Bioactive Profiling of Cowpea Pods via Optimized Extraction and Experimental–Computational Approaches

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Editor, 

Thank you for the opportunity to review the manuscript titled “Unveiling the Bioactive Potential of Cowpea Pods Through Optimized Extraction and Integrated Experimental-Computational Analysis.” This study investigates the optimized extraction of antioxidant-rich phenolic compounds from cowpea pods using ultrasound-assisted methods, supported by computational modeling. After careful consideration, I have a few major comments that I believe will help strengthen the manuscript.

Comments for Authors

Title

Please consider revising the title for clarity and brevity. A more concise version such as "Bioactive Profiling of Cowpea Pods via Optimized Extraction and Experimental–Computational Approaches" improves readability while retaining the scientific focus.

Abstract

Begin with a clearer statement of the value of cowpea pods as an underutilized by-product, emphasizing sustainability or waste valorization.

Clarify the flow between steps (extraction → fractionation → profiling → computational analysis). Transitional phrases would help the reader follow the workflow more smoothly.

Briefly state what was calculated in the DFT analysis (e.g., HOMO–LUMO gap, bond dissociation enthalpy) and which radicals or antioxidant mechanisms were studied.

Add a sentence at the end that links the findings to practical applications, such as their relevance for antioxidant-rich formulations in industry.

Keywords

Add "cowpea pods" and specific phenolics (e.g., morin, caftaric acid) to the keyword list for better indexing.

Introduction

There are repeated mentions of antioxidant potential and phenolic compounds (PCs) across multiple paragraphs. Consider merging or condensing sentences to avoid redundancy (e.g., paragraphs 2 and 3 can be partially merged).

Transitions between some sections (e.g., from extraction to chromatography, and from chromatography to DFT) could be made smoother to enhance logical flow and coherence.

While the rationale is strong, the specific knowledge gaps this study addresses is not sharply defined. For instance, is it the lack of information on cowpea pod fractions? Or limited studies linking extract composition to theoretical antioxidant predictions?

The conclusion of the introduction is informative but could be more assertive about the novelty and impact of combining fractionation with QTAIM and DFT to characterize cowpea pod bioactives.

Material and Methods

Consider specifying the harvest time or growth stage of the cowpea pods if available, as bioactive compound content can vary with maturity. Was any post-harvest treatment (e.g., desiccation method) performed before drying?

The expression “60 mesh sieve or 250 μm” could be better written as “60-mesh sieve (250 μm)” for clarity.

Revise “kept hermetically closed” to “stored in airtight containers” for better readability.

While HIU was applied, it is unclear whether the temperature during treatment was monitored or controlled. This is critical as high ultrasound energy can cause thermal degradation.

There is an inconsistency in variable labeling — two parameters are referred to as X1. This typographical oversight should be corrected to maintain the mathematical integrity of the design.

Although a Box-Behnken design (BBD) is applied, the text does not describe the design matrix, number of experiments, or levels per variable (other than "5 levels"). For reproducibility and statistical robustness, details such as the number of center points and replicates are essential.

The sentence “until the complete remove of the organic” is grammatically incorrect and lacks detail. Specify how ethanol removal was confirmed — e.g., by constant weight, odor check, or residual solvent analysis (e.g., GC-FID).

The 2-hour reaction time is longer than typical protocols (30–90 min) for the FC assay. Unless supported by kinetic data or literature validation, this extended duration could introduce overdevelopment artifacts.

You note “triplicate analysis” — clarify whether this refers to injection replicates, extraction replicates, or both.

Results

The impact of amplitude and extraction time is discussed purely statistically. Mechanistic reasoning for why certain conditions favor or do not favor phenolic release is missing.

The manuscript lacks Pearson correlation plots or regression validation visuals that could support the link between phenolic content and AOC.

Yield differences across fractions were noted, but no discussion was provided regarding matrix interactions or compound polarity influencing elution behavior. Consider briefly explaining why E2/W2, despite lower mass yields, show higher PC concentrations.

The role of molecular weight or polarity in elution profiles is not discussed, despite clear differences in compound distributions across fractions.

All compound identifications rely on retention times and external standards. While this is acceptable, the absence of mass spectral confirmation limits confidence in certain identifications, especially new findings (e.g., tyrosol, proanthocyanidin B1/B2).

The same conclusions regarding SPLET preference and morin superiority are repeated in multiple paragraphs. A more synthesized and concise narrative is needed.

Consider presenting a comparative table or visual chart that correlates computational AOC ranking with experimental AOC for easier comparison (e.g., morin > caffeic ≈ caftaric > p-coumaric).

The discussion is too data-heavy and lacks critical interpretation. Separating it into a standalone "Discussion" section would improve clarity and allow for deeper analysis, broader contextualization, and reflection on limitations. Currently, the integration of results and discussion limits the manuscript’s ability to connect findings to practical applications or theoretical implications.

Table 2

The column headers are not clearly separated, and the formatting appears cluttered. For example, "Model Experimental Error %" should be visually distinct for both TPC and AOC values.

The experimental AOC value (1.21 mmol TE/g) is slightly lower than predicted (1.34), yet the % error is listed as 9%, which seems a bit high for a desirability score of D=1.00. Consider rechecking this value or clarifying the CV% calculation method.

Table 3

Superscripts (letters) denoting significance (e.g., "A", "a", "b") are not clearly explained at the bottom of the table. A proper key (e.g., “Values with the same letter are not significantly different at p > 0.05”) should be explicitly stated.

For some values, such as TFC in E3 (18 ± 3a) and Eo (16 ± 8Aab), the variability overlaps, yet both are marked with "a" or "ab." This raises doubt on whether post-hoc test grouping was properly represented.

Conclusion

The conclusions largely reiterate the findings without outlining future directions, such as in vivo validation, formulation into functional foods or nutraceuticals, or evaluation of cost-effectiveness and industrial scalability.

The sustainability aspect (valorizing agricultural waste) is underemphasized. More emphasis on circular economy or waste-to-value transformation would improve relevance.

Author Response

Please find attached the detailed point-by-point response to the reviewers' comments.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Please see the attachment.

Comments for author File: Comments.pdf

Author Response

Please find attached the detailed point-by-point response to the reviewers' comments.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

Please, consider the following suggestions for manuscript revision:

L76 - I recommend the authors rephrase the part ''  for separating and enriching bioactive constituents''. Please, consider that the fractionation using Sephadex allow the selection of molecules from the input extract based on molecular weight, resulting in a fraction abundant in determined compound.

L114 - What do authors mean  with ''...on the bioactive power of cowpea pod extract''? Please, introduce the response variable (TPC, AOC?) here.

L120 - Please, write the unit used in ratio (g/ml?) after '' 1:15''

L143-146 - Please, avoid unnecessary repetition (L107-110); lines 150-168 - Include in one section after the 2.4 topic. Also, correct similar mistakes throughout the text (like in L-263-264, and so on). 

L265 - This part is related to the M&M section. Double check the need of including this.

L330-333 - This is not the correct justification. Ideally, authors might consider the target compounds and the matrix extracted to select the washing solvents.

Table 3 - Double check the yield calculations. As a sum, the authors might consider the yield of fractions to be compared to the input (W0 or E0)

Table 3 - Write the meaning of ''ND'' acronym.

L505-506 - The authors refer ''studies'', but only one study was cited.

3.5 - I recommend authors elaborate this section by improving its connection with the additional topics addressed. Additionally, highlight how the most abundant phenolics were accessed by this method. Did authors use HPLC analysis an an input? It is not clear. 

 

Author Response

Please find attached the detailed point-by-point response to the reviewers' comments.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have adequately addressed all of my comments; therefore, I have no additional remarks.

Author Response

We sincerely thank the reviewer for their positive feedback and appreciation of our revisions. We are grateful for your thoughtful comments, which helped improve the quality and clarity of the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

I sincerely thank the authors for their detailed response and the effort invested in revising and clarifying the manuscript. .

However, I respectfully maintain my original assessment that the manuscript does not align with the agronomic core of the journal Agronomy, even in the context of a Special Issue . While the topic of agricultural waste valorization is important and interdisciplinary, the study remains primarily analytical and focused on bioactive compound extraction, with only limited agronomic context and no evaluation of crop performance,, yield components, or production-related outcomes relevant to agricultural practice.

The authors note that they have previously published a similar article in Agronomy on cowpea extract profiling. I believe this further weakens the novelty of the current manuscript and reinforces the view that this line of work may be more appropriate for journals specialized in food chemistry, plant-derived bioactives, or sustainable materials. Publishing multiple similar studies in agronomy, with limited agronomic experimentation, may also raise concerns about scope dilution.

Finally, I understand that other reviewers may hold different views, which I fully respect. However, based on my interpretation   of the journal"s aims and standards, I continue to believe that this manuscript, in its current form, is not suitable for publication in Agronomy.

Author Response

We sincerely thank the reviewer for taking the time to evaluate our manuscript and for the thoughtful feedback provided. We truly appreciate your perspective.

We consider that the originality of this work lies in the integration of experimental and theoretical methodologies to explore the valorisation of cowpea pods, an underutilized agricultural by-product, thus contributing to the broader conversation on sustainable use of agro-waste.

We remain grateful for your comments, which have helped us refine and strengthen the manuscript.