-->PONE-D-25-60879-->-->Biochemical characterizations of leaves and fruits in Crataegus monogyna Jacq., C. pontica K.Koch, C. microphylla K.Koch, and C. pentagyna Waldst. & Kit. ex Willd-->-->PLOS One

Dear Dr. Khadivi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Two out of three reviewers noted the lack of biological replications during sample collection and metabolite extraction. In such a situation, many results obtained in the study are biased by small sample size and lead to over-explanation by PCA and over-interpretation of correlation and regression analysis. Some methodological errors and inconsistencies were also noted. The text itself is too lengthy in several places, but the discussion section lacks depth and meticulous literature analysis. Finally, the text presentation would benefit from having it proofread by a native English speaker or a professional editing agency. For example, Latin species names are presented under single quotation marks in many places, misleading the reader to present cultivars. Random word capitalization is also noted. L233-235 contain comma to present decimal places. Use 0.989 instead of .989 in Table 5.

==============================

Please submit your revised manuscript by Jun 19 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Academic Editor

PLOS One

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Reviewers' comments:

Reviewer's Responses to Questions

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1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: I Don't Know

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

-->5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: The manuscript addresses an important and timely topic, namely the comparative biochemical characterization of fruits and leaves of four Crataegus species. The range of analyzed secondary metabolites is broad, and the use of multivariate statistical approaches (PCA, HMA, MRA) represents a clear added value. The study fits well within the scope of PLOS ONE as a descriptive–comparative investigation with potential applied relevance. However, the manuscript suffers from serious methodological, statistical, and interpretative shortcomings, which substantially limit the reliability of several key conclusions in its current form. Major revisions are required before the manuscript can be considered for publication.

1. The writing style is at times overly repetitive and descriptive, particularly in the Results and Discussion section.

Several statements regarding “health benefits” are speculative and go beyond what can be concluded from in vitro antioxidant assays. The separation between results, interpretation, and application-oriented speculation is not always clear.

Some literature citations are discussed superficially, without critical comparison of methodologies.

2. Inconsistencies in units and literature comparisons: The manuscript compares results expressed in:mg/g DW, mg/100 g, mg/kg DW, without consistent conversion. This may mislead readers regarding agreement or disagreement with previous studies.

3. Pseudoreplication and extremely limited biological replication

This is the most serious issue of the study. Each species was collected from only one geographic location. Consequently, species identity is completely confounded with site-specific environmental conditions. As a result: conclusions regarding “species-specific differences” are methodologically unjustified, the application of ANOVA, Pearson correlations, PCA, and MRA with such limited biological replication is statistically problematic. The authors should: clearly state that the study is exploratory or pilot in nature, or restrict their conclusions to “sample-level comparisons” rather than “species-level differentiation”.

4. PCA explaining 100% of variance

A PCA in which three principal components explain exactly 100% of total variance strongly suggests: an extremely small number of observations, strong multicollinearity among variables, potential overparameterization. The manuscript lacks:

explicit information on the number of observations included in PCA,a scree plot, any form of validation of component stability.

In its current form, PCA should be interpreted only as a visualization tool, not as evidence of biologically meaningful differentiation.

5. Overinterpretation of correlations and regression analysis

Correlation coefficients approaching unity (e.g. r = 1.00**) are biologically implausible and most likely reflect: extremely low sample size, definitional or mathematical dependence among variables (e.g. total phenolics vs. phenolic subclasses).

Regarding multiple regression analysis: the ratio of observations to predictors is not reported, no diagnostic tests (normality, multicollinearity, VIF, residual analysis) are provided, the risk of overfitting is very high. Conclusions identifying “key predictors of antioxidant capacity” are therefore not statistically supported.

6. Recommendation: Major revision

The study is based on valuable experimental work and has clear publication potential. However, in its current form it does not meet the statistical and methodological rigor required to support its more ambitious conclusions, particularly those related to species-level differentiation and metabolic regulation. With substantial revisions and a more cautious interpretation of the data, the manuscript could become a solid exploratory contribution to the literature on Crataegus biochemistry.

Reviewer #2: the manuscript is well written and linguistially revised. The refernces are updated. The model is applicable to be taken in consideration and applied in breeding programs thus helping in developing active metabolites for functional food and medicinal applications.

Reviewer #3: This manuscript emphasizes the significance of hawthorn within the Rosaceae family. Despite the widely acknowledged cardioprotective properties of its fruits in numerous countries, the plant itself is frequently underappreciated and treated as a troublesome, thorny weed in temperate forests and scrublands. I consider the subject matter of this study to be valuable and worthy of publication. The manuscript was prepared quite carefully, but it has a few shortcomings. The tables and graphs were prepared with care, and I did not find any serious errors in the analysis of the presented data. Importantly, however, I was unable to find information regarding the number of extracts prepared or the number of replicates for each experiment. Perhaps this information is hidden somewhere, but it should be located in the sections generally designated for this purpose: in the description of the specific methodology, in the section devoted to statistics, or alongside the tables and figures. Without this, it is difficult to consider the presented results and statistical analysis as accurate. I also noticed a few errors and inaccuracies in the methodology. Some parts of the manuscript appear to be written in a rather wordy style and require editorial revisions.

I have compiled my additional comments into a list of points, which I have included below.

1. The authors wrote: ”Chemical properties of four species of Crataegus genus in Iran, including C. monogyna (collected from Dorood area of Lorestan province), C. pontica (collected from Hanivan area of Ilam province), C. microphylla (collected from Gachan area of Ilam province), and C. pentagyna (collected from Babol area of Mazandaran province) were evaluated” and shown the geographic locations on the map. Therefore, the next sentence, i.e. “The plant materials were collected from Lorestan, Ilam, and Mazandaran provinces” seems like an unnecessary repetition. The authors should reorganize Section 2.1.

2. The authors wrote: “After collecting and separating the waste materials, the samples were dried separately at room temperature away from the sunlight in the shade in the airflow and were converted into fine particles with a home grinder and passed through a sieve with 40 mesh.” By crushing the material and sifting it through a sieve, the authors obtain a finely ground material that is not only partially oxidized (which is very difficult to prevent) but also highly susceptible to further oxidation. How did the authors protect it from oxidative degradation? Did they store it in an inert atmosphere at –80 degrees? For how long? It is worth noting that material ground in this way should be used as soon as possible after grinding. The manuscript lacks information on storage.

3. The authors wrote: “All extracts were kept in a refrigerator at 4˚C until analysis.” This is the correct procedure. Although phenolic compound solutions should be stored at a much lower temperature to prevent the degradation of some of the compounds, storing them at 4 degrees Celsius prevents the precipitation of less soluble phenols. The point is that extracts can only be stored this way for a very short time. The authors did not specify exactly how long the extracts were kept at this temperature.

4. In the Folin–Ciocalteu method, the step described as “stopping the reaction after 3 minutes by adding sodium bicarbonate” is of limited significance under the conditions used, since further heating (to 100 degrees Celsius) and prolonged incubation bring the reaction to completion, thereby minimizing the impact of initial time differences.

5. The applied temperature (100°C) may promote partial degradation of thermolabile phenolic compounds, particularly flavonoids, including anthocyanins. The application of a lower temperature (e.g. 40–60°C) may be recommended in future studies to reduce thermal degradation of sensitive phenolics.

6. In the procedure of flavonoid quantification, the authors used ”one µL” of diluted extract.” Isn't there a mistake here? Does the final volume of 2 mL contain 1 μL of diluted sample? In the study cited by the authors (Zhishen et al. 2011), the authors used 500 μL. First of all, that’s not very much; second, mixing reagents in this way (1 + 1999) introduces a scale error.

7. Was the color of the resulting complex actually purple? In the cited study (Zhishen et al. 2011), it was yellow; in others, it was yellow-orange.

8. In the procedure for ortho-diphenol quantification, the authors wrote: “(...) spectrophotometer was zeroed with a blank, and 70% methanol was used as a blank” That is unnecessary repetition. Sentences with a similar structure can also be found in the methods for determining other phenols below.

9. In the determination of hydroxycinnamic acids and flavonols, the primary solvent is 2% hydrochloric acid; therefore, the sample should be zeroed with this solution, not with 70% methanol.

10. The sentence in line 183, i.e. “Put the red number in equation 1 and get the absorption rate of the diluted sample” should be removed.

11. The authors wrote:” The ability to remove DPPH of the samples was evaluated by the method proposed by Obied et al. [19] with minor changes”. This is a rather unfortunate (and incorrect) interpretation of the principle behind this measurement. DPPH radical is not removed but scavenged or simpler: reduced. I would also avoid using the incorrect term “DPPH removal.” (line 198).

12. A DPPH solution in methanol is purple, not blue (line 193). The color changes from purple through reddish to pale yellow. Therefore, the notation “purple → yellow to red” incorrectly indicates the direction of the change.

13. The HPLC methodology needs to be rewritten from scratch. I do not understand what is meant by ‘the calibration curve (...) was drawn at a wavelength of 220 to 400 nm’. This is an oversimplification. The description does not provide a detailed stepwise gradient profile, the total separation time, and the wavelength used for separating the test samples. There are no specifications for the column, chromatograph or detector. The authors have not stated where they obtained the standards. Analysis of the text does not prove that these were high-purity compounds.

14. There is an error in the description of the filter size, i.e. ‘0.2 mm syringe filter’. I believe it should have been written as ‘0.20 micrometers’.

15. In the description of the statistical analysis, specifically Pearson’s correlation – the significance level (I assume this is also p = 0.005) should also be stated.

16. In the description of the statistical analysis, the type of test is missing from the description of the one-way ANOVA, and in the description of Pearson’s correlation, the significance level (I believe also p = 0.05) should also be supplemented.

17. In line 236, the authors uses two the same phrases: “antioxidant capacity (5,80), and antioxidant capacity (1,26)”. A word (fruit/leaf) or symbol should appear here to distinguish between these two items.

18. In the text between lines 232 and 237, the numerical values in parentheses should be followed by a percentage symbol. Generally, when presenting values one after another, at least the first set of parentheses should include the percent sign. Parentheses containing numbers without percentages appear later in the Results section.

19. The authors wrote: “The biochemical contents of some Crataegus genus fruits are presented in Table 2.” This sentence is misleading because a plant’s “biochemical content” primarily refers to its essential components, and none are mentioned here. The next sentence clarifies the meaning and, in my opinion, is sufficient. I would omit the quoted sentence. Moreover, I would modify the title of Table 2 from “Biochemical contents of some Crataegus genus fruits” to “Phytochemical contents of some Crataegus genus fruits”.

20. The text between lines 254 and 278 essentially describes what we can see directly in Table 2. In my opinion, such a detailed description is unnecessary; the text here should be more concise.

21. The authors wrote: “In PCA, the original variables are transformed into a smaller set of uncorrelated components, known as principal components, that explain the maximum variance in the data. This method is widely employed for data reduction, pattern recognition, and identifying underlying structures in complex datasets [53]”. In my opinion there is no need to describe what PCA is as PCA’s are widely uses in recent scientific literature. This text should be condensed.

22. The following paragraphs on standardization and the details of PCA are helpful, as this information is not widely known. I would just suggest shortening them slightly.

23. I would advise the authors to describe other statistical tools (e.g., correlation matrix analysis) more concisely.

24. In Figures 2 and 3, the values shown in pale pink are almost invisible.

25. The p-values listed below Figures 2 and 3 should be written in italics.

26. The conclusion section should be no longer than 5–6 sentences, excluding the “introduction”. This one has 11 sentences and is too descriptive in the first part; it resembles a standard abstract too closely.

27. The authors may consider replacing hyphens (-) with en dashes (–) or the proper minus sign (−) in the tables and the regular text, where negative values are indicated, to maintain typographical consistency.

28. There should be a space before and after the equal sign (including when authors cite values in the text).

May 1, 2026

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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-->PONE-D-25-60879R1-->-->Biochemical characterizations of leaves and fruits in Crataegus monogyna Jacq., C. pontica K.Koch, C. microphylla K.Koch, and C. pentagyna Waldst. & Kit. ex Willd-->-->PLOS One

Dear Dr. Khadivi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jul 23 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:-->

• A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
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• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

As the corresponding author, your ORCID iD is verified in the submission system and will appear in the published article. PLOS supports the use of ORCID, and we encourage all coauthors to register for an ORCID iD and use it as well. Please encourage your coauthors to verify their ORCID iD within the submission system before final acceptance, as unverified ORCID iDs will not appear in the published article. Only  the individual author can complete the verification step; PLOS staff cannot  verify ORCID iDs on behalf of authors.

We look forward to receiving your revised manuscript.

Kind regards,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS One

Journal Requirements:

1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Additional Editor Comments:

The sampling procedure has remained insubstantially described (please see the Reviewer's comments below). A detailed sampling protocol should be submitted. Fig 2 and Fig 3 still contain comma instead full stop to define decimal places. Please correct.

[Note: HTML markup is below. Please do not edit.]

Reviewer's Responses to Questions

-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #3: (No Response)

**********

-->2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #3: Partly

**********

-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #3: I Don't Know

**********

-->4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #3: Yes

**********

-->5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #3: Yes

**********

-->6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #3: In my opinion, a very important point raised in the opening sentences of my review was not fully addressed. Like Reviewer 1, I pointed out deficiencies in the statistics: “Importantly, however, I was unable to find information regarding the number of extracts prepared or the number of replicates for each experiment. Perhaps this information is hidden somewhere, but it should be located in the sections generally designated for this purpose: in the description of the specific methodology, in the section devoted to statistics, or alongside the tables and figures. Without this, it is difficult to consider the presented results and statistical analysis as accurate”.

The authors included a sentence in the revised version: “Because each species was sampled from a single geographic location, species identity and environmental conditions may not be completely independent. Therefore, the present dataset primarily reflects sample-level biochemical variation under the specific ecological conditions of the collection sites”. I understand that there is only one sampling site. However, the authors still did not explicitly specify how many samples they collected from a single location, how many extracts they prepared, and in how many technical replicates they performed the individual biochemical assays. In Table 1, they provided standard deviations for all results, so the number of replicates, n, should be given below the table. This is a serious omission.

Most of my other comments have been incorporated, and the text has been modified and expanded. In my opinion, the revised manuscript is more substantive and more carefully presented. I have only two minor comments.

1. Typically, the name of the high-performance liquid chromatograph, the detector type, and the column manufacturer are provided. For those who use this technique on a daily basis, this information is essential.

2. I overlooked when reading the previous version: the molar extinction coefficient, epsilon, has its own unit, most often: dm³·mol⁻¹·cm⁻¹ (line 242). This should be added.

May 20, 2026

**********

-->7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #3: No

**********

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Biochemical characterizations of leaves and fruits in Crataegus monogyna Jacq., C. pontica K.Koch, C. microphylla K.Koch, and C. pentagyna Waldst. & Kit. ex Willd

PONE-D-25-60879R2

Dear Dr. Khadivi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #3: All comments have been addressed

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-->2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #3: Yes

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-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #3: Yes

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-->4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #3: Yes

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-->5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #3: Yes

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-->6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #3: The authors have addressed all of my comments. The number of replicates is now specified not only in the “Materials and Methods” section but also listed below the tables, which enhances the clarity of the results. As I suggested, the authors have provided a comprehensive description of the chromatograph used and its accessories. I have no further substantive comments regarding the manuscript. In my opinion, it can proceed to publication.

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-->7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review?  For information about this choice, including consent withdrawal, please see our Privacy Policy.-->

Reviewer #3: No

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