Emodin and the Anthraquinone Scaffold: Therapeutic Promise and Strategies to Overcome Translational Barriers
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis manuscript systematically reviews the extensive biological activities and mechanisms of action of emodin and related anthraquinone derivatives; comparatively evaluates the relative therapeutic potential of emodin and its analogues in several major disease models; summarizes their pharmacokinetic limitations, safety profiles, and toxicity concerns; and, in the “Conclusions” section, proposes and critically evaluates strategies—grounded in medicinal chemistry and pharmaceutics—to overcome these challenges.
In the past five years, reviews on emodin and other anthraquinone compounds have primarily reported advances in their therapeutic potential against brain-related disorders and cancers. In contrast, this manuscript provides a more comprehensive and systematic summary of current research on the full spectrum of biological activities exhibited by these compounds. Supported by a substantial number of references and thorough citation coverage, this review serves as a valuable resource for researchers in related fields.
The authors still need to make the following changes and improvements:
- The content of Figure 2 is nearly identical to that of Figure 1B; therefore, Figure 1B should be removed.
- Lines 297–310: Are there supporting references for this paragraph (including Figure 4)? Alternatively, is this intended as a summary of Sections 4.1–4.3? Additionally, regarding Figure 4, the caption states that it illustrates the regulatory effects of anthraquinone compounds on multiple signaling pathways; however, the ligand—anthraquinone—is not depicted in the figure. Please verify.
- Section 6.2 heading: Please revise the title by removing “Hepatotoxic Effects,” as this section describes gastrointestinal toxic side effects, not hepatotoxicity.
- Line 1217: Please include the chemical structure of compound 12r.
Author Response
Comment 1: This manuscript systematically reviews the extensive biological activities and mechanisms of action of emodin and related anthraquinone derivatives; comparatively evaluates the relative therapeutic potential of emodin and its analogues in several major disease models; summarizes their pharmacokinetic limitations, safety profiles, and toxicity concerns; and, in the “Conclusions” section, proposes and critically evaluates strategies—grounded in medicinal chemistry and pharmaceutics—to overcome these challenges.
In the past five years, reviews on emodin and other anthraquinone compounds have primarily reported advances in their therapeutic potential against brain-related disorders and cancers. In contrast, this manuscript provides a more comprehensive and systematic summary of current research on the full spectrum of biological activities exhibited by these compounds. Supported by a substantial number of references and thorough citation coverage, this review serves as a valuable resource for researchers in related fields.
Response: We thank the reviewer for the positive and constructive assessment of our manuscript. We appreciate the recognition of its comprehensive and systematic coverage of the biological activities, mechanisms of action, and therapeutic potential of emodin and related anthraquinone derivatives. We are grateful for the reviewer’s acknowledgment of the manuscript’s value to researchers in the field.
The authors still need to make the following changes and improvements:
Comment 2: The content of Figure 2 is nearly identical to that of Figure 1B; therefore, Figure 1B should be removed.
Response: We thank the reviewer for this helpful observation. In the original version, Figure 1B was intended to present summarized representative anthraquinone structures grouped according to their substituent patterns, while Figure 2 provided detailed individual structures for clarity and reference. However, according to your recommendation, we have removed Figure 1B and retained Figure 2, which more comprehensively illustrates the structures discussed in the manuscript.
Comment 3: Lines 297–310: Are there supporting references for this paragraph (including Figure 4)? Alternatively, is this intended as a summary of Sections 4.1–4.3? Additionally, regarding Figure 4, the caption states that it illustrates the regulatory effects of anthraquinone compounds on multiple signaling pathways; however, the ligand—anthraquinone—is not depicted in the figure. Please verify.
Response: We thank the reviewer for the valuable comment. The paragraph in Lines 297–310 was intended as a concise introductory summary to Sections 4.1–4.3, which detail the supporting studies and references. In addition, Figure 4 has been updated to include the anthraquinone scaffold, ensuring consistency with the caption and clearer representation of the moiety involved in the illustrated signaling pathways.
Comment 4: Section 6.2 heading: Please revise the title by removing “Hepatotoxic Effects,” as this section describes gastrointestinal toxic side effects, not hepatotoxicity.
Response: We thank the reviewer for pointing this out. We have accordingly revised the heading of Section 6.2 by removing “Hepatotoxic Effects” to accurately reflect the gastrointestinal toxic effects discussed in this subsection.
Comment 5: Line 1217: Please include the chemical structure of compound 12r.
Response: We thank the reviewer for the valuable comment. We have accordingly expanded the accompanying text to further elaborate on its key chemical features and structural modifications relative to the anthraquinone scaffold.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript is a review article discussing the therapeutic potential and translational challenges of emodin and other anthraquinone derivatives, focusing on their pharmacological properties, structural features, and formulation strategies to enhance bioavailability and efficacy.
The article is well written, and the topic is relevant in the field.
However, I do have some suggestions for authors, that could enhance the quality of this review:
- The authors did not include the methodology in the selection of the litearture. Is this a systematic review a narative or a scoping review? Also the time frame of literature coverage could be included
- Are there other similar reviews in the scientific database? What differentiates this review from the existing ones?
I suggest the acceptance of the manuscript after the clarification of its novelty, scope and methodology used.
Author Response
Comment 1: The manuscript is a review article discussing the therapeutic potential and translational challenges of emodin and other anthraquinone derivatives, focusing on their pharmacological properties, structural features, and formulation strategies to enhance bioavailability and efficacy.
The article is well written, and the topic is relevant in the field.
Response: We thank the reviewer for the positive evaluation of our manuscript. We appreciate the recognition of its relevance and clarity, as well as the acknowledgment of the importance of the topic within the field.
However, I do have some suggestions for authors, that could enhance the quality of this review:
Comment 2: The authors did not include the methodology in the selection of the litearture. Is this a systematic review a narative or a scoping review? Also the time frame of literature coverage could be included
Response: We thank the reviewer for this important comment. The present work is a narrative review rather than a systematic or scoping review. Nevertheless, to improve transparency, we have added a methodology Subsection 6.1 to clearly describe the search strategy, screening process, evidence stratification and scoring criteria, and we have built an additional Figure 5 as an evidence flow chart summarizing included and excluded studies across domains. While we do not present the manuscript as a formal PRISMA-compliant systematic review, we have structured the Section on the therapeutic potential of anthraquinones as an evidence-mapping review with semi-quantitative synthesis, which is now clearly stated and justified in Section 6.1.
Comment 3: Are there other similar reviews in the scientific database? What differentiates this review from the existing ones?
Response: We thank the reviewer for this important question. Reviews on emodin and related anthraquinones have largely focused on specific indications, most commonly brain-related disorders and cancers. In contrast, our review provides a broader, more systematic synthesis of the full spectrum of reported biological activities and mechanisms of action, and additionally integrates a focused discussion of pharmacokinetic limitations, safety considerations, and formulation/medicinal-chemistry strategies to improve translational potential. Supported by extensive and up-to-date referencing, we believe the manuscript offers a distinct, comprehensive resource for researchers in the field.
Comment 4: I suggest the acceptance of the manuscript after the clarification of its novelty, scope and methodology used.
Response: We thank the reviewer for the constructive feedback and positive recommendation. We appreciate the suggestion and have clarified the manuscript’s novelty, scope and methodological framework accordingly in the revised version.
Reviewer 3 Report
Comments and Suggestions for AuthorsAbstract:
-“Anthraquinone derivatives have demonstrated reproducible antiproliferative, anti‑inflammatory, metabolic, cardiovascular, antifibrotic, and immunomodulatory effects across diverse preclinical models”
“Reproducible” suggests robust and consistent effects. In reality:
- Many data are from in vitro and animal studies, often with non-physiological doses.
- Not all effects have been consistently replicated across laboratories or models.
Suggested softening: “have shown antiproliferative, anti-inflammatory … effects in preclinical studies.”
-“Emerging formulation strategies that decouple intrinsic bioactivity from pharmacokinetic limitations”
Be cautious with the slightly optimistic tone.
In preclinical studies, these systems improve bioavailability and targeting, but there is no clinical confirmation yet.
Suggestion: “emerging preclinical formulation strategies that aim to decouple …”.
Introduction:
-“Reproducible efficacy across tumor, inflammatory, and immunometabolic disease models”
The expression may sound stronger than the evidence allows.
Most of the data on emodin is preclinical (in vitro and animal models). Reproducibility across models is variable, and clinical evidence is limited.
It could be reformulated as something like “consistent preclinical efficacy.
- Nanoemulsions that reduce glucuronidation during absorption
Nanoformulations generally improve bioavailability by increasing solubility and absorption, but they do not necessarily “reduce glucuronidation” directly. Glucuronidation is a hepatic and intestinal enzymatic process; formulation may alter pharmacokinetics, but it does not directly inhibit the process (unless specific data support that claim).
It would be more appropriate to say that it “may reduce presystemic metabolism and improve systemic exposure.”
-Bioactivation pathways that generate more toxic metabolites (e.g., 5-hydroxyemodin)
5-Hydroxyemodin is described as an oxidative metabolite, but classifying it clearly as “more toxic” depends on the experimental context. Differential toxicity is not universally established.
It could be phrased as “potentially more reactive metabolites.”
Structural Features and SARs among Natural Anthraquinones
- “This presence of a rigid, polyaromatic quinone core …”
“This presence” should be corrected to “The presence.”
-“Rhein introduces a carboxylic acid group … reducing passive membrane permeability”
Correct in principle (ionized acids cross membranes more slowly), but effective in vivo permeability also depends on active transport and protein binding.
Add nuance: “which may reduce passive membrane permeability.”
-“Physcion … removes one site for direct conjugative metabolism (e.g., glucuronidation) at that position”
Correct for the O-methylated position, but it is important to remember that metabolism can occur at other sites on the core or on other functional groups.
Suggestion: “…removes one site susceptible to direct conjugative metabolism.”
-“Electron donating methoxy and methyl groups tend to weaken antibacterial potency compared to their hydroxyl or carboxyl analogues”
Correct based on some studies, but activity depends on the target and the organism tested.
Include “in certain bacterial assays” for precision.
- In the scheme of Figure 1B, change the substituents R1, R2, R3, and R4 to R1, R2, R3, and R4.
Natural Occurrence and Major Sources
-“Sugar conjugation does not necessarily abolish biological activity”
Correct, but the degree of activity strongly depends on the enzyme substrate and intestinal microbiota.
You could add: “depending on hydrolysis efficiency and biological context.”
-Line 149: “occurence” → “occurrence”
Line 172: “Cassiae semen or coffee senna group” → check taxonomic consistency; “Cassiae semen” is more of a botanical drug name than a taxonomic group.
- You have 2 numbered sections labeled with the number 3!
Physicochemical and pharmacokinetic characteristics of emodin
-“Extensive phase II metabolism … further reduce systemic exposure”
Minor grammatical adjustment: it should be “further reduces systemic exposure” (singular: metabolism).
-“Absolute oral bioavailability of emodin is approximately 2,8–3,2%”
The comma in 2,8–3,2% should be replaced with a point to follow the international standard: 2.8–3.2%.
-“Serum metabolites exhibiting higher free radical–scavenging capacity compared to intravenous dosing”
Interesting, but it depends on which specific metabolites are present and their concentrations.
You could specify that the comparison is relative.
Molecular and Cellular Mechanisms of Action
-ROS as messengers (lines 330–344)
Statement is accurate, but it’s worth emphasizing that cellular toxicity can occur if ROS exceed physiological thresholds.
-MAPK (lines 424–450)
Correct, but transient activation of ERK/JNK/p38 in some tumor models should be interpreted as a stress response rather than a universal proliferative signal.
-“For example, Trybus et. al. demonstrated that For example, Trybus et al. demonstrated …”
Remove the duplication.
-Extrinsic–intrinsic crosstalk
Appropriate, but it typically occurs in combination with chemotherapeutic agents or stress conditions.
Emphasize that this is more frequent in combinatorial or sensitized scenarios.
-p53 dependence
Could include that the effect in primary human cells still requires clinical confirmation.
-ROS as mediators of apoptosis
Appropriate. Could emphasize that ROS attenuation (e.g., with NAC) partially reduces apoptosis, reinforcing functional relevance.
-Hepatic gene expression
Suppression of PEPCK and G6Pase is reported in murine models; extrapolation to humans requires caution.
-11β-HSD1 inhibition potency
The statement of “nanomolar potency” should be accompanied by a specific reference or experimental context (in vitro vs. in vivo).
-Expressions like “broadly effective metabolic modulator” should be qualified as preclinical, since clinical data are limited.
Therapeutic Potential in Disease Models
-Statement of ferroptosis as the main mechanism of emodin in CRC:
The text suggests that ferroptosis is a central in vivo mechanism, but most studies are still preclinical, and the physiological role of ferroptosis in animal models is not fully equivalent to humans. This may represent an over-extrapolation.
-Tumor microenvironment modulation (TAMs and CXCL1/CAF):
Although reported, the cited models are specific to murine xenotransplants. Generalizing this as a “universal” effect of emodin in solid tumors may be scientifically risky, since human tumor microarchitecture is more complex.
-Emodin as a chemosensitizer in leukemias:
It is stated that it increases sensitivity to Ara-C in AML, but the data are preclinical; mentioning “reducing leukemic burden and improving survival” could imply clinical relevance, which is not established.
-Comparison among anthraquinones:
The text implies that aloe-emodin, rhein, and diacerein have significant antitumor activity in vivo, but for most, clinical data are lacking. This could be seen as an over-extrapolation.
-Diacerein as a clinical benchmark:
Correct for osteoarthritis, but the text mixes data from rheumatoid arthritis and osteoarthritis, which may cause confusion since the inflammatory mechanisms differ.
-“Emodin as a pleiotropic metabolic modulator”:
Many effects (WAT beiging, AMPK, PPARα/γ, SREBP1, lipogenesis, lipolysis) are reported in HFD and T2DM models, but there is no solid clinical evidence for all these effects.
The text may give the impression that emodin is already clinically validated, which is not the case.
-Comparison with rhein and diacerein:
For rhein, renoprotective effects and inhibition of ferroptosis/EMT in DM models are mentioned, but these are animal studies, and the text mixes them with general metabolic effects, potentially confusing the reader.
-Aloe-emodin as an α-glucosidase inhibitor:
The statement that it “may be more potent than emodin for postprandial glycemia” needs context, as most data come from in vitro assays or very high in vivo doses, without clinical equivalence.
-Neuroprotection in Parkinson’s and Alzheimer’s:
The text mentions ferroptosis, UQCRC1, β-amyloid, and tau, but all are acute or preclinical models, often with relatively high doses.
The presentation could lead to the interpretation of human efficacy, which is not supported.
-Rhein in epilepsy and stroke:
It is stated that rhein is protective in PTZ and MCAO models, but these effects are experimental, with no human translation. Listing multiple mechanisms (TLR4/NF-κB, NRF2/GPX4, glutamine metabolism) may be an overstatement for the same compound.
-Mixing preclinical and clinical data without clear distinction:
In several instances, the wording gives the impression of clinical confirmation for emodin (oncology, metabolism, neuroprotection), whereas most evidence is preclinical.
-Exaggerated multimodality:
The text lists dozens of distinct pathways and effects for each compound, which is scientifically unlikely to occur simultaneously in all contexts. This may give a false impression of a “miracle drug.”
-Safety, Toxicity, and Limitations
-“These risks reflect a class effect of emodin-type anthraquinones rather than an idiosyncratic property of emodin alone.”
It is not fully demonstrated that toxicity represents a true “class effect.”
Danthron is clearly carcinogenic.
Rhein and diacerein have a different clinical profile.
Chrysophanol and physcion show much lower toxicity.
It would be better to frame this as a “shared structural liability” rather than a consolidated “class effect.”
-“On-demand pharmacology rather than intrinsic organ toxicity”
Conceptually interesting formulation, but it is not an established toxicological consensus.
It may sound like speculative mechanistic interpretation.
-“LD50 of oral anthraquinone administered to SD rats was >5000 mg/kg”
It does not specify which anthraquinone.
It mixes “anthraquinone” (class) with “emodin.”
It may lead to the impression that emodin alone has such a high LD50 — which depends on source and purity.
Greater specificity is required.
-“56% unabsorbed and excreted in feces as prototype”
This value may vary significantly depending on: The species; The formulation; The dose
Presenting it as a fixed number may be overly assertive.
-“2–3-fold increase in colorectal cancer risk”
This is one of the most sensitive statements.
Many studies are unable to clearly separate the effect of the laxative from that of chronic constipation.
The evidence is inconsistent.
Some meta-analyses show a weak and non-significant association.
The statement may be considered too strong without emphasizing the methodological controversy.
-“Purified anthraquinone itself was non-mutagenic”
Which anthraquinone?
Many simple anthraquinones are not genotoxic.
Others are clearly mutagenic.
This may generate confusion.
Strategies to Improve Therapeutic Utility
-“Parenteral delivery … often exceeding 85% systemic availability”
The sentence suggests that parenteral anthraquinone formulations consistently achieve >85% systemic bioavailability.
IV = 100% by definition, but:
SC/IM do not consistently guarantee 85%.
It depends on formulation, solubility, and local precipitation.
Few robust data are published for emodin or aloe-emodin via parenteral routes.
This may be considered an overly assertive and non-universal figure.
-“Concurrent metabolic inhibition is needed to vitally improve bioavailability”
Conclusion is based on a specific animal model.
It suggests that the solution is to inhibit metabolism.
It does not consider alternatives such as: Targeted delivery; Prodrug strategy; Lymphatic transport
This may be considered a mechanistic oversimplification.
-Nanocarriers “consistently improve” solubility, PK, tumor accumulation, efficacy
The use of the word “consistently” is scientifically risky.
Not all nanocarriers robustly improve tumor accumulation. The EPR effect is highly variable.
Many results come from: Murine subcutaneous models; Highly vascularized tumors; These do not translate to humans.
This may be considered an overgeneralization.
-Emodin + quantum dots
Safety issue omitted: Quantum dots often contain heavy metals (Cd, Se).
Potential systemic toxicity is not discussed.
They are not clinically established.
Presented as a technological advance without discussion of toxicological limitations.
- SLNs and “wider therapeutic window”
Based on selective in vitro cytotoxicity.
Does not equate to a real in vivo therapeutic window.
Systemic toxicological data are lacking.
- After Section 7, it jumps directly to Section 9!!
Conclusions
-“Innovative formulation platforms … have demonstrated the capacity to improve solubility and permeability, attenuate first-pass metabolism, and preferentially enhance drug accumulation in diseased tissues”
Generalization: not all nanoformulations demonstrate preferential accumulation in diseased tissues in humans.
Many data come from murine or in vitro models.
This could be softened to: “preclinical studies suggest these platforms may improve …”.
-“Move from broadly active but ‘difficult’ natural products to carefully engineered agents, whose pleiotropic beneficial properties offer a genuine advantage over existing therapies”
“Genuine advantage over existing therapies” is strong and speculative.
No current clinical study demonstrates therapeutic advantage in humans.
Better phrased as “potential advantage” or “may provide opportunities for therapeutic optimization.”
Author Response
Comment 1: -“Anthraquinone derivatives have demonstrated reproducible antiproliferative, anti‑inflammatory, metabolic, cardiovascular, antifibrotic, and immunomodulatory effects across diverse preclinical models”. “Reproducible” suggests robust and consistent effects. In reality:
- Many data are from in vitro and animal studies, often with non-physiological doses.
- Not all effects have been consistently replicated across laboratories or models.
Suggested softening: “have shown antiproliferative, anti-inflammatory … effects in preclinical studies.”
Response: We thank the reviewer for this important clarification. We have explicitly emphasized that the reported activities were observed in preclinical models and have replaced the term “reproducible” with the more appropriate phrasing “consistently reported across multiple preclinical studies,” which better reflects the current evidence base without overstating robustness
Comment 2: -“Emerging formulation strategies that decouple intrinsic bioactivity from pharmacokinetic limitations”
Be cautious with the slightly optimistic tone.
In preclinical studies, these systems improve bioavailability and targeting, but there is no clinical confirmation yet.
Suggestion: “emerging preclinical formulation strategies that aim to decouple …”.
Response: Response: We appreciate the reviewer’s suggestion and agree that the original wording could imply premature translational success. We have revised the sentence to “emerging preclinical formulation strategies that aim to decouple intrinsic bioactivity from pharmacokinetic limitations,” that more appropriately reflects the current stage of evidence.
Introduction:
Comment 3: -“Reproducible efficacy across tumor, inflammatory, and immunometabolic disease models”
The expression may sound stronger than the evidence allows.
Most of the data on emodin is preclinical (in vitro and animal models). Reproducibility across models is variable, and clinical evidence is limited.
It could be reformulated as something like “consistent preclinical efficacy.
Response: We thank the reviewer for this constructive comment. To avoid overstating the strength of the evidence, we have accordingly revised the phrasing to “consistent preclinical efficacy across tumor, inflammatory, and immunometabolic disease models”.
Comment 4: - Nanoemulsions that reduce glucuronidation during absorption
Nanoformulations generally improve bioavailability by increasing solubility and absorption, but they do not necessarily “reduce glucuronidation” directly. Glucuronidation is a hepatic and intestinal enzymatic process; formulation may alter pharmacokinetics, but it does not directly inhibit the process (unless specific data support that claim).
It would be more appropriate to say that it “may reduce presystemic metabolism and improve systemic exposure.”
Response: We thank the reviewer for this valuable clarification. The wording has been revised to avoid implying a direct inhibition of glucuronidation and now states that nanoemulsion-based formulations “may reduce presystemic metabolism and enhance systemic exposure,” providing a more accurate description of the formulation effects.
Comment 5: -Bioactivation pathways that generate more toxic metabolites (e.g., 5-hydroxyemodin)
5-Hydroxyemodin is described as an oxidative metabolite, but classifying it clearly as “more toxic” depends on the experimental context. Differential toxicity is not universally established.
It could be phrased as “potentially more reactive metabolites.”
Response: Thank you for your note. To avoid overgeneralization regarding differential toxicity, we have revised the wording to refer to “potentially more reactive metabolites (e.g., 5-hydroxyemodin),” which more accurately reflects the context-dependent nature of their biological effects.
Structural Features and SARs among Natural Anthraquinones
Comment: 6: - “This presence of a rigid, polyaromatic quinone core …”
“This presence” should be corrected to “The presence.”
Response: We thank the reviewer for noting this error and have corrected the phrasing from “This presence” to “The presence” in the revised manuscript.
Comment 7: -“Rhein introduces a carboxylic acid group … reducing passive membrane permeability”
Correct in principle (ionized acids cross membranes more slowly), but effective in vivo permeability also depends on active transport and protein binding.
Add nuance: “which may reduce passive membrane permeability.”
Response: We appreciate the reviewer’s suggestion. The sentence has been revised to state that the carboxylic acid group in rhein “may reduce passive membrane permeability,” acknowledging the influence of additional factors such as active transport and protein binding on in vivo disposition.
Comment 8: -“Physcion … removes one site for direct conjugative metabolism (e.g., glucuronidation) at that position”
Correct for the O-methylated position, but it is important to remember that metabolism can occur at other sites on the core or on other functional groups.
Suggestion: “…removes one site susceptible to direct conjugative metabolism.”
Response: We appreciate the reviewer’s insightful comment and have refined the wording to indicate that physcion “removes one site susceptible to direct conjugative metabolism,”
Comment 9: -“Electron donating methoxy and methyl groups tend to weaken antibacterial potency compared to their hydroxyl or carboxyl analogues”
Correct based on some studies, but activity depends on the target and the organism tested.
Include “in certain bacterial assays” for precision.
Response: We thank the reviewer for this clarification and have revised the sentence to specify that the reduced antibacterial potency applies in certain bacterial assays, reflecting context-dependent activity
Comment 10: - In the scheme of Figure 1B, change the substituents R1, R2, R3, and R4 to R1, R2, R3, and R4.
Response: We thank the reviewer for the suggestion. However, Figure 1B has been removed in the revised manuscript in accordance with another reviewer’s recommendation due to substantial overlap with Figure 2; therefore, the requested modification is no longer applicable.
Natural Occurrence and Major Sources
Comment 11: -“Sugar conjugation does not necessarily abolish biological activity”
Correct, but the degree of activity strongly depends on the enzyme substrate and intestinal microbiota.
You could add: “depending on hydrolysis efficiency and biological context.”
Response: We thank the reviewer for the suggestion. The sentence has been revised accordingly.
Comment 12: -Line 149: “occurence” → “occurrence”
Response: We thank the reviewer for noting this typographical error and have corrected “occurence” to “occurrence” in the revised manuscript.
Comment 13: Line 172: “Cassiae semen or coffee senna group” → check taxonomic consistency; “Cassiae semen” is more of a botanical drug name than a taxonomic group.
Response: We thank the reviewer for this important clarification. To ensure taxonomic accuracy, we have revised the phrasing to refer to species of the genus Cassia, with the clarification that their seeds are “commonly referred to as Cassiae semen in traditional medicine”, thereby distinguishing between the botanical classification and the pharmacognostic drug name.
Comment 14: - You have 2 numbered sections labeled with the number 3!
Response: We thank the reviewer for identifying this numbering error and have corrected the duplicate section numbering in the revised manuscript.
Physicochemical and pharmacokinetic characteristics of emodin
Comment 15: -“Extensive phase II metabolism … further reduce systemic exposure”
Minor grammatical adjustment: it should be “further reduces systemic exposure” (singular: metabolism).
Response: We thank the reviewer for noting this grammatical issue and have corrected the sentence to read “further reduces systemic exposure” in the revised manuscript.
Comment 16: -“Absolute oral bioavailability of emodin is approximately 2,8–3,2%”
The comma in 2,8–3,2% should be replaced with a point to follow the international standard: 2.8–3.2%.
Response: We thank the reviewer for pointing out this formatting issue and have corrected the values to use decimal points, now reported as “2.8–3.2%,” in accordance with international standards.
Comment 17: -“Serum metabolites exhibiting higher free radical–scavenging capacity compared to intravenous dosing”
Interesting, but it depends on which specific metabolites are present and their concentrations.
You could specify that the comparison is relative.
Response: We thank the reviewer for this helpful suggestion. We have revised the sentence accordingly.
Molecular and Cellular Mechanisms of Action
Comment 18: -ROS as messengers (lines 330–344)
Statement is accurate, but it’s worth emphasizing that cellular toxicity can occur if ROS exceed physiological thresholds.
Response: We thank the reviewer for this important clarification. We have emphasized that ROS signaling is concentration- and context-dependent, noting that excessive ROS levels can induce cellular toxicity, and this aspect has been further elaborated earlier in the paragraph to provide appropriate physiological context.
Comment 19: -MAPK (lines 424–450)
Correct, but transient activation of ERK/JNK/p38 in some tumor models should be interpreted as a stress response rather than a universal proliferative signal.
Response: We thank the reviewer and have revised the text to clarify that MAPK activation in some tumor models may reflect a stress response mechanism.
Comment 20: -“For example, Trybus et. al. demonstrated that For example, Trybus et al. demonstrated …”
Remove the duplication.
Response: We thank the reviewer for identifying this duplication and have removed the repeated phrase in the revised manuscript.
Comment 21: -Extrinsic–intrinsic crosstalk
Appropriate, but it typically occurs in combination with chemotherapeutic agents or stress conditions.
Emphasize that this is more frequent in combinatorial or sensitized scenarios.
Response: We thank the reviewer for this helpful clarification and have revised the text to emphasize that extrinsic-intrinsic apoptotic crosstalk is predominantly observed in sensitized or combinatorial settings.
Comment 22: -p53 dependence
Could include that the effect in primary human cells still requires clinical confirmation.
Response: We thank the reviewer for this important point and have added a clarification indicating that the p53-dependent effects observed in primary human cells remain to be clinically validated.
Comment 23: -ROS as mediators of apoptosis
Appropriate. Could emphasize that ROS attenuation (e.g., with NAC) partially reduces apoptosis, reinforcing functional relevance.
Response: We thank the reviewer for this helpful suggestion. It has been clarified in the text.
Comment 24: -Hepatic gene expression
Suppression of PEPCK and G6Pase is reported in murine models; extrapolation to humans requires caution.
Response: We thank the reviewer for this important point. We never suggested direct translation to humans and have clearly specified that the reported suppression of PEPCK and G6Pase was observed in in vivo murine models.
Comment 25: -11β-HSD1 inhibition potency
The statement of “nanomolar potency” should be accompanied by a specific reference or experimental context (in vitro vs. in vivo).
Response: The statement has been revised to specify the experimental context and now reads: “In biochemical in vitro and in vivo studies, emodin inhibited human and murine 11β-HSD1 at nanomolar potency….”
Comment 26: -Expressions like “broadly effective metabolic modulator” should be qualified as preclinical, since clinical data are limited.
Response: In the original text, the statement was explicitly framed within the context of in vivo preclinical models, and no clinical efficacy was implied.
Therapeutic Potential in Disease Models
Comment 27: -Statement of ferroptosis as the main mechanism of emodin in CRC:
The text suggests that ferroptosis is a central in vivo mechanism, but most studies are still preclinical, and the physiological role of ferroptosis in animal models is not fully equivalent to humans. This may represent an over-extrapolation.
Response: We thank the reviewer for this important clarification. We did not intend to imply equivalence to human physiology, and no direct extrapolation beyond preclinical models was made. The text explicitly refers to mechanistic findings in in vitro systems and xenograft-based in vivo CRC models, emphasizing ferroptosis as a contributing pathway within experimental contexts rather than as a definitive clinical mechanism.
“In xenograft‑based CRC models, emodin inhibited tumor growth by activating ferroptosis‑related pathways; mechanistic studies found increased lipid peroxidation, downregulation of GPX4, activation of NCOA4‑mediated ferritinophagy and inhibition of NF‑κB signaling both in vitro and in vivo, establishing ferroptosis as a bona fide contributor to emodin’s antitumor activity rather than a purely in vitro phenomenon”.
Comment 28: -Tumor microenvironment modulation (TAMs and CXCL1/CAF):
Although reported, the cited models are specific to murine xenotransplants. Generalizing this as a “universal” effect of emodin in solid tumors may be scientifically risky, since human tumor microarchitecture is more complex.
Response: No generalization or extrapolation beyond the specific experimental system was stated. The text explicitly refers to findings from a murine bladder cancer model and describes a defined CAF-TAM interaction within that context, without implying universality across solid tumors or direct relevance to human tumor microenvironments. The original text reads: "In a murine bladder cancer model, emodin significantly inhibited tumor growth by reducing tumor-associated macrophage (TAM) accumulation, an effect mechanistically linked to selective suppression of CXCL1 secretion by cancer-associated fibroblasts (CAFs). Disruption of this CAF-TAM axis impaired TAM migration and was essential for emodin’s antitumor activity, as tumor growth inhibition was lost when CXCL1-deficient CAFs were present".
Comment 29: -Emodin as a chemosensitizer in leukemias:
It is stated that it increases sensitivity to Ara-C in AML, but the data are preclinical; mentioning “reducing leukemic burden and improving survival” could imply clinical relevance, which is not established.
Response: Our statement never suggested clinical efficacy, it clearly implies that the improved survival refers to an AML xenograft model. “…as well as in acute myeloid leukemia (AML) xenograft model, where it reduced leukemic burden and improving survival via inhibition of Akt and ERK signaling”.
Comment 30: -Comparison among anthraquinones:
The text implies that aloe-emodin, rhein, and diacerein have significant antitumor activity in vivo, but for most, clinical data are lacking. This could be seen as an over-extrapolation.
Response: We fully agree with the reviewer’s comment. As stated in the manuscript, we explicitly note that while aloe-emodin, rhein, and diacerein demonstrate in vivo anticancer activity, their evidence base is more limited and indication-specific compared with emodin, and no clinical extrapolation was done.
Comment 31: -Diacerein as a clinical benchmark:
Correct for osteoarthritis, but the text mixes data from rheumatoid arthritis and osteoarthritis, which may cause confusion since the inflammatory mechanisms differ.
Response: We thank the reviewer for this important point. No extrapolation between rheumatoid arthritis and osteoarthritis was intended. The manuscript first addresses general preclinical efficacy in chronic joint inflammation, then discusses specific in vivo polyarthritis models (with methotrexate as a reference comparator), followed by separate experimental osteoarthritis models, and finally distinct clinical studies in rheumatoid arthritis and knee and hip osteoarthritis. The progression was structured to clearly distinguish between disease models and clinical contexts, and the text has been reviewed to ensure this distinction remains explicit.
Comment 32: -“Emodin as a pleiotropic metabolic modulator”:
Many effects (WAT beiging, AMPK, PPARα/γ, SREBP1, lipogenesis, lipolysis) are reported in HFD and T2DM models, but there is no solid clinical evidence for all these effects.
The text may give the impression that emodin is already clinically validated, which is not the case.
Response: We thank the reviewer for this important observation. The text has been revised to refer to emodin as a potential pleiotropic metabolic modulator, and it is clearly stated that the described effects are derived from in vivo preclinical models.
Comment 33: For rhein, renoprotective effects and inhibition of ferroptosis/EMT in DM models are mentioned, but these are animal studies, and the text mixes them with general metabolic effects, potentially confusing the reader.
Response: We thank the reviewer for this helpful clarification. The text has been revised to avoid conflating renal protective mechanisms with general metabolic effects and to explicitly frame the findings within preclinical diabetic nephropathy models. “More recent work indicates that rhein inhibits ferroptosis and epithelial-mesenchymal transition in diabetic nephropathy via modulation of Rac1/NOX1/β-catenin signaling, improving histological outcomes in diabetic mice.”.
Comment 34: -Aloe-emodin as an α-glucosidase inhibitor:
The statement that it “may be more potent than emodin for postprandial glycemia” needs context, as most data come from in vitro assays or very high in vivo doses, without clinical equivalence.
Response: We thank the reviewer for this important clarification. The sentence has been revised to explicitly reflect the predominantly preclinical nature of the evidence and to distinguish between antihyperglycemic effects and direct α-glucosidase inhibition.
Comment 35: -Neuroprotection in Parkinson’s and Alzheimer’s:
The text mentions ferroptosis, UQCRC1, β-amyloid, and tau, but all are acute or preclinical models, often with relatively high doses.
The presentation could lead to the interpretation of human efficacy, which is not supported.
Response: Thank you for your note. We have carefully reviewed the neuroprotection section to ensure that all mechanistic findings are explicitly framed as arising from preclinical cellular and animal models, and no human efficacy is implied.
Comment 36: -Rhein in epilepsy and stroke:
It is stated that rhein is protective in PTZ and MCAO models, but these effects are experimental, with no human translation. Listing multiple mechanisms (TLR4/NF-κB, NRF2/GPX4, glutamine metabolism) may be an overstatement for the same compound.
Response: Thank you for your note. In the revised manuscript, all neuroprotective effects of rhein are explicitly framed as experimental and preclinical, with no implication of human translation. The mechanisms are described in relation to specific disease models, namely ischemic stroke and PTZ-induced epilepsy.
” In an experimental model of ischemic stroke, rhein dose‑dependently improved neurological scores, reduced infarct volume and preserved blood‑brain barrier integrity through activation of the NRF2/SLC7A11/GPX4 axis and limiting oxidative stress and ferroptosis [149]. Finally, in preclinical PTZ‑induced epilepsy, rhein delayed seizure onset, reduced seizure severity, duration and frequency, and ameliorated associated neurological deficits, concomitant with inhibition of TLR4-NF‑κB signaling”.
Comment 37: -Mixing preclinical and clinical data without clear distinction:
In several instances, the wording gives the impression of clinical confirmation for emodin (oncology, metabolism, neuroprotection), whereas most evidence is preclinical.
Response: We have carefully reviewed the manuscript to ensure a clear and consistent distinction between preclinical (in vitro and in vivo) findings and clinical evidence.
Comment 38: -Exaggerated multimodality:
The text lists dozens of distinct pathways and effects for each compound, which is scientifically unlikely to occur simultaneously in all contexts. This may give a false impression of a “miracle drug.”
Response: we appreciate the reviewer’s concern. However, our use of “multimodal” strictly refers to the context-dependent engagement of distinct mechanistic pathways across different experimental models and disease settings; we never implied that anthraquinones act as “miracle compounds” and pathway modulation is clearly framed within specific preclinical contexts.
Comment 39: -Safety, Toxicity, and Limitations
-“These risks reflect a class effect of emodin-type anthraquinones rather than an idiosyncratic property of emodin alone.”
It is not fully demonstrated that toxicity represents a true “class effect.” Danthron is clearly carcinogenic. Rhein and diacerein have a different clinical profile. Chrysophanol and physcion show much lower toxicity. t would be better to frame this as a “shared structural liability” rather than a consolidated “class effect.”
Response: In accordance with the recommendation, we have revised the wording to refer to a shared structural liability among emodin-type anthraquinones rather than a definitive class effect
Comment 40: -“On-demand pharmacology rather than intrinsic organ toxicity”
Conceptually interesting formulation, but it is not an established toxicological consensus.
It may sound like speculative mechanistic interpretation.
Response: Thank you for your note. To avoid speculative interpretation, we have revised the phrasing to a more neutral description of context-dependent biological activity.
Comment 41: -“LD50 of oral anthraquinone administered to SD rats was >5000 mg/kg”
It does not specify which anthraquinone.
It mixes “anthraquinone” (class) with “emodin.” It may lead to the impression that emodin alone has such a high LD50 — which depends on source and purity.Greater specificity is required.
Response: The cited study specifically reports the oral toxicity of the parent anthraquinone scaffold compound (unsubstituted anthracene-9,10-dione) in Sprague Dawley rats, and no extrapolation to emodin or substituted anthraquinones was done. We have revised the text to explicitly refer to the scaffold compound, clearly stating that the reported LDâ‚…â‚€ refers to anthraquinone itself and not to its substituted derivatives.
“Acute and Subchronic Oral Toxicity of Anthraquinone in Sprague Dawley Rats” doi: 10.3390/ijerph191610413
Comment 42: -“56% unabsorbed and excreted in feces as prototype”
This value may vary significantly depending on: The species; The formulation; The dose
Presenting it as a fixed number may be overly assertive.
Response: Thank you for your note. The text has been revised to specify that the reported proportion of unabsorbed emodin derives from a rat model and to avoid presenting it as a fixed or universally applicable value. “ Conversely, pharmacokinetic studies in rats indicate that limited intestinal absorption can result in substantially higher luminal concentrations of emodin following oral administration, with approximately 56% of the administered dose recovered unchanged in feces under experimental conditions, which likely contributes to the pronounced local gastrointestinal effects associated with chronic anthraquinone exposure”.
Comment 43: -“2–3-fold increase in colorectal cancer risk”
This is one of the most sensitive statements. Many studies are unable to clearly separate the effect of the laxative from that of chronic constipation. The evidence is inconsistent. Some meta-analyses show a weak and non-significant association.The statement may be considered too strong without emphasizing the methodological controversy.
Response: We fully agree with the reviewer comment. We have been careful to present the colorectal cancer risk data in a balanced and cautious manner, explicitly acknowledging the heterogeneity of findings, the limited statistical significance in meta-analyses, and the potential confounding effect of chronic constipation, as reflected in the correspondent paragraph:
“Dedicated in vivo studies, as well as epidemiological data suggest a 2-3-fold increase in colorectal cancer risk with long-term anthraquinone laxative use (senna, cascara, rhubarb), although much of the evidence derives from complex plant preparations rather than pure compound [157], [158]. Conversely, a recent systematic review and meta-analysis of randomized controlled trials and observational studies reported an increased odds ratio that did not reach conventional statistical significance (pooled OR 1.4-1.5), but the directionality sustains ongoing concern and the need for careful interpretation and evaluation of confounding factors, as chronic constipation itself may increase colorectal cancer risk [159].”
Comment 44: -“Purified anthraquinone itself was non-mutagenic”
Which anthraquinone? Many simple anthraquinones are not genotoxic. Others are clearly mutagenic. This may generate confusion.
Response: As previously stated, the sentence refers specifically to the parent anthraquinone scaffold compound (unsubstituted anthracene-9,10-dione) rather than to substituted anthraquinone derivatives. We have revised the wording to explicitly indicate this and to avoid any potential confusion
Comment 45: Strategies to Improve Therapeutic Utility
-“Parenteral delivery … often exceeding 85% systemic availability”
The sentence suggests that parenteral anthraquinone formulations consistently achieve >85% systemic bioavailability.
IV = 100% by definition, but:
SC/IM do not consistently guarantee 85%.
It depends on formulation, solubility, and local precipitation.
Few robust data are published for emodin or aloe-emodin via parenteral routes.
This may be considered an overly assertive and non-universal figure.
Response: We fully agree with the reviewer’s comment. The originally cited value (85%) was taken directly from the referenced study; however, to avoid implying a universal or consistent level of systemic availability across parenteral routes and formulations, we have revised the sentence to remove the specific percentage.
Comment 46: -“Concurrent metabolic inhibition is needed to vitally improve bioavailability”
Conclusion is based on a specific animal model.
It suggests that the solution is to inhibit metabolism.
It does not consider alternatives such as: Targeted delivery; Prodrug strategy; Lymphatic transport
This may be considered a mechanistic oversimplification.
Response: We thank the reviewer for this comment and have revised the sentence to clarify that the observation applies to a particular experimental model rather than representing a generalizable strategy.
Comment 47: -Nanocarriers “consistently improve” solubility, PK, tumor accumulation, efficacy
The use of the word “consistently” is scientifically risky.
Not all nanocarriers robustly improve tumor accumulation. The EPR effect is highly variable.
Many results come from: Murine subcutaneous models; Highly vascularized tumors; These do not translate to humans.
This may be considered an overgeneralization.
Response: We thank the reviewer for this important point and agree that the term “consistently” may overstates the current evidence. We have revised the wording by replacing it with “often” to more accurately reflect the variability observed across preclinical models and experimental conditions.
Comment 48: -Emodin + quantum dots
Safety issue omitted: Quantum dots often contain heavy metals (Cd, Se).
Potential systemic toxicity is not discussed.
They are not clinically established.
Presented as a technological advance without discussion of toxicological limitations.
Response: We thank the reviewer for raising this important safety consideration. We have clarified that the discussed nanoplatforms are carbon-based emodin-derived carbon dots, rather than conventional heavy-metal quantum dots, which are associated with systemic toxicity concerns. The text has been revised to explicitly distinguish these biocompatible carbon nanomaterials and to acknowledge that, despite promising preclinical efficacy, their long-term safety, biodistribution, and clinical translatability remain to be fully evaluated.
Comment 49: - SLNs and “wider therapeutic window”
Based on selective in vitro cytotoxicity.
Does not equate to a real in vivo therapeutic window.
Systemic toxicological data are lacking.
Response: We thank the reviewer for this important note. The sentence has been revised to avoid implying an in vivo therapeutic window and to explicitly frame the findings within in vitro selectivity data.
Comment 50: - After Section 7, it jumps directly to Section 9!!
Response: We thank the reviewer for identifying this numbering error. We have revised the manuscript to correct the section sequence and ensure consistent numbering throughout the text.
Comment 51: Conclusions
-“Innovative formulation platforms … have demonstrated the capacity to improve solubility and permeability, attenuate first-pass metabolism, and preferentially enhance drug accumulation in diseased tissues”
Generalization: not all nanoformulations demonstrate preferential accumulation in diseased tissues in humans.
Many data come from murine or in vitro models.
This could be softened to: “preclinical studies suggest these platforms may improve …”.
Response: Thank you for your note. According to your recommendation, we have revised the sentence to reflect that these effects are suggested by preclinical studies.
Comment 52: -“Move from broadly active but ‘difficult’ natural products to carefully engineered agents, whose pleiotropic beneficial properties offer a genuine advantage over existing therapies”
“Genuine advantage over existing therapies” is strong and speculative.
No current clinical study demonstrates therapeutic advantage in humans.
Better phrased as “potential advantage” or “may provide opportunities for therapeutic optimization.”
Response: We thank the reviewer for this constructive suggestion and have accordingly revised the wording by replacing “genuine advantage” with “potential advantage” to avoid speculative clinical implication.
Reviewer 4 Report
Comments and Suggestions for AuthorsReview Report
concerning the manuscript entitled „Emodin and the Anthraquinone Scaffold: Therapeutic Promise and Strategies to Overcome Translational Barriers”
by Rositsa Mihaylova and co-workers
Manuscript ID: molecules-4155344
General Comments:
The manuscript is a comprehensive and mostly well-organized review of emodin and the "anthraquinone scaffold," with a particular emphasis on translational barriers (ADME, first-pass metabolism, transporters, dose-dependent toxicity) and formulation/chemical strategies to circumvent them. The purpose and scope are clearly outlined in the abstract and introduction. A strength is the attempt at an "exposure-aware" interpretation (low bioavailability vs. high intestinal luminal concentrations) and the ambiguity of the context- and dose-dependent effects (protective vs. damaging). At the same time, the text requires refinement of the review methodology (transparency in the selection of references and criteria), more tabular syntheses, and editorial revision (visible typos and syntactic errors).
Major Comments:
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Figure 5 is based on a qualitative, point-based "scoring" of 0–4 and a description of keywords and criteria, but without full transparency. In my opinion, a list of included studies for each domain, the number of papers, rules for resolving contradictions, or even a table supporting the assigned results are missing.
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The text mentions key values: oral bioavailability of emodin ~2.8–3.2% and ~50–60% of the dose unabsorbed and excreted in feces, as well as the dominant role of glucuronidation/sulfation and the predominance of conjugated metabolites in plasma. However, it would be worthwhile to add a "PK dashboard" table (species, dose, route, Cmax/AUC/t½, free fraction, main metabolites, target tissues). This would significantly strengthen the translational message.
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Drug interactions are well described, but the "clinical relevance" should be clarified. The ICâ‚…â‚€ for CYPs is provided, along with a description of UGT/transporter modulation and possible interactions (e.g., P-gp/MRP2/3). However, I suggest adding a commentary on the exposures (plasma vs. gut) at which these interactions may be clinically feasible; separating the risk of systemic from intestinal interactions.
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Cremophor EL nanoemulsion, which reduces glucuronidation and increases permeability, is an interesting example, but at the same time, Cremophor has its own safety/tolerability limitations, which should be clearly discussed.
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The authors note that parenteral administration can result in very high availability (often >85%), but is limited by poor solubility and the risk of damage at the injection site. In my opinion, it would be worthwhile to add a short "developability" section (scalability, excipients, stability, CMC).
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The topic of diacerein as a clinically validated derivative (RA add-on, EULAR/ACR20) is valuable, as is the OA meta-analysis, but please clearly state in the section abstracts that this evidence is for diacerein/rhein, not emodin (to prevent the reader from automatically drawing conclusions).
Minor Comments:
- The bibliography frequently uses the notation "et al." Please replace it with the authors' first and last names.
- References are not prepared in accordance with editorial requirements. Please review these requirements and correct all references.
- Please use a single format for the title of cited references: either all words are capitalized or only the first word of the title.
- Numerous typos and similar errors, e.g., "Trybus et al. demonstrated that For example, Trybus...", "posess prominant," "efficacy and tolerability," etc.
- In several places, sentences are very long; consider shortening and more frequent use of tabular summaries (especially PK/toxicology/formulations).
- Inconsistent notation, e.g., "et al." vs. "et al," (µM vs. uM, etc.).
Recommendation: Minor revision. The manuscript demonstrates high substantive value, but refinement of the evidence assessment methodology, tabular syntheses, and linguistic corrections is needed.
Author Response
General Comments:
Comment 1: The manuscript is a comprehensive and mostly well-organized review of emodin and the "anthraquinone scaffold," with a particular emphasis on translational barriers (ADME, first-pass metabolism, transporters, dose-dependent toxicity) and formulation/chemical strategies to circumvent them. The purpose and scope are clearly outlined in the abstract and introduction. A strength is the attempt at an "exposure-aware" interpretation (low bioavailability vs. high intestinal luminal concentrations) and the ambiguity of the context- and dose-dependent effects (protective vs. damaging). At the same time, the text requires refinement of the review methodology (transparency in the selection of references and criteria), more tabular syntheses, and editorial revision (visible typos and syntactic errors).
Response: We thank the reviewer for the thorough and constructive evaluation of our manuscript and for the positive assessment of its scope, organization, and translational perspective, particularly the exposure-aware interpretation and emphasis on context-dependent effects. In response to the suggestions provided, we have clarified and expanded the review methodology to improve transparency (Section 6.1 of the revised manuscript), added a new evidence flow chart (Figure 5 in the revised file), and carefully revised the text to correct typographical and syntactic issues throughout.
Major Comments:
Comment 2: Figure 5 is based on a qualitative, point-based "scoring" of 0–4 and a description of keywords and criteria, but without full transparency. In my opinion, a list of included studies for each domain, the number of papers, rules for resolving contradictions, or even a table supporting the assigned results are missing.
Response: We thank the reviewer for this important remark. To improve transparency, we have added a methodology Subsection 6.1 to clearly describe the search strategy, screening process, evidence stratification and scoring criteria, and we have built an additional Figure 5 as an evidence flow chart summarizing included and excluded studies across domains. While we do not present the manuscript as a formal PRISMA-compliant systematic review, we have structured the Section on the therapeutic potential of anthraquinones as an evidence-mapping review with semi-quantitative synthesis, which is now clearly stated and justified in Section 6.1.
Comment 3: The text mentions key values: oral bioavailability of emodin ~2.8–3.2% and ~50–60% of the dose unabsorbed and excreted in feces, as well as the dominant role of glucuronidation/sulfation and the predominance of conjugated metabolites in plasma. However, it would be worthwhile to add a "PK dashboard" table (species, dose, route, Cmax/AUC/t½, free fraction, main metabolites, target tissues). This would significantly strengthen the translational message.
Response: Thank you for the constructive suggestion. In response, we now provide a focused PK summary table (Table 1) for emodin, detailing key parameters (dose, route, Cmax, AUC, t1/2, bioavailability, and dominating metabolites in plasma) from the available open‑access animal studies. The other anthraquinones were not tabulated because their quantitative PK datasets are sparse, derived from heterogeneous preparations (single compounds versus complex extracts) and often lack core parameters needed for meaningful comparison (risk of over‑interpretation of limited data).
Comment 4: Drug interactions are well described, but the "clinical relevance" should be clarified. The ICâ‚…â‚€ for CYPs is provided, along with a description of UGT/transporter modulation and possible interactions (e.g., P-gp/MRP2/3). However, I suggest adding a commentary on the exposures (plasma vs. gut) at which these interactions may be clinically feasible; separating the risk of systemic from intestinal interactions.
Response: We appreciate this important point and have clarified the likely clinical relevance of these interactions: “However,circulating free concentrations after oral dosing are usually far below these ICâ‚…â‚€ values, whereas intestinal and enterocyte exposures are likely much higher; thus, clinically meaningful drug interactions are more plausible at the level of the gut wall and biliary/renal transport than via systemic CYP inhibition, and will depend critically on formulation and dose”.
Comment 5: Cremophor EL nanoemulsion, which reduces glucuronidation and increases permeability, is an interesting example, but at the same time, Cremophor has its own safety/tolerability limitations, which should be clearly discussed.
Response: We thank the reviewer for the note and have revised the text to acknowledge the known safety and tolerability limitations of Cremophor EL while maintaining the mechanistic findings: … “however, its well-documented hypersensitivity reactions and tolerability concerns in paclitaxel formulations may limit clinical applicability despite its favorable pharmacokinetic effects”.
Comment 6: The authors note that parenteral administration can result in very high availability (often >85%), but is limited by poor solubility and the risk of damage at the injection site. In my opinion, it would be worthwhile to add a short "developability" section (scalability, excipients, stability, CMC).
Response: We thank the reviewer for this thoughtful suggestion. While we agree that developability considerations such as scalability, excipient selection, stability and CMC are important for translation, the diversity of formulation platforms and excipients that can be used for any compounds (including anthraquinones) is exceptionally broad, and their effects on bioavailability, safety, and stability are highly formulation-specific and difficult to generalize without compound- and system-specific experimental data. To avoid speculative or overly generic discussion, we have therefore focused the section on technological approaches that have been directly explored and supported by reported research studies. We believe this maintains scientific rigor while keeping the scope of the review appropriately evidence-based.
Comment 7: The topic of diacerein as a clinically validated derivative (RA add-on, EULAR/ACR20) is valuable, as is the OA meta-analysis, but please clearly state in the section abstracts that this evidence is for diacerein/rhein, not emodin (to prevent the reader from automatically drawing conclusions).
Response: We thank the reviewer for this important clarification and have ensured throughout the revised manuscript that all clinical evidence is explicitly attributed to diacerein, and not to emodin, thereby preventing any unintended extrapolation across anthraquinone derivatives.
Minor Comments:
Comment 8: The bibliography frequently uses the notation "et al." Please replace it with the authors' first and last names.
Comment 9: References are not prepared in accordance with editorial requirements. Please review these requirements and correct all references.
Comment 10: Please use a single format for the title of cited references: either all words are capitalized or only the first word of the title.
Response: We thank the reviewer for carefully noting these formatting issues. The reference list has been thoroughly revised to comply fully with the journal’s editorial requirements. We have replaced abbreviated author listings where required, ensured consistent formatting of author names, and standardized the capitalization style of article titles throughout the bibliography.
Comment 11: Numerous typos and similar errors, e.g., "Trybus et al. demonstrated that For example, Trybus...", "posess prominant," "efficacy and tolerability," etc.
Response: We thank the reviewer for highlighting these issues and have carefully proofread the manuscript to correct all typographical errors, duplicated phrases, and language inconsistencies throughout the text.
Comment 12: In several places, sentences are very long; consider shortening and more frequent use of tabular summaries (especially PK/toxicology/formulations).
Response: We thank the reviewer for this helpful suggestion. We have revised overly long sentences to improve clarity and readability and have incorporated additional tabular summaries (Table 1) to present complex information more concisely.
Comment 13: Inconsistent notation, e.g., "et al." vs. "et al," (µM vs. uM, etc.).
Response: Response: We thank the reviewer for noting these inconsistencies and have standardized all notations and abbreviations throughout the manuscript to ensure uniform formatting and clarity.
Comment 14: Recommendation: Minor revision. The manuscript demonstrates high substantive value, but refinement of the evidence assessment methodology, tabular syntheses, and linguistic corrections is needed.
Response: We thank the reviewer for the positive evaluation and recommendation. In response, we have refined the evidence assessment methodology, expanded tabular syntheses to improve data clarity, and carefully revised the manuscript to address linguistic and formatting issues throughout.
Round 2
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors have addressed all the questions and comments raised by the reviewers. They have also implemented all the suggested revisions. For this reason, the manuscript can be accepted for publication.
