Applications of Beehive Products for Wound Repair and Skin Care

Round 1

Reviewer 1 Report

This article presents a comprehensive overview of bee-derived products, with a particular focus on honey, and their potential applications in wound healing and skincare. However, given the composition of honey is not clearly defined in this manuscript, it is very difficult to judge how useful this review can guide the researchers in this field.

(1) The introduction could benefit from providing a comprehensive backdrop on bee products, along with a clear and precise definition of the composition of honey.

(3) The article is titled "Applications of Beehive Products for Wound Repair and Skin Care," yet a significant portion of the content delves into bee products like propolis and royal jelly, detailing their anti-inflammatory, antibacterial, and antioxidant properties.

(4) To offer more practical insights, a deeper exploration of how specific components within honey's composition impact the wound healing process in different contexts would greatly enrich the content's value for the readers.

(5) The incorporation of well-structured tables and figures could significantly enhance the presentation of key findings and insights.

see comments

Author Response

Dear Editor,

After meticulous consideration of the reviewers’ comments and suggestions, we are now resubmitting a duly revised version.

We wish to thank all the reviewers for contributing to a real improvement of the ms.

Reviewer 1

This article presents a comprehensive overview of bee-derived products, with a particular focus on honey, and their potential applications in wound healing and skincare. However, given the composition of honey is not clearly defined in this manuscript, it is very difficult to judge how useful this review can guide the researchers in this field.

We thank reviewer for comments and suggestions and for the real opportunity to improve the manuscript.

The introduction could benefit from providing a comprehensive backdrop on bee products, along with a clear and precise definition of the composition of honey.

Like physical properties, the chemistry of honey also varies depending upon the geography of the sample. There is no standard scale globally. Depending on the source(s) of nectar, honey can have a variable composition.

However, as indicated in the introduction, generally honey has a content of 80–85% carbohydrates, 15–17% water, 0.3% proteins, 0.2% ashes and minor quantities of amino-acids, phenols, pigments and vitamins (Bogdanov et al., 2008, Miguel et al., 2017). Beside these, other components are also found in minor concentration.

The carbohydrate components of honey contain various types of mono and discccharides. The average concentration of Fructose, Glucose, Sucrose and reducing sugars are 38.38%, 30.31%, 1.31% and 76.65% respectively. Irrespective of the origin or variety of honey, Fructose/Glucose ratio remains the same (i.e. 1.23).

For the sake of precision, we also cited David Ball on “The Chemical Composition of Honey” (J of Chemical Education, 2007) reporting the average composition of a sampling of 490 honey samples from around the United States (https://pubs.acs.org/doi/pdf/10.1021/ed084p1643).

We have also mentioned in the introduction other beehive products, than honey, that we describe later in the ms.

(3) The article is titled "Applications of Beehive Products for Wound Repair and Skin Care," yet a significant portion of the content delves into bee products like propolis and royal jelly, detailing their anti-inflammatory, antibacterial, and antioxidant properties.

Wound healing is a complex process with many interdependent pathophysiological and immunological mediators to restore the cellular integrity of damaged tissue.

Cutaneous wound healing is the repair response to a multitude of pathologies induced by trauma, surgery, and burn leading to the restoration and functionality of the compromised cells.

Many different methods have been developed to treat chronic and acute wounds, as most wounds are susceptible to infection from microbes and are difficult to treat.

However, many antimicrobial agents have become ineffective in wound treatment due to the emergence of multiple drug-resistant bacteria, and failures in current wound treatment methods have been widely reported.

For this reason, new therapies have been sought, one of which is the use of honey as a wound treatment agent.

The rediscovery of honey has recently gained clinical popularity for possible use in wound treatment and regenerative medicine.

Honey is a safe natural substance, effective in the inhibition of bacterial growth and the treatment of a broad range of wound types, including burns, scratches, leg ulcers, amputation, septic and surgical wounds, and wounds in the abdominal wall. Honey comprises a wide variety of active compounds, acting to improve the wound healing process as well as the antibacterial effects.

So, the aim of this review is to summarize recent studies on the wound healing properties of honey, considering also the other positive roles as antimicrobial agent and the immunodulatory ability.

Last but not least, other beehive products, such as propolis and royal jelly, are inserted due their increasing role and interesting as wound repair booster and for skin care.

(4) To offer more practical insights, a deeper exploration of how specific components within honey's composition impact the wound healing process in different contexts would greatly enrich the content's value for the readers.

We thank reviewer for the possibility to better explain the role of different honey’s components on wound healing.

In fact, as mentioned in the section “2. Honey and skin regeneration” the main driving force of honey for wound healing properties is the production of H₂O₂.

We have already demonstrated the crucial role of H₂O₂ as a wound closure booster. Certainly for the antibacterial action, in addition to the H₂O₂, other factors can contribute, such as the acidic pH, the rich presence of sugars and therefore the osmotic pressure, and the presence of peptides (such as defensin-1, as we have demonstrated in Bucekova et al 2017).

However, up to few years ago, no data was available about component(s) (other than antibacterial) of the honey able to induce/accelerate wound healing.

In Martinotti et al. (IJMS 2019), we demonstrated the involvement of H₂O₂ as a main mediator of honey regenerative effects on HaCaT cells, a spontaneously immortalized human keratinocyte line.

We confirmed that this extracellularly released H₂O₂ could pass across the plasma membrane through a specific aquaporin (i.e., AQP3). Once in the cytoplasm, H₂O₂ induces the entry of extracellular Ca²⁺ through TRPM2 and Orai1 channels by a ROS-dependent mechanism.

A huge number of evidence support this model. First, honey failed to elicit a detectable increase in [Ca²⁺]_i in the absence of extracellular Ca²⁺. Second, the Ca²⁺ response to honey was mimicked by 50 µM H₂O₂ and inhibited in a dose-dependent manner by catalase, an H₂O₂ scavenger.

Accordingly, honey samples release H₂O₂ that is produced by the enzyme glucose oxidase and is responsible for the antiseptic effect of honey dressing. Despite the production of H₂O₂ by honey, a harmful oxidizing effect is not observed on skin cells due to the honey polyphenolic component, which can antagonize this pro-oxidant action.

In agreement with this hypothesis, the use of generic fluorescent H₂O₂ indicators revealed local sites of H₂O₂ production within the cytoplasm, including ER, mitochondria and plasma membrane.

Moreover, genetic silencing of AQP3 with a selective RNAi prevented the increase in intracellular

ROS levels and abrogated the Ca²⁺ response to honey. Accordingly, AQP3 has recently been shown to mediate H₂O₂ uptake and accumulation within the cytosolic leaflet of the plasma membrane, thereby generating a signaling nanodomain that could gate adjacent H₂O₂-sensitive channels.

Conversely, AQP3 is not permeable to extracellular Ca²⁺, while there is not report about the recruitment of an intracellular signaling pathway leading to H₂O₂ production upon honey exposure.

We have also performed the confocal microscopy assay with an artificial honey (7.5 g sucrose, 37.5 g maltose, 167.5 g glucose, and 202.5 g fructose in 85 mL sterile water, able to simulate osmotic factors due to the high sugar level in honey) in order to test the effect on [Ca²⁺] dynamics. We did not observe any variations from control condition.

Moreover, in order to evaluate the role of enzymes, such as glucose oxidase present in honey, manuka honey sample were thermally treated by a conventional method, at 90°C for 60 min (Chua et al., International Food Research Journal 2014). We have then performed the confocal microscopy assay with the thermally treated manuka honey in order to test the effect on [Ca²⁺] dynamics. We did not observe any variations from control condition, suggesting the pivotal role of H₂O₂ production for honey-induced wound repair effects.

These data, therefore, strongly suggest that AQP3 mediates H₂O₂ entry into the cytoplasm, supporting the evidence of pivotal role of H₂O₂ as (main) mediator of wound healing properties.

(5) The incorporation of well-structured tables and figures could significantly enhance the presentation of key findings and insights.

We have modified the text of the ms and inserted a new figure (Figure 3).

Reviewer 2 Report

Authors wrote quite interesting draft on honey beehive for wound repairs, however, didn't include reports indicating products. Moreover, some specific comments are below to improve the readability of the draft.

Line no. 8, the highlighted statement is incomplete.

Line 118-122, could you please highlight in which cell the inflammatory  cytokines were tested. 

Also suggested to look for brief mechanism whether honey can affect polarisation of M1-M2 during the process of inflammation investigation and add in the manuscript.

Line no 131-133. at which proportion of honey the wound repair was demonstrated? would be more interesting if reported.

Suggested to include a figure indicating various mechanisms involved in wound repair and then high light the mechanism involve with honey for same.

It would be more interesting to see the summarise antioxidant and antimicrobial results such as MIC/MBC or zone of inhibition in tabular form.

Suggested to work out on conclusions to bring summarise conclusions.

Thanks and Good luck 

Comments for author File: Comments.pdf

Author Response

Dear Editor,

After meticulous consideration of the reviewers’ comments and suggestions, we are now resubmitting a duly revised version. We wish to thank all the reviewers for contributing to a real improvement of the ms.

Authors wrote quite interesting draft on honey beehive for wound repairs, however, didn't include reports indicating products. Moreover, some specific comments are below to improve the readability of the draft.

Line no. 8, the highlighted statement is incomplete.

We have modified the statement.

Line 118-122, could you please highlight in which cell the inflammatory cytokines were tested.

We have modified the statement.

Also suggested to look for brief mechanism whether honey can affect polarisation of M1-M2 during the process of inflammation investigation and add in the manuscript.

We have inserted a statement concerning the polarization of M1-M2. Very few data are available on the honey ability to induce polarization of macrophages on M1-M2 during inflammation process. It was showed that there is not a specific modulation on the expression of gene markers of macrophage polarization with the honey treatment.

Line no 131-133. at which proportion of honey the wound repair was demonstrated? would be more interesting if reported.

We have reported the concentration of honey utilized.

In fact, calcein-AM assay data showed similar, low cytoxicity levels for all honeys tested. Based on these data, in subsequent experiments we used honey doses of 0.1% (v/v), a value that is below the EC₀₅ of each honey, and has been already used in an analogous study on keratinocytes.

Suggested to include a figure indicating various mechanisms involved in wound repair and then high light the mechanism involve with honey for same.

We have inserted a new figure (Figure 3) to describe the effects of honey on the different phases of wound healing.

It would be more interesting to see the summarise antioxidant and antimicrobial results such as MIC/MBC or zone of inhibition in tabular form.

We have summarized the main effects of honey as antimicrobial agents, with particular attention to manuka honey.

In fact, up to now, there are very few studies investigating traditional antimicrobial potential of honey from different countries and comparison among honey are also difficult due to different composition of honey samples.

Suggested to work out on conclusions to bring summarize conclusions.

We have modified the conclusion section, inserting also a new figure (Figure 3).

Thanks and Good luck

We hope for a positive acceptance of our ms.

Round 2

Reviewer 1 Report

The original manuscript has undergone a revision aimed at enhancing the article's clarity. However, the revised version still presents certain issues.

(1) The supplementary content introduces chemical components tied to honey's biological functions, such as flavonoids, phenolic compounds, and defensin-1. Regrettably, there is a lack of pertinent literature references. Notably, the emphasis remains solely on the antibacterial function, with a notable absence of discussion on honey components pivotal for anti-inflammatory, anti-oxidative, and wound healing functions.

(2) The supplementary sections fail to align cohesively with the article's core theme. Instances like lines 156-161 appear disconnected from the article's context. Moreover, various supplementary details, including lines 67-73, 94-95, and 170, contribute little to the overall article composition, thereby muddling its logical progression.

(3) Although a concluding image has been incorporated, its specific details and significance remain undisclosed.

see comments

Author Response

The original manuscript has undergone a revision aimed at enhancing the article's clarity. However, the revised version still presents certain issues.

(1) The supplementary content introduces chemical components tied to honey's biological functions, such as flavonoids, phenolic compounds, and defensin-1. Regrettably, there is a lack of pertinent literature references. Notably, the emphasis remains solely on the antibacterial function, with a notable absence of discussion on honey components pivotal for anti-inflammatory, anti-oxidative, and wound healing functions.

We thank reviewer for the opportunity to clarify these points. For the wound healing properties of honey, we have extensively discussed in the section 2 “Honey and skin regeneration”, the pivotal role played by H₂O₂. We have cited the literature supporting this statement as well as Figure 2 depicted the molecular mechanism of honey action on keratinocytes as characterized in Martinotti et al. [33].

For the antibacterial activities, we discussed from line 74 to 113, paying attention to components (such as H₂O₂, sugar content, osmolarity, pH, defensin-1) recognized to contribute to honey antibacterial effects.

Furthermore, Honey has been suggested as an immune-modulatory agent with dual role: (1) anti-inflammatory activities through downregulating the inflammatory transcription factors (NF-κB and MAPK) and/or suppressing the production of pro-inflammatory cytokines, and (2) stimulate the production of inflammatory mediators such as prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2). However, there is no precise information on components of honey that may primarily play this role. Surely it is the set of components, already present in honey, which also carries out this activity.

Honey is a natural ready-to-eat product rich in flavonoids. Some authors (please see Silva et al, Food Research International 2021) proposed that flavonoids mitigate inflammatory processes, and thus it could currently support studies of anti-inflammatory potential of honeys. Flavonoids found in honey have demonstrated antioxidant properties and ability to inhibit pro-inflammatory enzymes such as COX, LOX, iNOS, and pro-inflammatory mediators, including nitric oxide, cytokines and chemokines. Transcriptional factors such as NF-κB are also modulated by flavonoids, controlling the expression of several inflammatory mediators.

However, given the relationship between flavonoid structure and the anti-inflammatory activity, other minor compounds present in honey can also play an important role, even in low concentrations.

We have briefly inserted this information on ms, line 150-152.

(2) The supplementary sections fail to align cohesively with the article's core theme. Instances like lines 156-161 appear disconnected from the article's context. Moreover, various supplementary details, including lines 67-73, 94-95, and 170, contribute little to the overall article composition, thereby muddling its logical progression.

We thank reviewer for the opportunity to clarify these points.

In particular, statements in line 156-161 were suggested by reviewer 2 that asked us to discuss and add in the manuscript mechanism whether honey can affect polarisation of M1-M2 during the process of inflammation investigation.

Moreover, the details concerning the cell line (in 151-152 line) were suggested by reviewer 2 (“could you please highlight in which cell the inflammatory cytokines were tested”).

For the statement on line 67-73, we replied to reviewer that in first turn of revision asked us to provide “a clear and precise definition of the composition of honey”. So, to support the fact that is not possible to define a precise composition of a honey sample, we provided information and data coming from a large survey on honey from US demonstrating this honey’ composition variations.

On line 94-95, we replied to reviewer 1 and 2 questions. In particular, reviewer 2 asked us to describe antimicrobial properties of honey.

(3) Although a concluding image has been incorporated, its specific details and significance remain undisclosed.

Figure 3 has been inserted by suggestion of reviewer 2, showing the different roles of honey during wound healing and in particular during the different phases of wound closure.

Reviewer 2 Report

The authors have reflected all the suggestion, and I recommend to editors for consideration of manuscript in present form for publication 

Author Response

The authors have reflected all the suggestion, and I recommend to editors for consideration of manuscript in present form for publication.

We thank reviewer for the suggestions that really improved the ms.

Round 3

Reviewer 1 Report

The authors have successfully addressed the comments.