained in the text: how is it calculated? Why is this called a "ratio"? And why are the much higher values on days 6, 7, 8 , 9 in Fig. 3 not commented upon? What does this all mean?
The discussion is way too broad and speculative based on these very thin, tenuous, results.
We learn nothing about the mechanisms of action of verbascoside that was not already known, the alleged lipophilicity of ES2 is not measured, only surmised and it does not contribute to any interesting effects. It is even somehow illogical to expect lipophilicity to have a positive effect in a cell culture where no cutaneous barrier has to be overcome.
I do not see how this paper could be improved to be suitable for Cosmetics. Finding out how verbascoside interacts with keratinocytes and with dermal fibroblasts, with 3D skin models (biopsies) at a genetic level, at protein levels, would require a total rethinking of the experiments. Animal tests should be strictly avoided when writing for cosmetic applications.
The text would need, independent of the scientific content (or its absence) a good review by a native English speaking scientist. Nothing shocking, but still too many grammatical and orthographic errors persist in the text.
Reviewer 3 Report
Abstract: edit to provide specific information on the experiments, its results, and conclusions based on it
Methods: Cell culture/cell treatments – state how long the cells were in 2% serum before the treatments, and clarify if the treatments were in 10% serum or 2% serum.
Results: Use different symbols (eg*, #) to differentiate the significant differences (p<0.5) of each (verbascoside or ES2) from control (eg*) and from each other (verbascoside and ES2) (eg #) Cell Viability – the LDH release is a measure of toxicity, not cell viability. Edit Fig 1a Y axis to (% of control), and express Fig 1b data as % of control (not % of triton X100).
Introduction/Discussion: Organize on the specific background to the experiments performed, and its results. Place the antioxidant or anti-inflammatory mechanism as possible inferences, without focusing on it since there is no data in the manuscript for it.
Author Response
Abstract: edit to provide specific information on the experiments, its results, and conclusions based on it
We thanks the reviewer for the good insights and we have now modified the abstract accordingly to the suggestions.
Methods: Cell culture/cell treatments – state how long the cells were in 2% serum before the treatments, and clarify if the treatments were in 10% serum or 2% serum.
The requested info are now added to the revised text.
Results: Use different symbols (eg*, #) to differentiate the significant differences (p<0.5) of each (verbascoside or ES2) from control (eg*) and from each other (verbascoside and ES2) (e.g. #) Cell Viability – the LDH release is a measure of toxicity, not cell viability. Edit Fig 1a Y axis to (% of control), and express Fig 1b data as % of control (not % of triton X100).
thanks for the suggestions but since we always compare ES2 to verbascoside we did not add any further symbol to the figure.
Introduction/Discussion: Organize on the specific background to the experiments performed, and its results. Place the antioxidant or anti-inflammatory mechanism as possible inferences, without focusing on it since there is no data in the manuscript for it.
Thanks for the comment. We would like to mention that discussion session should also take into consideration pathways showed in other work that can be somehow related to the results presented in the study although are not data from the present work. We think that including other similar study that can be associated to the work can stimulate further discussion in future studies.
