Identification of Active Anti-Inflammatory Compounds in Sweet Potato Storage Roots Extracted with Ethanol via LC-Q-TOF-MS

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors successfully employed an integrated LC/MS Q-TOF approach to identify anti-inflammatory compounds in sweet potato ethanol extracts, specifically β-sitosterol and campesterol, and validated their bioactivity in a relevant cell-based assay. The application of high-resolution mass spectrometry, coupled with computational tools (MS-DIAL, MS-FINDER) and rigorous confirmation against authentic standards, is a major strength. The research objective is clearly addressed, the methodology is appropriate, and the data robustly support the conclusion that these phytosterols contribute to the observed anti-inflammatory effects. However, the manuscript requires minor revisions to enhance its clarity, accuracy, and overall presentation before it is acceptable for publication. The primary concerns are the correction of a critical error in Figure 1's caption, refinement of language and terminology consistency, and the provision of additional methodological details (e.g., rationale for concentration selection in bioassays). Once these specific points are addressed, I believe the manuscript will be a valuable contribution to the field.

 

1. Lines 9–13: The sentence "However, the causative compounds responsible for the anti-inflammatory effect have not yet been identified" clearly articulates the research gap and establishes a solid foundation for the study.
2. Lines 14–15: For the phrase "using liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC/MS Q-TOF)", it is recommended to unify the instrument abbreviation to a more commonly used format such as LC-Q-TOF-MS and provide its full name upon first mention.
3. Lines 25–27: The result statement "In contrast, lauroyl diethanolamide did not similarly suppress LPS-induced IL-6 production" is clear. However, it is advisable to briefly explain the significance of this negative result in the abstract’s conclusion section to make the conclusion more comprehensive.
4. Lines 48–51: The sentence "Our previous findings demonstrated that... however, the specific bioactive compounds responsible for this activity have not yet been elucidated" effectively bridges previous research and justifies the necessity of the present study.
5. Line 65: The research objective stated in "The present study aimed to identify constituents..." is clear. It is recommended to add a sentence before this line to specify the integrated mass spectrometry-based approach to be adopted (e.g., mentioning tools like MS-DIAL or MS-FINDER), thereby highlighting the technical roadmap.
6. Lines 90–93: There is a critical error in the figure legend of Figure 1. The legend describes "lauroyl diethanolamide (A, B), lauroyl diethanolamide (C, D), and campesterol (E, F)". Based on the figure content and context, this is obviously a typo and should be corrected to "lauroyl diethanolamide (A, B), β-sitosterol (C, D), and campesterol (E, F)". This is a key error requiring priority correction.
7. Lines 100–103: The description of retention time (RT) differences (e.g., "-0.04 to 0.02 min") meets conventional requirements. However, it is suggested to cite the ±25 min standard proposed by Pérez-Ortega et al. in the main text or table notes to provide direct literature support for data reliability.
8. Lines 122–125: The table footnote of Table 1 states "The brackets for the product ions represent the relative intensity ratio". This information is crucial for interpreting the table and is presented in a clear and understandable manner.
9. Lines 136–138: The result statement "β-Sitosterol suppressed IL-6 production at concentrations equivalent to those present in SP-EtOH-Ex... Similarly, campesterol suppressed..." is accurate. It is recommended to replace "equivalent to" with the more rigorous term comparable to, as the concentrations used in cell experiments represent a range rather than an exact match.
10. Line 140: The figure legend of Figure 4, "Lau, Sit, and Cam values indicate concentration", is somewhat ambiguous. It is advisable to revise it to "Concentrations of Lau (lauroyl diethanolamide), Sit (β-sitosterol), and Cam (campesterol) are indicated on the x-axis (ng/mL or μg/mL)" to avoid misinterpretation.
11. Lines 175–178: The sentence "These findings highlight the importance of using multiple tools to enhance the identification accuracy" provides an excellent summary of the advantages of integrating multiple analytical tools.
12. Lines 195–197: The statement "These reports agree with the results obtained in this study" adequately demonstrates consistency with literature data. It is suggested to further specify the exact points of consistency between the study’s data and literature values (e.g., key fragment ions) instead of using the general term "agree".
13. Lines 221–231: The discussion of the study’s limitations is comprehensive and sincere, reflecting a spirit of scientific self-criticism and serving as a highlight of this section.
14. Lines 234–237: The sentence "Briefly, sweet potato storage roots used in this study were washed in water, and freeze dried, and then pulverized" has room for improvement in conjunction usage. It is recommended to revise it to "were washed in water, freeze-dried, and then pulverized".
15. Lines 237–238: The sentence "All sweet potatoes used in this study were of the cultivar Beniharuka" presents key information but is prone to being overlooked in its current position. It is strongly recommended to move the cultivar information to the beginning of Section 2.1 "Annotation of Nine Compounds" or the sample description section, as cultivar is a primary factor influencing chemical composition.
16. Lines 286–290: The detailed description of data processing parameters (e.g., "MS1 tolerance, 0.01 Da") ensures the reproducibility of the experiments.
17. Throughout the manuscript: The formatting of terminology needs standardization. For example, "lipopolysaccharide(LPS)" is sometimes written with a space and sometimes without (e.g., Lines 11 vs. Line 44). It should be uniformly revised to lipopolysaccharide (LPS).
18. Lines 304–308: The long sentence "Chromatographic separation was performed using... for all remaining standards" is recommended to be split into two shorter sentences to enhance readability.

Author Response

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Reviewer 2 Report

Comments and Suggestions for Authors

This study aimed to identify anti-inflammatory components in ethanol extracts of sweet potato storage roots using LC/MS Q-TOF, with a focus on the identification and functional validation of β-sitosterol and campesterol. Although the experimental design is complete and the methodology is standardized, the study shows significant deficiencies in scientific novelty, innovation, and academic contribution. The main issues are as follows:

1. Lack of novelty and innovation
   • (1) The presence of phytosterols (including β-sitosterol and campesterol) in sweet potatoes has been extensively documented, e.g., Food Science and Human Wellness2024, DOI: 10.26599/fshw.2022.9250225; Journal of Agricultural and Food Chemistry 2018, DOI: 10.1021/acs.jafc.8b03305; Trends in Food Science & Technology 2021, DOI: 10.1016/j.tifs.2021.07.001.
   • (2) The anti-inflammatory activity of phytosterols is well-established.
     The mechanisms of β-sitosterol and campesterol have been widely studied in various cellular and animal models, involving signaling pathways such as NF-κB, MAPK, and PPARγ (e.g., Journal of Biochemical and Molecular Toxicology2025, DOI: 10.1002/jbt.70287; Antioxidants 2023, DOI: 10.3390/antiox12020391; Frontiers in Pharmacology 2024, DOI: 10.3389/fphar.2023.1346054; Pakistan Journal of Health Sciences 2023, DOI: 10.54393/pjhs.v4i05.792). This study only verifies their effect on inhibiting IL-6 in LPS-stimulated RAW264.7 cells and does not provide any new mechanistic or target insights, essentially representing a “repeated validation of known compounds in a known model.”
   • (3) Insufficient exploration of sweet potato specificity.
     Sweet potatoes contain various known anti-inflammatory components besides phytosterols, such as anthocyanins, carotenoids, and polysaccharides. This study does not investigate potential synergistic effects between phytosterols and other constituents, nor does it justify why sweet potato was selected as a distinctive source of phytosterols.
2. Limited scientific contribution; fails to advance the field
   • The study essentially follows a routine “compound identification + activity validation” workflow, without proposing new hypotheses, methods, or insights. The techniques employed (LC/MS Q-TOF, MS-FINDER, MassBank) are standard in metabolomics and do not offer methodological innovation.
3. Severe lack of mechanistic investigation
   • Only IL-6 was measured as an inflammatory marker, while other key mediators such as TNF-α, IL-1β, COX-2, and iNOS were not examined. No signaling pathway or molecular mechanism experiments were conducted. Consequently, the study cannot address the core scientific question: “How do these phytosterols exert their anti-inflammatory effects?”
4. Preliminary experimental design
   • Only a single cell line (mouse RAW264.7 macrophages) was used, and the sample sources were limited (one cultivar from two regions). Moreover, no positive controls (e.g., dexamethasone or established anti-inflammatory phytosterol preparations) were included, making it difficult to evaluate the potency of the observed activity. Overall, the experimental approach remains superficial.
5. Lack of food science and nutritional perspective
   • Although positioned within food chemistry, the study does not integrate food science or nutritional viewpoints. It fails to experimentally explore or discuss the potential application and challenges of using sweet potato-derived phytosterols in functional foods.

Author Response

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Reviewer 3 Report

Comments and Suggestions for Authors

The submission reports the results of a study aimed at identifying active anti-inflammatory compounds in sweet potato storage roots extracted with ethanol via Q-TOF LC/MS.
In my opinion, the work is well-structured, the experimental approach is correct, and the results are accurately reported and discussed.
I suggest a very minor revision:

• Reference 7 is not complete. (Nutrients 2024 Feb 18;16(4):563.  doi: 10.3390/nu16040563.)
• Could authors better specify how many chromatographic runs were performed for each extract?

Author Response

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Reviewer 4 Report

Comments and Suggestions for Authors

In the presented study, the Authors attempted to identify active compounds present in a fraction obtained from an ethanol extract of sweet potato storage roots. As a result, three compounds were successfully identified, quantitatively determined in the studied fraction, and subsequently subjected to biological activity assays. The identification of active compounds in plant material is an important task that aligns with current research trends. However, the presented study has certain shortcomings and gaps. Moreover, the results reported in the manuscript do not allow some conclusions to be drawn. Below, I present my suggestions and comments.

Comment 1: Lines 66–67 – When defining the aim of the study, the Authors indicate the identification of active constituents in the extract: “The present study aimed to identify constituents within the ethanol extract of sweet potato storage roots that contribute to their anti-inflammatory effects.” They also refer to their previous studies on the extract (lines 10–11; lines 49–51) and state that “the specific bioactive compounds responsible for this activity have not yet been elucidated.” However, these fragments of text, together with the description of the studied sample (SP-EtOH-Ex), are misleading. In the present manuscript, the term “SP-EtOH-Ex: ethanol extracts of sweet potato storage roots” is introduced, but this material is not an extract; it is only a fraction of an extract. The manuscript does not justify why an attempt was made to identify active compounds based solely on a fraction of the extract rather than the whole extract. There is also no justification for focusing on this particular fraction (collected over 9 minutes). As a result, the authors attempt to identify compounds present in the fraction and to determine whether they may contribute to the activity of that fraction not extract. This should be clearly justified and described in the discussion. And it should be clearly indicated in the text that a faction is being examined.

Comment 2: A major problem for the potential reproducibility of the study is the lack of detailed information regarding the extraction procedure. No data are provided on the amount of plant material or the volume of solvent used for extraction. There is also no information on the extraction technique applied, the extraction time, or the process temperature—factors that are critical and directly influence the variability of the extract composition.

Comment 3: Lines 135–136: In the text and in Figure 4, it is stated that the activity of sitosterol against IL-6 was tested at concentrations of 1, 5, and 10 µg/ml, which correspond to 1000, 5000, and 10,000 ng/ml. Thus, the lowest tested concentration was 1000 ng/ml. Given that the concentration of sitosterol in the tested fraction (SP-EtOH-Ex) was 2.527–4.850 ng/ml, the statement that “Sitosterol suppressed IL-6 production at concentrations equivalent to those present in SP-EtOH-Ex” is not correct. There is no information indicating that such low concentrations of sitosterol were tested or was active.

Comment 4: As a result, the authors’ statement (line 29), “These results suggest that the β-sitosterol and campesterol in sweet potato storage roots contribute to their anti-inflammatory effects,” as well as the statement in the conclusion (lines 358–359), is only partially supported by the presented data, because the authors did not provide evidence that sitosterol is active at concentrations as low as those present in the extract. Thus, how many active ingredients have been identified?

Comment: 5:  In Figure 4, ordinal numbers are provided for the “extracts” (fractions); however, the concentration at which their activity was tested should be added. From the Methods section, it can be inferred that this concentration was 10 µg/ml. Was the extract activity tested at the same concentration at which it was subjected to the quantitative analysis of individual constituents?

Comment 6: Lines 361–363: The Authors state that “Overall, the edible portion of sweet potatoes holds promise as a raw material for the development of new and effective functional components with anti-inflammatory properties.” This conclusion is very general and imprecise. What do the authors mean by the term “functional components”? This needs to be specified, especially since the study does not provide any information on the content of the compounds identified as active when calculated per unit mass of the plant material. The only information provided concerns their content in a fraction of the extract, but it is not known from how much plant material the extract was obtained. This means that the Reader cannot assess whether the plant material is realistically a good source of anti-inflammatory substances.

Comment 7: Lines 344–350: Information is missing on how the substances and the fraction used for analysis were dissolved—specifically, in which solvent and whether the same solvent was used for all samples. It is also unclear whether this aspect was taken into account in comparison with the control. Appropriate information should be added.

Comment 8: Sometimes commas are used instead of dots in the notation of numbers, for example in Table 2 and in the abstract. This should be change to dots

Author Response

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Reviewer 5 Report

Comments and Suggestions for Authors

Article
Identification of active anti-inflammatory compounds in sweet potato storage roots extracted with ethanol via LC/MS Q-TOF

A brief summary
The work seems interesting, but there are a few questions regarding methodological issues.

Broad comments
1. The authors identified several compounds present in the ethanol extract from sweet potatoes that are thought to be responsible for the anti-inflammatory effects of this extract.

2. It has been confirmed that β-sitosterol and campesterol are present in ethanol extract from sweet potato roots and have been shown to contribute to their anti-inflammatory effect.

3. On the one hand, phytosterols are widely reported to possess anti-inflammatory properties; on the other hand, however, the question arises as to how much of these compounds were present in the tested extract. See point 4.

4. Some notes regarding the methodology.
a. Please explain why 100% ethanol was used as the extractant (line 235). Plant sterols are lipophilic compounds. They dissolve in anhydrous ethanol (100%), but poorly or moderately, and the degree of solubility depends on the conditions. In 100% ethanol, they dissolve better than in hydrated ethanol (70–96%), but much worse than in non-polar solvents.

b. Both this text and the cited work (https://doi.org/10.3390/nu16040563) lack information on the type and parameters of extraction. The solubility of sterols is affected by temperature – heating significantly improves dissolution, the form of sterols – free sterols dissolve less easily than their esters, and concentration – saturation is quickly reached at higher concentrations.

c. There is also no information on whether the sweet potatoes were pilled or/and chopped before freeze-drying, what model of freeze-dryer was used, or what freeze-drying parameters were employed. The decision to freeze-dry the sweet potatoes before analysis is also unclear. No information is available on what equipment was used to grind the freeze-dried material or how finely it was ground (pulverized).

d. Is it possible that no additional compounds, such as colourants (line 42), were found in the extract (even ethanol)? After all, it is a red variety.

5. The conclusions presented refer to the research results previously outlined. It is noteworthy that the results are presented in an extremely concise manner, particularly given their substantial size.

6.The selection of literature appears to be appropriate.

7. Additional comments:

Table 2 Please explain why the content of lauroyl diethanolamide and β-sitosterol is given to three decimal places, while campesterol is given to two. Please replace the commas with full stops.

Author Response

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Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

accept

Reviewer 2 Report

Comments and Suggestions for Authors

In their response to the review comments, the authors primarily provided explanations and justifications for the original scope of the study, rather than substantively enhancing its depth, breadth, or novelty through the addition of new experimental data. For instance, they have added clarifying background statements and discussion, and have more clearly defined the study's focus: 1) it targets a specific chromatographic fraction (51–60 min) previously confirmed to possess unique anti-inflammatory activity, rather than a whole-root extract; 2) it represents the first Level 1 identification of compounds (phytosterols) within this active fraction; and 3) the research objective is explicitly framed as "identifying the active constituents," not as exploring novel mechanisms.

However, the authors' response does not fundamentally address the core concerns raised in the review—namely, the lack of innovation, limited scientific contribution, and absence of mechanistic investigation. It must be acknowledged that the authors have conducted a solid and technically sound confirmatory analysis. Nevertheless, the scientific findings themselves are not sufficiently novel. In essence, the manuscript does not make a substantive new contribution to scientific knowledge, such as revealing a new structure, elucidating a new mechanism, establishing a novel functional link, or providing a profound interpretation of known constituents in a significantly new context.

Therefore, I maintain a reserved stance regarding the suitability of this work for publication in its current form. Should the editor deem its technical demonstration valuable enough for publication, the text must undergo the substantial revisions outlined above to more accurately reflect the study's actual contribution.

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript has been sufficiently improved. In my opinion, it can be accepted for publication in its current form.