Review Reports - Polyphenols, Polysaccharides, and Their Complexes from <i>Aronia melanocarpa</i> in the Chemoprevention of Colorectal Cancer

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1) Lack of Critical Analysis of Clinical Data: The most significant shortcoming is the almost complete lack of discussion of human clinical studies. Most of the evidence cited is from in vitro or animal models. Although the authors briefly mention systematic reviews that include human data, the text does not provide a critical analysis of the methodological quality, sample size, or results of existing clinical trials specifically focused on CRC prevention.
2) Potential Conflicting Data in Tables: Tables 1 and 2 provide numerical data for compound content. However, the units of measurement differ (DW, FW), and some ranges of values (e.g., for total polyphenols in Table 2: 28-1022 mg/100 g) are extremely broad without sufficient explanation of the reasons (differences in varieties, extraction methods, analysis). This may confuse the reader. A clearer unification or explanatory footnote is needed. 3) The authors briefly mention that high doses of polyphenols can exhibit pro-oxidant properties and induce apoptosis in cancer cells. This important phenomenon deserves a more in-depth discussion: what are the potential risks, threshold values, and does this apply to whole extracts in the human body?
4) The review paints an overwhelmingly positive image of chokeberry. Potential adverse effects with long-term high-dose intake, such as the impact on iron absorption due to tannins, or drug interactions, are not discussed. A safety section is needed for a complete picture.
5) References in the text to Figure 1 and Tables are sometimes unclear.

Author Response

Comment 1:

Lack of Critical Analysis of Clinical Data: The most significant shortcoming is the almost complete lack of discussion of human clinical studies. Most of the evidence cited is from in vitro or animal models. Although the authors briefly mention systematic reviews that include human data, the text does not provide a critical analysis of the methodological quality, sample size, or results of existing clinical trials specifically focused on CRC prevention.

Response 1:

We agree that the initial version of the manuscript did not provide an in-depth discussion of human clinical studies in the context of CRC prevention. We have revised the manuscript and added a new section 6.3 entitled “Clinical Trials on Aronia melanocarpa in Cancer Prevention” (pages 14-16). In this section, we critically evaluate the existing clinical evidence, which coansist investigate pathways and biomarkers relevant to CRC pathophysiology (e.g., systemic inflammation, oxidative stress, lipid metabolism, immune modulation). We also discuss the limitations of extrapolating findings from non-CRC clinical populations, highlight methodological constraints (sample size, study duration, heterogeneity of interventions), and identify the key gaps that must be addressed to establish stronger clinical evidence specific to CRC prevention.

Comment 2:

Potential Conflicting Data in Tables: Tables 1 and 2 provide numerical data for compound content. However, the units of measurement differ (DW, FW), and some ranges of values (e.g., for total polyphenols in Table 2: 28-1022 mg/100 g) are extremely broad without sufficient explanation of the reasons (differences in varieties, extraction methods, analysis). This may confuse the reader. A clearer unification or explanatory footnote is needed.

Response 2:

We thank the Reviewer for drawing attention to this important issue. In response, Table 1 has been revised and Table 2 has been removed to streamline and clarify the presentation of the data. Explanatory footnotes have been added to Table 1 indicating that all values are reported as in the original studies (pages 3-5). The footnotes also explain that the wide ranges observed for some compounds reflect natural variability related to ripening stage, environmental conditions, extraction procedures, and analytical methods used across studies. For these reasons, inter-study comparisons should be interpreted with caution. The table has been thoroughly checked for internal consistency, and the added notes should help minimize potential confusion for readers.

Comment 3:

The authors briefly mention that high doses of polyphenols can exhibit pro-oxidant properties and induce apoptosis in cancer cells. This important phenomenon deserves a more in-depth discussion: what are the potential risks, threshold values, and does this apply to whole extracts in the human body?

Response 3:

We appreciate valuable comment regarding the pro-oxidant properties of polyphenols at high doses. We agree that this phenomenon is important and requires further clarification. In our response, we have expanded the discussion in the "Study limitations and Future Perspectives" section (page 16; lines 745-765) to include an overview of pro-oxidant mechanisms, a discussion of the dose thresholds (concentrations) observed in in vitro studies, and the limitations of transferring these observations to in vivo conditions (the role of metabolism, plasma proteins, gut microbiota, and extract composition). We also addressed the phenomenon of hormesis—that low doses often exhibit cytoprotective/antioxidant effects, while high doses can induce oxidative stress and apoptosis, which has both therapeutic (e.g., in oncology) and toxicological implications

Comment 4:

The review paints an overwhelmingly positive image of chokeberry. Potential adverse effects with long-term high-dose intake, such as the impact on iron absorption due to tannins, or drug interactions, are not discussed. A safety section is needed for a complete picture.

Response 4:

Thank you for pointing this out. We agree with this comment and we have added a safety section in the conclusion as a separatly paragraph. The newly included text discusses key limitations related to long-term or high-dose intake of Aronia melanocarpa, including the potential for modest reductions in non-heme iron absorption due to tannin content, reports of mild gastrointestinal discomfort at higher doses, and the theoretical possibility of interactions with drug-metabolizing enzymes. Those partagraph is on page 17; lines 805-813.

Comment 5:

References in the text to Figure 1 and Tables are sometimes unclear.

Response 5:

With the aim of improving the clarity of the manuscript, we have prepared a new figure (Figure 1) illustrating the modulatory effects of aronia polyphenol–polysaccharide complexes on the gut microbiota and the bioavailability of polyphenols in the context of CRC risk (page12) . We have removed Tables 2 and 3, and Table 1 has been revised and supplemented (pages 3-5). We hope that the information presented in Figure 1 and in Table 1 is now clearer and therefore more accessible.

We thank the Reviewer for these suggestions and we believe that the added corrections will strengthen our manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Reviewer comments

The study explores the potential of Aronia melanocarpa (black chokeberry) as a complementary strategy for colorectal cancer (CRC) prevention and treatment. The authors highlighted that conventional chemotherapy has limitations, creating a need for natural products that can enhance therapeutic outcomes. They describe A. melanocarpa fruits, leaves, and pomace as rich in polyphenols and bioactive polysaccharides that exert strong antioxidant and anti-inflammatory effects. They report that these compounds can modulate apoptosis pathways and specifically target cancer cells. They also note that A. melanocarpa supplementation may reduce inflammatory markers such as IL-6 and TNF-α, influencing the tumor microenvironment. They conclude that A. melanocarpa holds promise as part of integrative approaches for CRC prevention and adjunct therapy.

Decision: Minor revision

Line should be modfied and the end of section the authors needs to present the text that describe afterward “Systematic reviews confirm that chokeberry supplementation reduces markers of inflammation and oxidative stress while enhancing the activity of the body’s endogenous antioxidant enzymes [19,23].”
Is the table 3 need an individual attention, I think they can merge it
More related studies needed to specify whether these findings are from in vitro, in vivo, or clinical studies and comment on the strength of the evidence?
The introduction is clear, but it should better connect global CRC trends with the choice of melanocarpa as a chemo preventive focus.
Please updates authors elaborate on what makes melanocarpa uniquely suited for CRC prevention compared to other polyphenol-rich plants?
It would be nice the authors clarify the specific pathways that explain the proposed synergy between polyphenols and polysaccharides in the discussion section
They can prepare a figure for describing mechanistic detail or supporting evidence explaining how polyphenol–polysaccharide complexes enhance bioavailability and gut micro biota modulation in CRC prevention?
The conclusion discusses the valorization of pomace and leaves could the authors clarify how these findings might translate into practical applications or clinical relevance?
Limitations of the study is also needed to clarify the points of using it.

Author Response

Comment 1:

Line should be modfied and the end of section the authors needs to present the text that describe afterward “Systematic reviews confirm that chokeberry supplementation reduces markers of inflammation and oxidative stress while enhancing the activity of the body’s endogenous antioxidant enzymes [19,23].”

Response 1:

Thank you for this helpful observation. In response, we have revised the relevant section to improve coherence, and the indicated sentence has now been moved to the end of the section, as recommended. This placement now serves as a clear concluding statement summarizing the evidence discussed. The revised paragraph can be found at the end of Section 1.3, on page 3.

Comment 2:

Is the table 3 need an individual attention, I think they can merge it.

Response 2:

Thank you for this observation. After careful consideration, we agree that Table 3 does not require separate presentation. In the revised manuscript, Table 3 has been removed.

Comment 3:

More related studies needed to specify whether these findings are from in vitro, in vivo, or clinical studies and comment on the strength of the evidence?

Response 3:

Thank you for this valuable comment. In response, we have expanded the 1.3 section on related studies to clearly specify whether each cited finding originates from in vitro, in vivo, or clinical research (page 3; lines 97-112). Additionally, we have included a brief evaluation of the strength of the evidence for each category of studies. These modifications improve the transparency and context of the reported results.

Comment 4:

The introduction is clear, but it should better connect global CRC trends with the choice of melanocarpa as a chemo preventive focus.

Response 4:

Thank you for this valuable suggestion. In response, we have added sentences in section 1.2, “The Role of Chemoprevention in CRC” to better connect global CRC trends with the rationale for focusing on Aronia melanocarpa as a chemopreventive agent. The last paragraph of section 1.2 now also underscores that the synergistic interactions among multiple bioactive compounds naturally present in whole food matrices, such as A. melanocarpa, enhance their chemopreventive potential (page 2; lines 82-86).

Comment 5:

Please updates authors elaborate on what makes melanocarpa uniquely suited for CRC prevention compared to other polyphenol-rich plants?

Response 5:

In response, we have expanded the discussion in paragraph 1.3 „Significance of Aronia melanocarpa in Preventive Medicine” to more clearly explain the features that distinguish Aronia melanocarpa from other polyphenol-rich plants in the context of CRC prevention. This section now highlights its unique polyphenolic profile, particularly the high content of anthocyanins and procyanidins, as well as the evidence supporting its superior antioxidant, anti-inflammatory, and antiproliferative properties compared with other sources. These additions provide a clearer justification for focusing on A. melanocarpa in CRC-related research (pages 2-3; lines 93-96).

Comment 6:

It would be nice the authors clarify the specific pathways that explain the proposed synergy between polyphenols and polysaccharides in the discussion section.

Response 6:

In response, we have expanded the discussion section by adding a dedicated explanation in the concluding paragraphs that clarifies the mechanistic pathways underlying the proposed synergy between polyphenols and polysaccharides. The added text emphasizes how these complementary actions improve polyphenol bioavailability and collectively contribute to anti-inflammatory and anti-tumorigenic effects relevant to CRC prevention (page 17; lines 783-787). This addition directly addresses the reviewer’s concern and provides a clearer mechanistic context for the synergistic interactions discussed in the manuscript.

Comment 7:

They can prepare a figure for describing mechanistic detail or supporting evidence explaining how polyphenol–polysaccharide complexes enhance bioavailability and gut micro biota modulation in CRC prevention?

Response 7:

Thank you for this valuable suggestion. In response, we have prepared a new figure (Figure 1.) illustrating how polyphenol-polysaccharide complexes enhance bioavailability and modulate the gut microbiota in the context of CRC prevention (page 12).

Comment 8:

The conclusion discusses the valorization of pomace and leaves could the authors clarify how these findings might translate into practical applications or clinical relevance?

Response 8:

Thank you for this helpful comment. We now explain more clearly how aronia pomace and leaves can be used in practical and clinically relevant ways as ingredients in functional foods or nutraceuticals in the context of CRC prevention. Their high content of polyphenols and fiber, as well as their low cost and year-round availability, make them promising materials for future dietary and clinical applications. The changes have been incorporated in the conclusion section, at the end of the second paragraph, on page 17; lines 796-799.

Comment 9:

Limitations of the study is also needed to clarify the points of using it.

Response 9:

We thank the Reviewer for this suggestion. In response, we have added a dedicated subsection titled “Study Limitations and Future Perspectives” (pages 15-16). This section summarizes the main limitations of the current evidence, including the low bioavailability of Aronia polyphenols, variability in composition due to cultivar, cultivation, and processing differences, and challenges related to bitterness and stability in food applications. It also highlights the limited and variable clinical data, short intervention periods, and potential safety considerations such as gastrointestinal effects and prooxidant activity at high doses. By including this subsection, we provide a balanced perspective and clarify the constraints that should be considered when interpreting the findings.

We thank the Reviewer for these suggestions and we believe that the added corrections will strengthen our manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

This review article comprehensively summairzed the chemical profile and chemopreventive mechanisms of Aronia melanocarpa (black chokeberry) components against Colorectal Cancer (CRC). The integration of information on polyphenols (anthocyanins, quercetin), polysaccharides (pectins, arabinogalactans), and their synergistic complexes is timely and highly valuable for integrative strategies aimed at CRC prevention. The manuscript excels in detailing the molecular pathways modulated by aronia extracts, including NF-κB inhibition, apoptosis induction, and ROS neutralization.

However, the reporting of compositional data contains critical inconsistencies between the summary tables and the detailed text, which undermines the quantification foundation of the manuscript. Therefore, a recommendation a Revision is necessary to ensure accuracy and scientific rigor.

The core scientific content is strong, but the authors must resolve significant internal inconsistencies related to the chemical composition data provided in the tables and text

The quantitative data for anthocyanins is contradictory across the manuscript:

Table 1 reports the Total Anthocyanin content in Whole Fruit as being in the range of 2000–4000 mg/100 g DW.

Section 2.1 (Anthocyanins) cites a study where a specific extract of cyanidin-3-O-galactoside reached 917.31 mg/g DW total anthocyanins, which equates to over 90,000 mg/100g DW. The authors must clarify this massive discrepancy and ensure the typical range in Table 1 accurately reflects whole fruit/berry data.

The concentrations of flavonols in the whole fruit must be standardized:

Table 1 (Flavonols) reports the range for Whole Fruit as 100–400 mg/100 g DW.

Section 2.1 (Flavonols) states that concentrations in fruits range from ~12 to 44 mg/100 g DW.

This nearly tenfold difference requires correction to ensure the tables and text present a consistent quantitative assessment of these key compounds (e.g., quercetin and kaempferol derivatives).

The presence of lignans (e.g., secoisolariciresinol) needs consistent description.

Table 1 reports Lignans as "Not detected/significant" or "Not detected" across all parts (Fruit, Juice, Leaves, Pomace).

Table 2 lists Lignans in Fruits as "Present in small amounts" and in Leaves as "Likely present.The authors should unify the description, perhaps clarifying that while they are generally not major sources, trace components are indeed present, and amend the "Not detected" notation in Table 1 to reflect the trace presence noted elsewhere.

While the polysaccharide section (Section 4) excellently details mechanisms (e.g., PI3K/Akt and NF-κB inhibition) and the generation of SCFAs (butyrate), the discussion relies heavily on general plant polysaccharides. Strengthen Section 4 by explicitly highlighting which of these mechanisms have been demonstrated specifically using Aronia melanocarpa polysaccharides or extracts rich in these fractions (like pomace) when discussing CRC-related pathways, or clearly state the gap in such specific mechanistic data if it exists.

Author Response

Comment 1:

The core scientific content is strong, but the authors must resolve significant internal inconsistencies related to the chemical composition data provided in the tables and text

- The quantitative data for anthocyanins is contradictory across the manuscript:

Table 1 reports the Total Anthocyanin content in Whole Fruit as being in the range of 2000–4000 mg/100 g DW.

- The concentrations of flavonols in the whole fruit must be standardized: Table 1 (Flavonols) reports the range for Whole Fruit as 100–400 mg/100 g DW.

Section 2.1 (Flavonols) states that concentrations in fruits range from ~12 to 44 mg/100 g DW.

This nearly tenfold difference requires correction to ensure the tables and text present a consistent quantitative assessment of these key compounds (e.g., quercetin and kaempferol derivatives).

- The presence of lignans (e.g., secoisolariciresinol) needs consistent description.

Table 1 reports Lignans as "Not detected/significant" or "Not detected" across all parts (Fruit, Juice, Leaves, Pomace).

Response 1:

We thank the Reviewer for carefully examining the compositional data and for highlighting the inconsistencies between the summary tables and the detailed text. We fully agree that accurate quantitative reporting is essential for scientific rigor. In response, we have undertaken a comprehensive verification of all compositional values throughout the manuscript.

To address these concerns, the entire dataset has been re-evaluated, and the numerical values have been cross-checked against the original publications. The previously separate tables have now been consolidated into a single, unified Table 1 to eliminate redundancy and improve clarity. We also explicitly emphasize in both the table and the text that substantial differences in extraction procedures (e.g., solvent composition, purification steps, and analytical techniques) significantly influence the reported concentrations of phenolic compounds, which explains some of the variation observed across studies.

Regarding the anthocyanin values, we thank the Reviewer for pointing out the apparent discrepancy. We would like to clarify that the value of 917.31 mg/g DW does not refer to the whole Aronia fruit but to a purified anthocyanin-rich fraction obtained using NKA-9 macroporous adsorption resin. This purification step selectively concentrates anthocyanins while removing sugars, organic acids, and non-phenolic constituents, leading to values that can be approximately tenfold higher than those found in whole fruit. The total anthocyanin content in the whole aronia fruit is now clearly stated in Table 1 and in the revised text.

We have revised the manuscript to explicitly distinguish between whole-fruit composition and data derived from purified extracts to prevent any future misinterpretation. These corrections and clarifications should resolve the inconsistencies noted by the Reviewer and strengthen the quantitative foundation of the manuscript.

Comment 2:

Response 2:

We thank the Reviewer for this insightful comment. Section 4 was intended to present general mechanisms described for plant polysaccharides, rather than findings specific to Aronia melanocarpa. As most existing studies on chokeberry utilize polyphenol-rich extracts rather than isolated polysaccharide fractions, the polysaccharide-specific effects on biological pathways cannot yet be clearly delineated. These limitations, including the predominance of polyphenol-focused data and the lack of studies testing polysaccharide fractions independently, are further addressed in Chapter 7 “Study Limitations and Future Perspectives” (page 16; lines 758-765). We believe that these clarifications adequately address the Reviewer’s concern and strengthen the accuracy and transparency of the manuscript.

We thank the Reviewer for these suggestions and we believe that the added corrections will strengthen our manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors took all my comments into account and tried their best to improve the article. I believe that in its current form the article is worthy of publication.

Reviewer 3 Report

Comments and Suggestions for Authors

The revisions have clearly improved the manuscript, and I now recommend acceptance