Promising Support Coming from Nature: Antioxidant and Anti-Inflammatory Potential of Castanea sativa Wood Distillate on Skin Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this study, the authors explored the potential applicability of natural product Castanea sativa wood distillate (WD) in in medical human care. They investigated the effects of WD on inflammation, oxidative stress, cell viability, endothelial permeability, and wound healing in various skin cell models. The presented data supporting anti-inflammatory and anti-oxidative properties of WD. This is an interesting study and opening up new direction to consider WD in future research. So, this article can be accepted for publication.    

1.     The Caspase 3 activity increased in all the cells as concentration of WD increased. However, in HaCaT cells decreased at 0.33% WD. What could be the reason?

2.     Quality of images in Fig. 1I is rather poor.

3.     For quick understanding and interest of readers, please convert Table 1 into a Figure.

 

Author Response

Comments and Suggestions for Authors

In this study, the authors explored the potential applicability of natural product Castanea sativa wood distillate (WD) in in medical human care. They investigated the effects of WD on inflammation, oxidative stress, cell viability, endothelial permeability, and wound healing in various skin cell models. The presented data supporting anti-inflammatory and anti-oxidative properties of WD. This is an interesting study and opening up new direction to consider WD in future research. So, this article can be accepted for publication.   

We greatly appreciate the interest and comments of the reviewer, aimed at enhancing the scientific work presented on the wood distillate. 

1. The Caspase 3 activity increased in all the cells as concentration of WD increased. However, in HaCaT cells decreased at 0.33% WD. What could be the reason?

We thank the reviewer for this interesting question. Indeed, cleaved caspase 3 is slightly reduced at a concentration of 0.33% compared with the previous concentration 0.2% of WD. However, this concentration shows a significantly high increase when compared to control condition and to the lower WD concentrations. Each cell type has a different sensitivity to the distillate, as demonstrated in both this and the previous paper (Filippelli 2021 [17]), so it could be assumed that in HaCaT there is no perfect dose response course, or alternatively, the higher concentration triggers other molecular mechanisms involved in cell death that occur parallel to caspase cleavage 3 (for example: Necrosis, Caspase 8 mediated pathways and Caspase independent mechanisms: https://www.frontiersin.org/journals/oncology/articles/10.3389/fonc.2020.616952/full; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9106709/)

2. Quality of images in Fig. 1I is rather poor.

We thank the reviewer for this suggestion and apologize for the quality of the images. Unfortunately, cleaved caspase is a particularly complex marker to be detected in a clear way. However, we have increased the quality of the figures, in particular we have improved the image quality of the Comet assay in Figure 1 I, and cleaved caspases in Figure 1 A and C.

3. For quick understanding and interest of readers, please convert Table 1 into a Figure.

We appreciate this suggestion, allowing to value the data obtained. The complete panels of the immunofluorescence analysis for COX-2, mPGES-1, ICAM and VCAM are already listed in the supplementary materials section (figure S2,3). However, we have added to the table (now Table 2) indicators/arrows that graphically represent the positive or negative modulation of these markers, in relation to the control condition and the condition of induced inflammation (exposure to MIX).

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors

This is an interesting and well written article.

1.      You mention the crude composition of WD in table 2. You also mention that the complete composition is reported in Celletti et al.[ref20]. Is this a study by your group on the same composition? If yes and if the composition refers to the same ingredient please mention it further in the discussion.

WD can vary a lot in individual ingredients and for a concrete conclusion on its bioactivity, a correlation between composition and efficacy should be made. In line 386 you state “The richness in tannins, phenols, and polyphenols in WD suggested potential..” In table 2 one cannot see tannins, and one can notice a rather poor concentration in phenols. Again if reference 20 corresponds to the same material please use some data in the discussion.

If not, the thorough analysis should be made in order to increase the impact of the work. Since you mention – line 373

“In the whole the presented work aims to highlight the potential

applicability of this natural product in medical human care and the need of deeply investigate the promising support coming from bio-derivates in new field of applicability, improving the sustainability of bio-pharma industry” a potential pharma application in an industrial level, the exact chemical analysis of the distillate is essential.

Also the first step to an optimized formulation “A deeper investigation and an optimized formulation of Castanea sativa”  is beginning by standardization of the WD composition.

2.      Can you explain the choice of concentrations in 3.1? How do you go from 0.04 to 0.07 and then 0.2?

Comments on the Quality of English Language

Please revise for minor mistakes

Author Response

Comments and Suggestions for Authors

Dear authors

This is an interesting and well written article.

We greatly appreciate the opinion, interest and comments of the reviewer, aimed to value the scientific data presented on the wood distillate. 

 

1. You mention the crude composition of WD in table 2. You also mention that the complete composition is reported in Celletti et al.[ref20]. Is this a study by your group on the same composition? If yes and if the composition refers to the same ingredient please mention it further in the discussion.

WD can vary a lot in individual ingredients and for a concrete conclusion on its bioactivity, a correlation between composition and efficacy should be made. In line 386 you state “The richness in tannins, phenols, and polyphenols in WD suggested potential..” In table 2 one cannot see tannins, and one can notice a rather poor concentration in phenols. Again if reference 20 corresponds to the same material please use some data in the discussion.

If not, the thorough analysis should be made in order to increase the impact of the work. Since you mention – line 373 “In the whole the presented work aims to highlight the potential

applicability of this natural product in medical human care and the need of deeply investigate the promising support coming from bio-derivates in new field of applicability, improving the sustainability of bio-pharma industry” a potential pharma application in an industrial level, the exact chemical analysis of the distillate is essential.

Also the first step to an optimized formulation “A deeper investigation and an optimized formulation of Castanea sativa”  is beginning by standardization of the WD composition.

The product used in this and the previous paper [ref. 17] is the same of the paper by Celletti and co-workers. We believe that the positive effects are related to phenols, polyphenols and tannins as reported for other similar products reported in the literature (For example: Bark, Flower, Inner Skin, Kernel and Leaf Extracts of Castanea Crenata doi:10.3390/antiox6020031; Panax ginseng doi: 10.1016/j.jgr.2017.11.004. Centella Asiatica Extract, Aqueous Extract of Aloe Vera, Fraxinus extract and distillate, Wild Bitter Melon Extract: doi:10.1155/2021/6671129. And others: doi:10.3390/antiox9070573. doi:10.3390/molecules21050559. doi:10.1186/s43094-021-00202-w. doi:10.2174/1389201019666171219105920. )

 While phenols and polyphenols levels are reported in Celletti et al, and again reported in table 1, the content of tannins is described by the vendor in the product composition (0.13%). To determine exactly the tannin species in a complex matrix as WD remains an unresolved question ( https://doi.org/10.1111/j.1365-2435.2010.01826.x). More difficult will be the recognition of the (multiple) molecular species responsible for the biological responses described in our paper. This remains a big challenge which only further extensive research will reveal.

On the other hand, the physicochemical characteristics evaluated in previous studies (Celletti) are consistent with those reported by the manufacturer (Maresca). Therefore, the composition of the WD is standardized, and the levels of potentially active compounds remain stable over time. Indeed, the producer company makes blends, which it analyzes to ensure compositional homogeneity. The producer company makes blends, which it analyzes to ensure compositional homogeneity. These characteristics are guaranteed by the company that deals with the distillate of wood and that performs periodic internal analysis to ensure the stability and standardization of the product, necessary for other uses in the agrifood company where WD is already on the market.

2. Can you explain the choice of concentrations in 3.1? How do you go from 0.04 to 0.07 and then 0.2?

The concentrations chosen and tested in this work are consistent with the concentrations of our previous study [ref. 17], on their turn based on the concentrations suggested for the different uses of the WD in agriculture, from 0.04 to 0.5 % (corresponding to 1:2800 - 1:200 dilutions). The analysis started as a study of the effects on operators whose tissues might have occasional exposure to the wood distillate during its use. In fact, they correspond to the concentration range suggested by the datasheet of WD for different uses. The concentrations from 0.05% to 0.07% correspond to open field cultivation, tree cultivation, horticulture, flower growing, seed germination, indoor plant treatment, respectively. From this we further diluted by a factor of 2 to evaluate even lower concentrations [0.04% and 0.02%].

 Comments on the Quality of English Language

Please revise for minor mistakes

We thank the reviewer for the careful analysis of the manuscript, we have performed an extensive and detailed revision of the English.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for the answers.

Please make it clearer in the text that the composition is similar to Celletti et al.[ref20] and provide a more thorough composition overview

Comments on the Quality of English Language

Please make a final revision

Author Response

Please make it clearer in the text that the composition is similar to Celletti et al.[ref20] and provide a more thorough composition overview.

Thank you for your suggestions and review. We have included the extensive profiling of WD in the "Materials and Methods" section, instead of the summarized composition, and we have highlighted the components of interest, such as tannins, phenols, and polyphenols, in the discussion. We have also emphasized that the WD analyzed by Celletti is produced by the same company using a standard procedure, and that the company guarantees the stability of the WD composition over time.

We revised English again to satisfy the requirement of the reviewer