Mushroom-Derived Compounds as Inhibitors of Advanced Glycation End-Products

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

the manuscript "Mushroom-Derived Compounds as Inhibitors of Advanced Glycation End-Products" (applsci-4077707) by the authors: Filip Šupljika , Monika Kovačević , Mojca Čakić Semenčić, is related to the important general fields of fundamental biology and physiology, biotechnology and large-scale cultivation, in particular - inhibitors of advanced glycation end-products (AGEs). It is aimed as the following: “to summarize the research on the AGE-inhibitory effect of mushrooms and their extracts and to highlight their potential as a rich source of natural compounds capable of inhibiting the formation of AGEs, thus offering therapeutic promise for the treatment of AGEs-related diseases, especially diabetes and age-related diseases”.

In general, I do not doubt the technical quality of the work and feel that there is a sufficient impact on a broader readership, but there are not enough data. This topic is in the frame of the journal scopes. It is important and positive that the subject matter has been treated in depth. The manuscript is relevant for the field, and clearly written. All the cited references are relevant. This manuscript is scientifically sound and it design is appropriate to test the author’s hypothesis. Positive sites of the manuscript are the following. An analysis of a large number of existing studies led the authors to conclude that several mushroom species are a source of natural compounds capable of inhibiting AGE formation. Six mushroom species in particular (Lignosus rhinocerus, Ganoderma capense, Auricularia auricula, Pleurotus ostreatus, Boletus snicus, and Inonotus obliquus) are particularly promising, demonstrating pronounced inhibitory activity in vitro. The authors attribute this activity to the antioxidant effects of polysaccharides and phenolic compounds found in high concentrations in these mushrooms. The authors briefly discuss the most likely mechanisms of action of polysaccharides and phenolic compounds but do not provide schematics of the actual chemical processes or specific reactions. Numerous in vitro studies provide reliable evidence of the potential of these compounds, but further in vivo studies, and ultimately, clinical trials, are needed to confirm their therapeutic efficacy. This review provides valuable material for identifying avenues for further research into the potential of these mushrooms as a valuable resource for the development of safe and effective natural AGE inhibitors. Mushroom extracts can serve as the basis for medicinal products for the prevention and treatment of a wide range of chronic diseases associated with aging and metabolic disorders.

There are following comments on this manuscript:

1. The part “Featured Application” (page 1, line 12, left). It is important to provide abbreviation of the “AGEs” (as “advanced glycation end-products”) in the part “Featured Application”
2. The part “Abstract” (page 1). It is important to present more details to prove the authors’ ideas concerning “mushroom-derived compounds as inhibitors advanced glycation end-products” in the part “Abstract”. It will be reasonable to exchange the word “sugars” (page 1, line 37) to the word “carbohydrates” which is more precise nomenclature from the chemical point of view.
3. The part 2. “Advanced glycation end-products (AGEs) and inhibition strategies” (page 4, since page 91). It is important to present the basis and particular details concerning AGEs’ classification. In addition, it will be reasonable to exchange the words “chemical characteristics” to the word “chemical structures” in the title of the Figure 2. “Classification of advanced glycation end products according to their chemical characteristics” (page 4, lines 130-131).
4. The part 4. “Specific mushroom species and their AGE-inhibiting properties” (page 6, since page 212). It is important to present the particular details concerning “Active Fraction/ Compound(s)” in such columns in the Table 1. “Antiglycation activities of mushroom species” (page 8, line 319) , as well as in the text. For example, concerning “active fraction or specific compounds” - about “…the GC70 fraction of these polysaccharides…” on the page 6 (line 230); about – “…an anti-oxidative proteoglycan named FYGL…” (lines 237-238); etc.
5. Part 5. “Mechanisms of Action of Mushroom-Derived AGE Inhibitors” (page 6, starting on page 212). In this section, the authors attempted to systematize and discuss the mechanisms of action of mushroom-derived AGE inhibitors. However, several questions arise. From the brief section 5.1. “Antioxidant Activity”, it is unclear why: 1) “polysaccharides from Ganoderma capense exhibit the ability to neutralize both DPPH and hydroxyl radicals [39]”; 2) “The antiglycation activity of Pholiota nameko is also due to the antioxidant activity of its constituent polysaccharides [46].” It is well known that polysaccharides have a significantly lower antioxidant activity than proteins, which are not mentioned at all in this section. If the authors would like to convince review readers of their ideas based on the data presented in references [39,46, etc.], they should show diagrams of actual chemical processes or, even better - specific reactions, and discuss all stages of these mechanisms. This should be the central part of section 5.1. “Antioxidant activity”, even if this section is significantly expanded, since this section should be the central (main) section of the entire review.

Similar requirements should be imposed on each subsequent section from 5.2 to 5.8.

6. I consider it insufficient to present all of these points in a single Figure 4 (“Main mechanisms of action of mushroom-derived AGE inhibitors”). Moreover, this figure contains significant inaccuracies. For example, at the end of the first line, a schematic diagram of the protein structure is shown with the –NH₂ functional group, which, for some reason, is covalently bound to the inhibitor. In this case, it should appear as –NH– or –NH₂⁺–.

At the second line, a free radical is shown as (HO^•). But there are following “reactive oxygen species” (ROS) known: hydroxyl radical (HO^•-), superoxide radical (O₂^•-), hydroperoxyl radical (HOO^•-), lipid radical (L^•), lipid peroxyl radical (LOO^•), peroxyl radical (ROO^•), lipid alkoxyl radical (LO^•), nitrogen dioxide radical (NO₂^•), nitric oxide radical (NO^•), thiyl radical (RS^•). These (ROS) species, as can be seen, have different chemical structures and, accordingly, participate in different reactions leading to different chemical pathways, which would be useful to discuss “in light of the mechanisms” of action of mushroom-derived AGE inhibitors.

In conclusion:  all these points 1-6 (mentioned above) are important to fulfill before the publication of this manuscript.  

Comments for author File: Comments.pdf

Author Response

Comment: 

The part “Featured Application” (page 1, line 12, left). It is important to provide abbreviation of the “AGEs” (as “advanced glycation end-products”) in the part “Featured Application”

Response:

We agree. The "Featured Application" section on page 1 has been revised to include the full name followed by the abbreviation: "advanced glycation end-products (AGEs)".

Comment:

 The part “Abstract” (page 1). It is important to present more details to prove the authors’ ideas concerning “mushroom-derived compounds as inhibitors advanced glycation end-products” in the part “Abstract”. It will be reasonable to exchange the word “sugars” (page 1, line 37) to the word “carbohydrates” which is more precise nomenclature from the chemical point of view.

Response: 

Thank you for this suggestion. We have revised the Abstract to include the specific details you requested, such as potent mushroom species , bioactive fractions (FYGL, polysaccharides), and specific chemical mechanisms . Following your advice, the term "sugars" was replaced with "carbohydrates". Please note that, in accordance with the journal's editorial guidelines, the Abstract is strictly limited to 200 words. We have carefully balanced the need for additional technical data with this word count constraint to ensure the abstract remains both informative and compliant with the journal's requirements.

Comment:

The part 2. “Advanced glycation end-products (AGEs) and inhibition strategies” (page 4, since page 91). It is important to present the basis and particular details concerning AGEs’ classification. In addition, it will be reasonable to exchange the words “chemical characteristics” to the word “chemical structures” in the title of the Figure 2. “Classification of advanced glycation end products according to their chemical characteristics” (page 4, lines 130-131).

Response: 

Thank you for this suggestion. We have revised Section 3 (former 2) to include a more detailed explanation of the classification criteria for AGEs. We now explicitly discuss the categories based on fluorescence and cross-linking capacity, including examples.

Comment: 

The part 4. “Specific mushroom species and their AGE-inhibiting properties” (page 6, since page 212). It is important to present the particular details concerning “Active Fraction/ Compound(s)” in such columns in the Table 1. “Antiglycation activities of mushroom species” (page 8, line 319) , as well as in the text. For example, concerning “active fraction or specific compounds” - about “…the GC70 fraction of these polysaccharides…” on the page 6 (line 230); about – “…an anti-oxidative proteoglycan named FYGL…” (lines 237-238); etc.

Response:

We have revised Section 4 and Table 1 to provide more technical specificity regarding the active fractions.

Comment: 

Part 5. “Mechanisms of Action of Mushroom-Derived AGE Inhibitors” (page 6, starting on page 212). In this section, the authors attempted to systematize and discuss the mechanisms of action of mushroom-derived AGE inhibitors. However, several questions arise. From the brief section 5.1. “Antioxidant Activity”, it is unclear why: 1) “polysaccharides from Ganoderma capense exhibit the ability to neutralize both DPPH and hydroxyl radicals [39]”; 2) “The antiglycation activity of Pholiota nameko is also due to the antioxidant activity of its constituent polysaccharides [46].” It is well known that polysaccharides have a significantly lower antioxidant activity than proteins, which are not mentioned at all in this section. If the authors would like to convince review readers of their ideas based on the data presented in references [39,46, etc.], they should show diagrams of actual chemical processes or, even better - specific reactions, and discuss all stages of these mechanisms. This should be the central part of section 5.1. “Antioxidant activity”, even if this section is significantly expanded, since this section should be the central (main) section of the entire review.

Similar requirements should be imposed on each subsequent section from 5.2 to 5.8.

Response: We sincerely thank the Reviewer for the constructive critique regarding the mechanistic depth of our discussion. We fully agree that providing a clear chemical basis for the observed biological activities is essential for a high-quality review. Section 6 (former 5) has been significantly enhanced and completely restructured to serve as the central mechanistic core of the paper. We have moved beyond general descriptions to provide a rigorous discussion of actual chemical processes. Specifically, we have integrated formal chemical equations to illustrate the Hydrogen Atom Transfer (HAT) mechanism, explaining how polysaccharides and phenolics stabilize free radicals. Furthermore, we have clarified the role of nucleophilic attack on electrophilic carbonyl carbons, providing the specific reaction pathways for the trapping of reactive dicarbonyl species (RCS). Addressing the Reviewer’s important point about proteins, we have now explicitly included protein-based and enzymatic mechanisms, such as the Glyoxalase I pathway, and discussed the structural protection offered by proteoglycans. We also expanded on the role of uronic acid content and molecular weight, explaining how these structural features facilitate metal chelation and stabilize the glycation cascade. By distinguishing between direct radical scavenging and indirect antioxidant effects, and by providing specific chemical reactions for each stage, we believe the revised Section 6 now offers the comprehensive mechanistic clarity requested.

Comment: I consider it insufficient to present all of these points in a single Figure 4 (“Main mechanisms of action of mushroom-derived AGE inhibitors”). Moreover, this figure contains significant inaccuracies. For example, at the end of the first line, a schematic diagram of the protein structure is shown with the –NH₂ functional group, which, for some reason, is covalently bound to the inhibitor. In this case, it should appear as –NH– or –NH₂⁺–.

Response: 

We acknowledge the Reviewer’s rigorous assessment and have corrected the technical inaccuracies in our manuscript. Specifically, we have updated our schematics to correctly represent the covalent bonding to the protein amino group as –NH– (or –NH₂⁺–), ensuring chemical valency is respected. Furthermore, we have replaced the generic "ROS" terminology with specific species. Figure 4 has been deconstructed into three detailed panels (4a, 4b, and 4c) to more precisely illustrate radical scavenging, carbonyl trapping, and signaling modulation.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

My main comment on this review concerns the lack of a methodology that would support the selection of publications and the synthesis of the review. It is necessary to state which databases were searched, in what time period, and who conducted the review. It may be worth introducing a PRISMA algorithm/flow diagram to improve the transparency and clarity of the review. At present, this is a major weakness for me and it does not allow me to accept the manuscript in its current form.

I would also note that, for a review, using only 53 references is relatively few.

The manuscript must include data on the bioavailability of the compounds, as this will determine their usefulness and therapeutic effect.

Please clearly distinguish which studies discussed crude extracts and which discussed isolated single compounds. This is important; please do not compare these directly unless a given study includes standardization.

In Table 1, please add the experimental model (this is essential) and also indicate the final outcome (a quantitative indicator of potential efficacy).

In Section 5.6, you discuss additivity or synergy. Are you referring to the same targets/mechanisms of action, or different ones?

Are there any available RCT studies that confirm the claims? It may be worth highlighting them. In my view, the review should be structured differently, with a clear separation of in vitro studies, animal studies, and, if available, human studies.

The authors cite a lot of older literature. In my opinion, given the progress in the AGEs field, this is difficult to understand.

Please discuss safety issues in more detail; this is important for practice.

Please cite original studies rather than other reviews, especially when discussing AGEs. Reference no. 26 (Bakris et al.) should be replaced with a more recent one; it is 20 years old and there has been significant progress.

Detailed comments:

1. Please improve the abstract. In its current form, it does not say much about the content of the paper. Please specify the focus and the main mechanisms.
2. In line 52 and then in line 59 you cite many references. In my opinion, after each effect listed in line 58, such a citation should appear, rather than presenting them collectively at the end.
3. Please revise the aim of the study and the conclusion/summary. A summary is not an aim in itself; reviews are expected to address a specific issue in a clear, synthetic, and evidence-based manner.
4. In Chapter 2, in my opinion a schematic visualization of the targets for the inhibitors would be crucial for understanding the text. Please also indicate which compounds are key, which are used in practice, and which are used as markers.
5. In Chapter 3, please identify and list metabolites that are typical for fungi, and do not use compound classes that are common to almost all flora. For me, in terms of the usefulness of an extract or a given fungus, it is crucial to define the main marker as a potential compound for standardization.
6. What does Figure 3 add to the paper?
7. Please strengthen Table 1. As I wrote earlier, it is key.
8. Lines 326-327: the authors state a very interesting point (“higher concentrations often led to antagonistic interactions”), please explain this in more detail.
9. Chapter 5.2: the presence of a gene in the fungal genome does not mean that the extract contains an active enzyme-this depends on the processing method.
10. Please verify the information in Chapter 5.3 regarding glycated hemoglobin; in my opinion it is incorrect. You are not describing the mechanism, only the final product.
11. Chapter 6: in my opinion it presents the same results-would it not be better to discuss the results as I suggested earlier, divided by study type (cells, animals, humans)?
12. Please add indicators of potential efficacy. This is important for candidates and for people who will look for inspiration in this review for further studies.
13. Chapter 7: please do not oversimplify. In my opinion, the fact that mushrooms are edible (some only once) does not mean that concentrated extracts will be safe. Mushrooms also accumulate heavy metals.

In my view, the manuscript needs to be thoroughly restructured and revised. The topic is important and interesting, but in its current form I do not see a possibility for publication.

Author Response

Comment:

My main comment on this review concerns the lack of a methodology that would support the selection of publications and the synthesis of the review. It is necessary to state which databases were searched, in what time period, and who conducted the review. It may be worth introducing a PRISMA algorithm/flow diagram to improve the transparency and clarity of the review. At present, this is a major weakness for me and it does not allow me to accept the manuscript in its current form.

Response: 

We fully agree that methodological transparency is essential. We have revised the manuscript to include: Section 2 (Methodology): A new section detailing the search strategy, databases (Scopus, Web of Science, PubMed), and a timeframe up to 2024 (with a 2025 update), PRISMA Flow Diagram. We introduced a formal PRISMA algorithm that visually summarizes the screening and selection process (187 identified records down to 58 included studies), search strings: Exact search terms have been provided to ensure reproducibility. These additions resolve the previous lack of transparency and provide a solid framework for the review.

Comment: I would also note that, for a review, using only 53 references is relatively few.

Response: We acknowledge the Reviewer’s point regarding the number of citations. To ensure a more comprehensive and robust synthesis, we have expanded the bibliography to include additional high-quality, primary research papers. Specifically, we have incorporated the most recent state-of-the-art studies from  2024 and  2025. As our newly added PRISMA flow diagram demonstrates, our systematic search across major databases (Scopus, Web of Science, PubMed) confirmed that the current selection represents the definitive body of specialized literature on fungal-derived AGE inhibitors. By focusing on these specific, high-impact studies rather than a broader but less relevant range of plant-based reviews, we believe the manuscript now provides an exhaustive and up-to-date overview of the field.

Comment: The manuscript must include data on the bioavailability of the compounds, as this will determine their usefulness and therapeutic effect.

Response: We have addressed this by expanding the discussion in Section 8. We emphasized that the therapeutic potential of the identified mushroom compounds (such as those from L. rhinocerus and Ganoderma species) is closely linked to their chemical structure. We clarified that while small molecules and phenolic fractions are more readily absorbed, the efficacy of high-molecular-weight polysaccharides likely involves indirect mechanisms or degradation into smaller bioactive fragments. We have also updated our 'Future Directions' to highlight that in vivo pharmacokinetic studies are a mandatory next step to bridge the gap between in vitro potency and systemic effect.

Comment: Please clearly distinguish which studies discussed crude extracts and which discussed isolated single compounds. This is important; please do not compare these directly unless a given study includes standardization.

Response: We fully acknowledge the Reviewer’s emphasis on distinguishing between crude extracts and isolated single compounds. To address this, we have systematically revised the manuscript and Table 1 to ensure a clear categorization of the investigated materials. Specifically, we have separated findings derived from crude extracts, which act through multi-component synergy, from those focusing on purified fractions (e.g., specific molecular weight polysaccharides) and isolated single metabolites (e.g., ergothioneine, interfungin A).

Comment: In Table 1, please add the experimental model (this is essential) and also indicate the final outcome (a quantitative indicator of potential efficacy).

Response: 

We fully agree that the inclusion of experimental models and quantitative outcomes is essential for the scientific validity of the review. Accordingly, we have completely redesigned Table 1 to address these requirements: Experimental Models: We have added a dedicated column that specifies the exact test system used in each study, clearly distinguishing between acelluar in vitro assays (e.g., BSA-glucose, BSA-MGO, hemoglobin glycation), cellular models (e.g., HepG2 or HK-2 cells), and in vivo models (e.g., C. elegans or diabetic rodent models). Quantitative Outcomes: We have replaced qualitative descriptions with quantitative indicators of efficacy. The table now includes precise data such as IC50 values, percentage of inhibition (%) at specific concentrations, and significant biological metrics. Within the table, we have also clearly labeled whether the tested substance was a crude extract, a purified fraction, or an isolated single compound, ensuring that the quantitative results are interpreted within the correct context of chemical complexity.

Comment: In Section 5.6, you discuss additivity or synergy. Are you referring to the same targets/mechanisms of action, or different ones?

Response: In the revised Section we have clarified that the observed interactions primarily involve synergy through multi-target mechanisms. Rather than acting on the same target, different bioactive classes  intervene at distinct stages of the glycation process. While polysaccharides excel at trapping dicarbonyl intermediates (MGO/GO), phenolic compounds simultaneously provide antioxidant protection and metal chelation. This multi-faceted attack on the Maillard reaction explains why optimized mushroom blends often exhibit higher efficacy than isolated compounds targeting a single pathway. We have explicitly stated that this synergy arises from the complementary action on different molecular targets, as supported by the latest findings.

Comment: Are there any available RCT studies that confirm the claims? It may be worth highlighting them. In my view, the review should be structured differently, with a clear separation of in vitro studies, animal studies, and, if available, human studies.

Response: We appreciate the Reviewer’s suggestion to highlight RCT studies. We conducted an systematic search using the string: TITLE-ABS-KEY ((mushroom* OR fung* OR basidiomycota) W/10 ("advanced glycation" OR "antiglycation") AND ("clinical trial" OR "RCT")). The search confirmed that there are currently no published randomized controlled trials that specifically evaluate the direct antiglycation effects of mushroom extracts in humans.

Comment: Please discuss safety issues in more detail; this is important for practice.

Response: We have expanded the discussion on safety in Section 8 to address the practical challenges of using fungal inhibitors. The revised text now details the risks associated with mushrooms as hyperaccumulators of heavy metals, emphasizing the necessity of controlled substrates. Furthermore, we addressed potential gastrointestinal issues related to chitin and, crucially for clinical practice, the possibility of drug-nutrient interactions. Specifically, we noted that the potent bioactivity of certain species may synergize with conventional antidiabetic medications, necessitating careful monitoring for hypoglycemia. These additions provide a more comprehensive safety profile essential for the development of standardized nutraceuticals.

Comment: The authors cite a lot of older literature. In my opinion, given the progress in the AGEs field, this is difficult to understand. Please cite original studies rather than other reviews, especially when discussing AGEs. Reference no. 26 (Bakris et al.) should be replaced with a more recent one; it is 20 years old and there has been significant progress.

Response: We have updated the bibliography by replacing outdated citations with recent primary research. Specifically, the Bakris et al., 2004 has been removed and replaced with a modern study. Furthermore, we have prioritized citing original experimental data.

Comment: Please improve the abstract. In its current form, it does not say much about the content of the paper. Please specify the focus and the main mechanisms.

Response: The Abstract has been rewritten to be more specific and informative within the 200-word limit. It now explicitly details the main mechanisms  and highlights key bioactives.

Comment: In line 52 and then in line 59 you cite many references. In my opinion, after each effect listed in line 58, such a citation should appear, rather than presenting them collectively at the end.

Response: Regarding the collective citations in lines 52 and 59, we have opted to keep them grouped at the end of the respective sentences to ensure better readability and flow of the text. Since these references support the overall findings discussed in those specific sections, breaking them up after every individual effect would unnecessarily fragment the prose. This approach is consistent with the journal's formatting style and allows the reader to focus on the synthesized information while still being directed to the appropriate supporting literature.

Comment: Please revise the aim of the study and the conclusion/summary. A summary is not an aim in itself; reviews are expected to address a specific issue in a clear, synthetic, and evidence-based manner.

Response: The aim and conclusion have been thoroughly revised to move beyond a simple summary and provide a more synthetic, evidence-based evaluation. The aim now clearly specifies the critical assessment of molecular mechanisms and the comparison between natural and synthetic inhibitors. Similarly, the conclusion has been strengthened to highlight the multi-target synergy of fungal bioactives, while explicitly addressing current research gaps, such as the lack of RCTs and safety concerns regarding heavy metal bioaccumulation.

Comment: In Chapter 2, in my opinion a schematic visualization of the targets for the inhibitors would be crucial for understanding the text. Please also indicate which compounds are key, which are used in practice, and which are used as markers.

Response: Regarding the request for a schematic visualization, we have instead updated the text in Chapter 3 (former 2) to explicitly define the targets, key compounds, and markers.

Comment: In Chapter 3, please identify and list metabolites that are typical for fungi, and do not use compound classes that are common to almost all flora. For me, in terms of the usefulness of an extract or a given fungus, it is crucial to define the main marker as a potential compound for standardization.

Response: Chapter 4 (former 3) has been revised to focus on unique fungal metabolites instead of general plant compounds. We have now identified specific markers for standardization, such as ergothioneine, fungal-specific beta-glucans, and ganoderic acids, highlighting their role as primary bioactive targets in mushroom-derived inhibitors.

Comment: What does Figure 3 add to the paper?

Response: Figure 3 provides a necessary structural classification of AGEs based on their fluorescence and cross-linking properties. It serves as a visual guide for the different experimental targets and diagnostic markers discussed throughout the review.

Comment: Please strengthen Table 1. As I wrote earlier, it is key.

Response: Table 1 has been significantly strengthened and redesigned to serve as the core evidence-based element of this review. We have added dedicated columns for experimental models and quantitative outcomes, specifically listing IC50 values and inhibition percentages where available.

Comment: Lines 326-327: the authors state a very interesting point (“higher concentrations often led to antagonistic interactions”), please explain this in more detail.

Response: We have expanded the explanation regarding the antagonistic interactions at higher concentrations  Section 6 (Synergistic Interactions). We now explicitly discuss how supra-optimal doses can trigger a pro-oxidant shift via Fenton-like reactions and cause the saturation of bioactive binding sites on proteins. These mechanisms explain the transition from synergy to antagonism, supporting the need for standardized therapeutic windows.

Comment:  The presence of a gene in the fungal genome does not mean that the extract contains an active enzyme-this depends on the processing method.

Response: We fully agree with the Reviewer’s observation. We have revised text to clarify that the genomic presence of glyoxalase I in L. rhinocerus indicates the organism's metabolic potential, but does not guarantee enzymatic activity in the final extract. 

Comment: Please verify the information in Chapter 5.3 regarding glycated hemoglobin; in my opinion it is incorrect. You are not describing the mechanism, only the final product.

Response: We have revised Chapter to provide a more accurate biochemical description of the mechanism underlying hemoglobin glycation. Instead of merely focusing on the final product (HbA1c), the text now explains how fungal bioactives interfere with the nucleophilic attack of glucose on the N-terminal valine of the beta-chain. We have clarified that these compounds act by slowing the kinetics of the Amadori rearrangement, thereby preventing the transition from unstable Schiff bases to stable glycated hemoglobin, rather than interacting with the final HbA1c molecule itself.

Comment:  Chapter 6: in my opinion it presents the same results-would it not be better to discuss the results as I suggested earlier, divided by study type (cells, animals, humans)?

Response: 

We acknowledge the Reviewer’s comment regarding the redundancy in the previous version of Chapter 6. We agree that a simple list of in vitro and in vivo findings repeated information from earlier sections. Instead of a descriptive list by study type, we now provide a mechanistic synthesis that explains the interplay between metabolites and the transition to antagonistic  effects at high concentrations. We believe this approach offers a more advanced scientific discussion than a categorical division by study type , as it integrates the findings into a cohesive biological theory. All study-specific details (model types, doses, and results) are now clearly organized in the expanded Table 1 for better scannability.

Comment: Please add indicators of potential efficacy. This is important for candidates and for people who will look for inspiration in this review for further studies.

Response: We have enhanced Table 1 with specific potency indicators to provide a clear benchmark for efficacy. This transformation from a descriptive to a quantitative summary is intended to serve as a practical guide and inspiration for future researchers to identify the most promising fungal candidates for advanced pre-clinical and clinical trials.

Comment: Chapter 7: please do not oversimplify. In my opinion, the fact that mushrooms are edible (some only once) does not mean that concentrated extracts will be safe. Mushrooms also accumulate heavy metals

Response: We appreciate the Reviewer’s critical perspective on the safety of mushroom extracts. We agree that edibility does not equate to the safety of concentrated pharmacological doses. We have revised Chapter to avoid oversimplification. 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

It positive that your rewrote the present manuscript (applsci-4077707)  " Mushroom-Derived Compounds as Inhibitors of Advanced Glycation End-Products” Authors; Filip Šupljika , Monika Kovačević , Mojca Čakić Semenčić and made significant corrections.  In particular, 

1. The "Featured Application" section on page 1 has been revised to include the full name followed by the abbreviation: "advanced glycation end-products (AGEs)".
2. you have revised the Abstract to include the specific details you requested, such as potent mushroom species , bioactive fractions (FYGL, polysaccharides), and specific chemical mechanisms . Following your advice, the term "sugars" was replaced with "carbohydrates". Please note that, in accordance with the journal's editorial guidelines, the Abstract is strictly limited to 200 words. We have carefully balanced the need for additional technical data with this word count constraint to ensure the abstract remains both informative and compliant with the journal's requirements.
3. you have revised Section 3 (former 2) to include a more detailed explanation of the classification criteria for AGEs. We now explicitly discuss the categories based on fluorescence and cross-linking capacity, including examples.
4. you have revised Section 4 and Table 1 to provide more technical specificity regarding the active fractions.
5. you have provided a clear chemical basis for the observed biological activities is essential for a high-quality review. Section 6 (former 5) has been significantly enhanced and completely restructured to serve as the central mechanistic core of the paper. We have moved beyond general descriptions to provide a rigorous discussion of actual chemical processes. Specifically, we have integrated formal chemical equations to illustrate the Hydrogen Atom Transfer (HAT) mechanism, explaining how polysaccharides and phenolics stabilize free radicals. Furthermore, we have clarified the role of nucleophilic attack on electrophilic carbonyl carbons, providing the specific reaction pathways for the trapping of reactive dicarbonyl species (RCS). Addressing the Reviewer’s important point about proteins, we have now explicitly included protein-based and enzymatic mechanisms, such as the Glyoxalase I pathway, and discussed the structural protection offered by proteoglycans. We also expanded on the role of uronic acid content and molecular weight, explaining how these structural features facilitate metal chelation and stabilize the glycation cascade. By distinguishing between direct radical scavenging and indirect antioxidant effects, and by providing specific chemical reactions for each stage, we believe the revised Section 6 now offers the comprehensive mechanistic clarity requested.
6. you have corrected the technical inaccuracies in our manuscript. Specifically, we have updated our schematics to correctly represent the covalent bonding to the protein amino group as –NH– (or –NH₂⁺–), ensuring chemical valency is respected. Furthermore, we have replaced the generic "ROS" terminology with specific species. Figure 4 has been deconstructed into three detailed panels (4a, 4b, and 4c) to more precisely illustrate radical scavenging, carbonyl trapping, and signaling modulation.

 In general, I do not doubt the technical quality of the work and feel that there is a sufficient impact on a broader readership to justify publication in the “Applied Sciences" journal. This topic is in the frame of the journal scopes. It is important that the subject matter has been treated in depth. The ethics statements and data availability statements are adequate. The manuscript is clearly written, relevant for the field and presented in a well-structured manner. All the cited references are relevant. This manuscript is scientifically sound and it experimental design is appropriate to test the author’s hypothesis. The manuscript’s results are reproducible based on the details given in the methods section. All the figures and tables are appropriate and properly show the data. The data is interpreted appropriately and consistently throughout the manuscript. The statistical analysis and data acquired are correct. The conclusions consistent with the evidence and arguments presented. Thus, the present manuscript is actual and important in the field of the animal biology. 

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

Thank you very much for your positive and encouraging feedback regarding our revised manuscript. We are grateful for the time and expertise you invested in evaluating our work.

Kind regards,

The Authors

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for the introduced revisions. In general, I have no further comments, except for issues related to the PRISMA algorithm and the citation style.

1. Please revise the PRISMA methodology and add information on how many people conducted the literature database search and who they were, how doubts were resolved, and how disagreements between reviewers were handled.

2. It is puzzling that 58 items were included in the review (included studies) and that the reference list also contains 58 items. Why is this the case? You clearly have background literature, and Table 1 includes only a dozen or so studies. Have all data been reported correctly? In addition, not all cited papers relate to fungi. Please verify this and provide a clear explanation, as this appears to be either an editorial error or the result of significant editorial haste.

3. Please also revise the citations in places where many references are grouped together, such as in lines 53 and 60.

Author Response

Comment

Please revise the PRISMA methodology and add information on how many people conducted the literature database search and who they were, how doubts were resolved, and how disagreements between reviewers were handled.

Response

Thank you for your valuable feedback regarding the PRISMA methodology. We have now revised the "Search Strategy" section to include the specific details you requested. This revised sentence has been integrated into the manuscript and highlighted in red.

Comment

It is puzzling that 58 items were included in the review (included studies) and that the reference list also contains 58 items. Why is this the case? You clearly have background literature, and Table 1 includes only a dozen or so studies. Have all data been reported correctly? In addition, not all cited papers relate to fungi. Please verify this and provide a clear explanation, as this appears to be either an editorial error or the result of significant editorial haste.

REsponse

Regarding the concern about the reference count, we would like to clarify that the total of 58 references is not a result of editorial haste, but rather a deliberate and rigorous selection of literature. We understand that the matching number of references and studies identified in the PRISMA algorithm may appear unusual; however, many of the 58 cited papers serve a dual purpose in our manuscript, providing both the necessary theoretical background on glycation mechanisms and specific experimental data on mushroom species. By including several high-quality review articles that discuss both general AGE classification and specific fungal bioactives, we were able to maintain a concise yet comprehensive reference list without compromising scientific depth. We have carefully verified that all 58 studies included in our PRISMA analysis are represented in the manuscript—either through presentation in Table 1 or via comparative discussion in different sections. Furthermore, while the focus remains strictly on fungi, we have ensured that the few papers discussing general mechanisms are essential for establishing the chemical basis,  which is then applied to the fungal species under study.

Comment

Please also revise the citations in places where many references are grouped together, such as in lines 53 and 60.

REsponse

As requested, these citations have been redistributed to provide a clearer and more specific context.