Botanical Origin-Dependent Phytochemical Profiles and Pharmaceutical Properties of Medical-Grade Honeys: Transdermal Delivery and Antibacterial Efficacy in a Wound Fluid Model

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Article entitled "BOTANICAL ORIGIN-DEPENDENT PHYTOCHEMICAL PROFILES AND PHAR-2 MACEUTICAL PROPERTIES OF MEDICAL-GRADE HONEYS: TRANSDERMAL DE-3 LIVERY AND ANTIBACTERIAL EFFICACY IN A WOUND FLUID MODEL" by Nowak A. et al., it covers comprehensively the phytochemical profiles, antioxidant capacity, transdermal penetration, wound-healing potential, and antibacterial efficacy of three commercially available medical-grade honeys (MGHs) of different botanical origins: manuka (MH), chestnut (ChH), and multifloral (MFH), using artificial honey as a control. It is quite well written and has minor revision that must be addressed before its publication : 

The introduction certainly includes relevant references describing the biological mechanisms through which honey-derived products may potentially exert positive effects on the wound healing process. However, regarding the rationale for this study, the evidence on how honey improves wound healing is presented only in qualitative terms. This reduces the scientific rigor of the manuscript. The authors should provide quantitative data on this topic and, ideally, results from non-inferiority studies comparing honey with the gold-standard treatment for wounds.  The methods employed are suitable for evaluating each of the beneficial properties attributed to honey. I suggest improving some sections, perhaps by adding a graphical overview of the entire experiment. The conclusions are presented in a highly relevant and appropriate manner, fully consistent with the findings reported in this study. Given the distinct differences among the three honey types, we need to advance these results to a clinical context. As an extra suggestion, I recommend including quantitative data from previously published studies on the effects of honey in wound healing in the Introduction section to strengthen the scientific justification.

Author Response

Comment: The introduction certainly includes relevant references describing the biological mechanisms through which honey-derived products may potentially exert positive effects on the wound healing process. However, regarding the rationale for this study, the evidence on how honey improves wound healing is presented only in qualitative terms. This reduces the scientific rigor of the manuscript. The authors should provide quantitative data on this topic and, ideally, results from non-inferiority studies comparing honey with the gold-standard treatment for wounds.  The methods employed are suitable for evaluating each of the beneficial properties attributed to honey. I suggest improving some sections, perhaps by adding a graphical overview of the entire experiment. The conclusions are presented in a highly relevant and appropriate manner, fully consistent with the findings reported in this study. Given the distinct differences among the three honey types, we need to advance these results to a clinical context. As an extra suggestion, I recommend including quantitative data from previously published studies on the effects of honey in wound healing in the Introduction section to strengthen the scientific justification.

Reply: Thank you very much for your overall positive evaluation of our manuscript. Based on your comments and suggestions, we added quantitative data regarding the clinical efficacy of honey in wound care in the introduction section. The following paragraph was added:

“The wound-healing efficacy of topically applied honey has been evaluated in several recent meta-analyses [3-5]. An updated meta-analysis of eight randomised controlled trials encompassing 906 patients demonstrated that honey dressings significantly accelerated wound healing time by a mean of 17.13 days and increased the wound healing rate by 18.31% compared to control treatments in the treatment of chronic wounds [4].In obstetric wounds, a meta-analysis of five RCTs including 353 patients found no statistically significant difference in final wound closure between honey and placebo groups, although honey significantly reduced pain intensity during treatment, highlighting that the clinical performance of honey is wound type-dependent and likely influenced by the biological activity of the specific honey product used [5].”

Regarding the graphical overview of the methods used in the manuscript, we would not like to increase the number of Figures. We already reached large numbers of Figures and Tables and some of them were included in the Supplementary material.

Reviewer 2 Report

Comments and Suggestions for Authors

Here are the major comments/suggestions:

1. The study largely reproduces well-established observations that the biological activities of medicinal honeys are strongly dependent on botanical origin. The antioxidant, antibacterial, and wound-healing properties of manuka and other honeys are already extensively documented. Although the assessment of transdermal penetration adds some incremental value, the overall conceptual advance remains challenging.
2. The manuscript does not sufficiently articulate its novelty or clearly distinguish its contribution from prior studies. Please highlight the novelty in the introduction.
3. The role of MGO in manuka honey should be more critically addressed, particularly in relation to wound-healing applications. While elevated MGO levels are linked to strong antibacterial activity, they may also exert cytotoxic effects and promote glycation, which is particularly relevant in diabetic wound environments. A more balanced discussion incorporating both beneficial and potentially adverse effects is required. In addition, the rationale for selecting only three honey types is insufficiently justified.
4. Although MGO is discussed as a key bioactive constituent, it is not quantitatively assessed in this study, creating a disconnect between discussion and experimental evidence.
5. The authors should clarify whether additional phytochemical classes, including peptides and polyamine-derived phenolamides (e.g., spermidine-, putrescine-, and spermine-conjugates reported in pollen and honey), were considered. If excluded, the analytical scope should be explicitly defined. Restricting the analysis primarily to phenolic acids and flavonoids may limit the comprehensiveness of the chemical profiling and weaken the interpretation of structure-activity relationships.
6. The manuscript combined compositional profiling with multiple bioassays without a clearly defined mechanistic hypothesis. Although activity differences are reported, no robust linkage between specific phytochemicals and biological effects is established.

Author Response

Comment 1: The study largely reproduces well-established observations that the biological activities of medicinal honeys are strongly dependent on botanical origin. The antioxidant, antibacterial, and wound-healing properties of manuka and other honeys are already extensively documented. Although the assessment of transdermal penetration adds some incremental value, the overall conceptual advance remains challenging.

Reply: Thank you for your comment. We agree that there is a large body of evidence on the biological properties of different honey types; however, we used gamma-irradiated medical-grade honey and tested its wound-healing activity. Some activities, such as antibacterial activity and the ability to generate hydrogen peroxide, were characterized under a simulated wound environment using artificial wound fluid. To highlight the novelty of our manuscript, we added a paragraph at the end of the introduction section:

„Despite the extensive research on honey bioactivity, several critical knowledge gaps relevant to the clinical application of MGHs remain unaddressed. First, the transdermal penetration of phenolic acids from certified, commercially available MGHs has not been systematically investigated. Second, the antibacterial efficacy of MGHs against wound pathogens has been mostly assessed under ideal in vitro conditions, which do not reflect the complex biochemical environment of infected wounds. The effect of artificial wound fluid exudate on both antibacterial activity and H₂O₂ accumulation in MGHs of different botanical origins has not been comparatively evaluated. Third, while the fatty acid composition of honey is rarely reported, its potential role as an endogenous penetration enhancer, facilitating the transdermal delivery of hydrophilic phenolic compounds, represents a mechanistically novel concept. The present study addresses these gaps by integrating phytochemical profiling with transdermal delivery assessment and wound-environment-adapted antibacterial testing in three commercially registered MGHs.”

Comment 2: The manuscript does not sufficiently articulate its novelty or clearly distinguish its contribution from prior studies. Please highlight the novelty in the introduction.

Reply: Thank you for your comment. Please see the above answer, which addresses the novelty and importance of our work.

Comment 3: The role of MGO in manuka honey should be more critically addressed, particularly in relation to wound-healing applications. While elevated MGO levels are linked to strong antibacterial activity, they may also exert cytotoxic effects and promote glycation, which is particularly relevant in diabetic wound environments. A more balanced discussion incorporating both beneficial and potentially adverse effects is required. In addition, the rationale for selecting only three honey types is insufficiently justified.

Reply: Thank you for your comment and suggestions. We are aware of the detrimental effects of MGO, even in manuka honey, where it significantly decreases the activity of all enzymes and peptides through crosslinking. It is disputable whether high concentrations of MGO in manuka honey can have a negative effect on the regeneration and reepithelialization phase of wound healing. Clinical studies using manuka honey in wound care have not shown any negative effects on human skin cells or their function. However, we added more information about the potentially harmful effects of MGO in manuka honey on wound healing:

"The high MGO content characteristic of MH warrants a more nuanced interpretation in the context of wound healing. MGO is the primary driver of antibacterial activity of MH, but it is also a highly reactive dicarbonyl compound capable of glycating proteins and peptides, forming advanced glycation end-products (AGEs) that may impair cellular function and delay tissue regeneration ([44]). This is particularly relevant in diabetic wound environments, where endogenous MGO accumulation already contributes to glycation-mediated tissue damage. Furthermore, we showed that MGO in MH negatively impacts the enzymatic and antibacterial activities of GOX and defensin-1, respectively [14,15]. In addition, it may exert concentration-dependent cytostatic effects on fibroblasts and keratinocytes [45,46]. The reduced fibroblast migration observed in the scratch assay for MH in the present study may partly reflect such effects at the tested concentration. These considerations do not diminish the clinical value of MH-based MGH, but underscore the importance of concentration optimisation and careful patient selection — particularly in diabetic wound care — when choosing between MGH types”.

The selection of manuka, chestnut, and multifloral honey-based MGHs was based on the current availability of commercially certified medical-grade honey products registered as medical devices on the European market. To date, only a limited number of honey-based products have obtained MGH certification globally, and the three products selected represent the principal botanical types among those commercially available, thereby covering the spectrum of known antibacterial mechanisms — MGO-dependent (manuka), H₂O₂-dependent with high polyphenol content (chestnut), and multifloral as a representative of broadly sourced honey-based wound care products.

Comment 4: Although MGO is discussed as a key bioactive constituent, it is not quantitatively assessed in this study, creating a disconnect between discussion and experimental evidence.

Reply: Thank you for your comment. Indeed, MGO is one of the most important bioactive compounds of manuka honey. Based on the scientific literature, MGO content in blossom and honeydew honeys is very low and negligible compared to that in manuka honey (up to 100-times less). However, the exact concentration of MGO in MGH derived from manuka honey was not determined in our study, which represents a study limitation. We added the following paragraph at the end of the discussion section:

"A limitation of the present study is the absence of quantitative MGO determination in the MH sample. Although MGO is discussed as a key bioactive constituent, its concentration was not directly measured, which creates a disconnect between the mechanistic interpretation and the experimental data. In the present study, a certified commercial product based on MH was used, with a unique manuka factor (UMF) of at least 15+.

Comment 5: The authors should clarify whether additional phytochemical classes, including peptides and polyamine-derived phenolamides (e.g., spermidine-, putrescine-, and spermine-conjugates reported in pollen and honey), were considered. If excluded, the analytical scope should be explicitly defined. Restricting the analysis primarily to phenolic acids and flavonoids may limit the comprehensiveness of the chemical profiling and weaken the interpretation of structure-activity relationships.

Reply: Thank you for your valuable comments. We did not focus on other phytochemicals in MGHs, what can also be considered as a limitation of the present study. We added this limitation at the end of the discussion section:

"The phytochemical profiling in this study was intentionally focused on phenolic acids and flavonoids, as these represent the predominant skin-penetrating bioactive compounds relevant to the transdermal delivery objective of the study. However, honey contains additional bioactive constituents not assessed here, including bee-derived proteins and peptides (e.g., defensin-1, glucose oxidase, apisimin, MRJPs) and polyamines, such as spermidine, putrescine, and spermine conjugates. Despite the negligible polyamine concentration in honey (Vukasinovic et al., 2024), these compounds may contribute to the observed biological activities, and their exclusion limits the comprehensiveness of the chemical profiling. "

Comment 6: The manuscript combined compositional profiling with multiple bioassays without a clearly defined mechanistic hypothesis. Although activity differences are reported, no robust linkage between specific phytochemicals and biological effects is established.

Reply: Thank you for this valuable comment. We added a few statements about our hypothesis in the introduction and discussion sections in order to improve our linkage between the bioactive molecules and biological effects described in the manuscript:

“Specifically, we hypothesised that: (i) the fatty acid composition of MGHs would modulate the transdermal penetration of phenolic acids through disruption of the stratum corneum lipid architecture; (ii) the antibacterial efficacy of MGHs would be maintained or decreased in the wound fluid environment due to interactions between honey constituents and wound exudate components; and (iii) H₂O₂ accumulation capacity in wound fluid would differ significantly among MGH types, reflecting differences in GOX activity and phytochemical modulation."

“Taken together, these findings support our hypothesis that fatty acid composition functions as an endogenous penetration enhancer in MGHs, providing a mechanistic basis for the observed differences in phenolic acid skin bioavailability across botanical types.”

Reviewer 3 Report

Comments and Suggestions for Authors

This study aims to comprehensively characterize the wound-healing potential of three commercially available medicinal-grade honeys (MGHs), with particular emphasis on their antioxidant capacity, the accumulation of selected phytochemicals in the skin, and their skin penetration rates. Furthermore, the antibacterial efficacy of the MGHs against wound pathogens, as well as their ability to generate H₂O₂ in the presence of wound fluid exudate, was investigated. However, several modifications should be made to improve the manuscript, as outlined below:

• In the Introduction, the authors discuss only the benefits of Manuka honey (MH), while the properties and therapeutic potential of Chestnut honey (ChH) and Multifloral honey (MFH) are not sufficiently addressed.
• The main objective and novelty of the study should be clearly stated in the Introduction section.
• The antioxidant activities (DPPH and ABTS assays) of the MGHs should be compared with Trolox or another appropriate positive control.
• The IC₅₀ values for the antioxidant activities are missing and should be included.
• To confirm the identification of fatty acids in the MGHs, the authors should calculate the retention indices (RI).
• The meaning of “CAS” in Table S1 should be clearly defined.
• The Cytotoxicity section should be further developed and discussed in greater detail.
• The authors should present the inhibition zone diameters of the different MGHs prior to conducting the MIC tests.
• The MBC (Minimum Bactericidal Concentration) tests are missing and should be included.
• The discussion section should be strengthened by comparing the obtained results with findings from previous studies.
• The HPLC chromatograms are missing and should be provided, either in the main manuscript or as supplementary material.

Author Response

Comment 1: In the Introduction, the authors discuss only the benefits of Manuka honey (MH), while the properties and therapeutic potential of Chestnut honey (ChH) and Multifloral honey (MFH) are not sufficiently addressed.

Reply: Thank you for your comment, and we fully agree that the Introduction would benefit from a more balanced presentation of all three honey types. However, we respectfully note that this asymmetry reflects the current state of the scientific literature rather than a selective bias in our manuscript. Manuka honey is by far the most extensively studied MGH in clinical settings, whereas published clinical evidence for chestnut- and multifloral-honey-based MGHs is extremely limited, and robust clinical studies remain lacking. We added this information to the Introduction part. Please see our revised manuscript.

Comment 2: The main objective and novelty of the study should be clearly stated in the Introduction section.

Reply: Thank you for the suggestion. The main object, hypothesis, and novelty have been updated in the Introduction.

Comment 3: The antioxidant activities (DPPH and ABTS assays) of the MGHs should be compared with Trolox or another appropriate positive control.

Reply: Thank you for your suggestion. We added Trolox equivalent values to Table 1. Please see our revised manuscript.

Comment 4: The IC₅₀ values for the antioxidant activities are missing and should be included.

Reply: Thank you for the comment. In this study, antioxidant activity was assessed using RSA% and expressed as Trolox equivalents under established experimental conditions, enabling direct comparison of the tested medical-grade honeys. Using RSA% along with Trolox equivalents already provides a quantitative assessment of antioxidant activity and is commonly used to comparatively characterize the antioxidant potential of honeys. Determining IC₅₀ values ​​is typically used in studies designed to assess concentration-dependent antioxidant effects based on a dose-response relationship. In this study, antioxidant activity was intentionally assessed using a single fixed concentration under standardized experimental conditions to enable direct comparison of the free radical scavenging potential of the tested medical-grade honeys, which are directly applied to the skin in this form and concentration. Therefore, RSA% values, along with Trolox equivalents, were considered the most appropriate parameters for the comparative assessment of antioxidant activity in the experimental model used, reflecting real-world application conditions.

Comment 5: To confirm the identification of fatty acids in the MGHs, the authors should calculate the retention indices (RI).

Reply: Thank you for your comment. Retention indices (RI) have now been added to Table S1 to provide additional support for compound identification. Literature RI values obtained from the NIST Chemistry WebBook database have been included. These values correspond to nonpolar capillary columns (DB-5/HP-5 type), which are comparable to the chromatographic conditions used in this study. Additionally, the description of the RI approach has been clarified in the Materials and Methods section, indicating that the concept of linear retention indices follows the methodology of van den Dool and Kratz (1963). Furthermore, the abbreviation “CAS” has now been defined in Table S1 as the Chemical Abstracts Service registry number. 

Comment 6: The meaning of “CAS” in Table S1 should be clearly defined.

Reply: Thank you for your comment. We defined the shortcut in Table S1.

Comment 7: The Cytotoxicity section should be further developed and discussed in greater detail.

Reply: Thank you for your comment. We rewrote the paragraph describing the results from cytotoxicity testing. We also improved the discussion section regarding cytotoxicity results. Please see our revised version of the manuscript.

Comment 8: The authors should present the inhibition zone diameters of the different MGHs prior to conducting the MIC tests.

Reply: Thank you for your suggestion. We respectfully disagree with this suggestion because the radial diffusion assay is not suitable for determining the antibacterial potential/activity of honey, a complex matrix containing bioactive molecules of varying molecular weights. Radial diffusion assay can be used as an alternative first-screening test for unknown honey samples. However, we used medical-grade honeys, selected for their high antibacterial potential. In addition, the results from honey samples testing by radial diffusion assay are often misleading and do not provide an overall view of total honey antibacterial activity.

Comment 9: The MBC (Minimum Bactericidal Concentration) tests are missing and should be included.

Reply: Thank you for your comment. We acknowledge that MBC values would provide additional information on bactericidal activity. However, for honey and honey-based products, the literature is well established that MBC values are either identical to or only marginally higher than MIC values, reflecting the concentration-dependent bactericidal nature of honey. This has been consistently demonstrated in our previous studies and by multiple independent research groups. The determination of MBC for honey is therefore considered to add limited additional discriminatory value beyond MIC in this context.

Comment 10: The discussion section should be strengthened by comparing the obtained results with findings from previous studies.

Reply: Thank you for your comments and suggestions. We have improved our discussion section, also based on other reviewers’ comments. We also added a paragraph dedicated to studying limitations. Please see our revised discussion section in the manuscript.

 Comment 11: The HPLC chromatograms are missing and should be provided, either in the main manuscript or as supplementary material.

Reply: Thank you for your suggestion. We added HPLC chromatographs as a supplementary material. Please see our revised manuscript (Figure S2).

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Here are the minor comments/suggestions:

1. Please correct it as “polyamines and polyamine-derived phenolamides (e.g., spermidine-, putrescine-, and spermine-conjugates reported in pollen and honey).

Author Response

Comment 1: Please correct it as “polyamines and polyamine-derived phenolamides (e.g., spermidine-, putrescine-, and spermine-conjugates reported in pollen and honey).

Reply: Thank you for your comment. We corrected it in the revised manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

All suggested modifications have been incorporated into the manuscript, except for the retention time, which is still missing from Table S1.

Author Response

Comment 1: All suggested modifications have been incorporated into the manuscript, except for the retention time, which is still missing from Table S1.

Reply: Thank you for your comment. We added the retention times to the revised Table S1.