Untargeted Metabolomic Analyses and Antilipidemic Effects of Citrus Physiological Premature Fruit Drop

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have investigated the in vitro antilipidemic effect of citrus physiological premature fruit drop and on oleic acid-induced steatosis in HepG2 and 3T3-L1 cells. They also supported their study by untargeted metabolomic analyses. The manuscript is interesting and valuable. However, there are some considerations that should be taken into consideration.

1)     β and O (in chemical names; as in lines 161, 201, 202) and the expressions: via, in vitro, and p (denoting probability value in the text and figures’ legends) as well as plant names (in line 295 and 307) should be italic please check.

2)     In line 396, the solvent composition should be written (methanol: water = 4:1, v/v)

3)     The authors should explain how they detected that the identified compounds were upregulated or downregulated, did they quantify them?

4)     The authors should add the software used to generate the heatmap and graphs.

5)     Regarding figures 3 and 4, the authors should provide clearer figures.

6)     Concerning the metabolic profiling, the molecular mass should be written in 4 digits, the mass error should be calculated, the molecular formula should be added, as well as mass fragments. In addition, the authors should provide the chromatograms (even as supplementary data).

7)      In table 1 ,  the column named (regulate), is a bit confusing, I cannot figure out the compound is upregulated or down regulated in which cultivar. Therefore, I recommend separating it from table 1 and constructing another table depicting the upregulation or downregulation of the identified compounds in DDPD and HYPD for much better understanding.

 

Author Response

The authors have investigated the in vitro antilipidemic effect of citrus physiological premature fruit drop and on oleic acid-induced steatosis in HepG2 and 3T3-L1 cells. They also supported their study by untargeted metabolomic analyses. The manuscript is interesting and valuable. However, there are some considerations that should be taken into consideration.

We appreciated that the reviewer finds our study of value.

Major points:

1.β and O (in chemical names; as in lines 161, 201, 202) and the expressions: via, in vitro, and p (denoting probability value in the text and figures’ legends) as well as plant names (in line 295 and 307) should be italic please check.

Thank you. We revised it accordingly.

2.In line 396, the solvent composition should be written (methanol: water = 4:1, v/v).

We appreciate the reviewers' suggestion regarding the solvent composition in line 396. We have carefully reviewed and revised the manuscript accordingly, and we have updated the text to read (methanol: water = 4:1, v/v) (Materials and Methods, Page 14, Line 456).

3.The authors should explain how they detected that the identified compounds were upregulated or downregulated, did they quantify them?

We feel sorry for the absence of an explanation how we detected that the identified compounds were upregulated or downregulated. In this study, we used the partial least squares discriminant analysis (PLS-DA) model to calculate variable importance for projection (VIP) values to identify differentially expressed metabolites between Citrus aurantium L. 'Daidai' physiological premature fruit drop (DDPD) and Citrus aurantium 'Changshan-huyou' physiological premature fruit drop (HYPD) sample groups. Furthermore, to ensure structural elucidation accuracy, they established specific criteria for selecting differential metabolites, including a Fragmentation Score greater than 50, VIP values exceeding 1.00, and a significance level with a p-value less than 0.05. We have also added this information to the revised manuscript (Results, Page 5, Line 182-187).

4.The authors should add the software used to generate the heatmap and graphs.

We thank the reviewer for this comment. We have added the information about the software used to generate the heatmap and graphs (Materials and Methods, Page16, Line 522-524).

5.Regarding figures 3 and 4, the authors should provide clearer figures.

Thank you. We revised it accordingly.

6.Concerning the metabolic profiling, the molecular mass should be written in 4 digits, the mass error should be calculated, the molecular formula should be added, as well as mass fragments. In addition, the authors should provide the chromatograms (even as supplementary data).

We thank the reviewer for pointing out this problem. As the reviewer suggested, we have made modifications to this portion of the information in Table 1, and the chromatograms in untargeted metabolomics were provided as supplementary materials.

7.In table 1, the column named (regulate), is a bit confusing, I cannot figure out the compound is upregulated or down regulated in which cultivar. Therefore, I recommend separating it from table 1 and constructing another table depicting the upregulation or downregulation of the identified compounds in DDPD and HYPD for much better understanding.

We thank the reviewer for this question. As mentioned earlier, we conducted screening of differential metabolites in citrus physiological premature fruit drops based on the VIP values from the PLS-DA model. Therefore, in order to better demonstrate the degree of differences among different metabolites in DDPD and HYPD, we arranged the differential metabolites in descending order of VIP values. As pointed out by the reviewer, the terms "upregulation" and "downregulation" can potentially lead to reader misunderstandings. Hence, we added a footnote below Table 1 to provide explanations for the meanings of "up" and "down".

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The title of the paper seems complicated and hard to understand. The authors should make it simple. The title is not an abstract! The abstract on the other hand is very general. It should present data and major results. The authors need to improve the abstract by adding valuable information for the readers.

On row 67, explain what CPFD means. Even if the abbreviation was presented in the abstract, it should be explained again in the main manuscript.

Row 89, explain what HYPD, DDPD, CJPD, OGPD, and XYPD mean. The same goes for the capitation of Figure 1. Each abbreviation should be detailed. Please do the same for each figure legend. Each figure should be treated as independent from the manuscript. If a reader looks only at a figure, he should be able to understand it without reading the whole paper.

Row 89, HYPD (144.00 mg/g) should be written as HYPD (144.00 ± statistical error). The same for the section 112-115. The type of statistical error evaluation should be presented in the figures capitation.

Row 104 and elsewhere in the paper, present all the abbreviations like DPPH and ABTS.

Row 132, move from row 454 “Principal component analysis (PCA)”

Figure 3 needs important improvement. It has a poor quality and is barely visible.

Row 148, authors present a “score”, but they don’t add any details on what it means and how is was calculated.

In table 1, explain what regulate and VIP mean. M/z should have more decimals.

I don’t understand the order of the compounds in table 1. The authors should use the retention time or m/z.

Row 187, explain what PC (16:0/0:0) means. Or the other PCs. The same for MGDG, HODE and HOTrE

Row 191, the authors presented 11(R)-HEDE. Why not the S isomer? Or the racemic? On what data do they base the assessment of the optic isomers? Please check all the other ones.

The presence of some compounds looks strange in my view. For example nitrophenol and 2-naphthylamine. Please comment on this.

In 4.1. section the authors should add the botanical analyses that certified that the fruirs are exactly from the declared species and that the plant material is homogen. They should add the time of harvest.

Row 369, please detail on the tissues of the plant that were used. Exocarp, albedo, flavedo, seeds? The authors should present if the plant material was cut in pieces or grinded up.

Row 403, add details on database search and identification protocol.

The quality of all the figure should be improved.

The discussion section needs improvement. It is just a description of the results, and not a critical analysis of them. Please detail the weak points of the study, highlight possible shortcomings or sources of error. Describe how the results could be used in future studies. The discussion section present in a synthetic way all the results produced. How are the results correlated? Chemical composition and biological effects?

The conclusions are very general. Please be specific and exact. For example, “stronger antioxidant activity than mature fruits” should be replace with something like “with x % higher” or any other clear quantitative measure.

 

  

 

 

Comments on the Quality of English Language

needs correction.

Author Response

1.The title of the paper seems complicated and hard to understand. The authors should make it simple. The title is not an abstract! The abstract on the other hand is very general. It should present data and major results. The authors need to improve the abstract by adding valuable information for the readers.

We thank the reviewer for this critique. We have addressed the reviewer's comment by simplifying the title and improving the abstract of the paper. We understand that the title should be easy to understand, and have made necessary changes to make it simpler and clearer for the readers.

2.On row 67, explain what CPFD means. Even if the abbreviation was presented in the abstract, it should be explained again in the main manuscript.

Thank you. We revised it accordingly.

3.Row 89, explain what HYPD, DDPD, CJPD, OGPD, and XYPD mean. The same goes for the capitation of Figure 1. Each abbreviation should be detailed. Please do the same for each figure legend. Each figure should be treated as independent from the manuscript. If a reader looks only at a figure, he should be able to understand it without reading the whole paper.

We thank the reviewer for pointing out this problem. We have provided detailed explanations for the abbreviations in the legend in the revised manuscript.

4.Row 89, HYPD (144.00 mg/g) should be written as HYPD (144.00 ± statistical error). The same for the section 112-115. The type of statistical error evaluation should be presented in the figure’s capitation.

We appreciate your attention to detail. We have carefully considered your suggestion regarding the presentation of statistical errors in our Results.

5.Row 104 and elsewhere in the paper, present all the abbreviations like DPPH and ABTS.

Thanks for the reviewer's valuable feedback. We have addressed all of the reviewer's concerns and made the necessary revisions throughout the paper, including presenting all the abbreviations like DPPH and ABTS.

6.Row 132, move from row 454 “Principal component analysis (PCA)”

Thank you. We revised it accordingly.

7.Figure 3 needs important improvement. It has a poor quality and is barely visible.

We sincerely appreciate the valuable feedback provided by the reviewer. We have taken the reviewer's comment into serious consideration. The quality of the figure has been enhanced to ensure better visibility and clarity.

8.Row 148, authors present a “score”, but they don’t add any details on what it means and how was calculated.

Thank you for bringing this concern to our attention. In untargeted metabolomics, the Fragmentation Score is a metric that measures the quality of metabolite structure elucidation. It is used to assess the degree of matching and reliability of metabolite ion fragments in mass spectrometry data(Lin et al., 2020).

When a metabolite undergoes fragmentation analysis by a mass spectrometer, a series of ion fragments is generated. These spectra can be compared with known metabolite databases to determine the structure of the metabolite. The Fragmentation Score is calculated based on the degree of matching between the ion fragments and known metabolite structures, thereby evaluating the confidence in metabolite structure elucidation.

Typically, a higher Fragmentation Score indicates a higher accuracy in metabolite structure elucidation and more reliable matching results. Therefore, in untargeted metabolomics studies, the use of Fragmentation Score can assist in identifying differential metabolites and further understanding their relative abundance changes under different conditions(Zuo et al., 2021). We have provided additional explanation regarding the presence of this score in the revised manuscript (Results, Page5, Line 184-187).

9.In table 1, explain what regulate and VIP mean. M/z should have more decimals.

In Table 1, the term "regulate" represents the direction of metabolite abundance changes (up-regulate or down-regulate) in DDPD relative to HYPD, as indicated in line 189. To facilitate reader comprehension, we have emphasized this point again in the table caption. "VIP" stands for variable importance for projection, which is the weighted sum of squares of the projection of each variable in the model. In this study, we employed the partial least squares discriminant analysis (PLS-DA) model to calculate the VIP values for variable importance projection, enabling us to identify metabolites that exhibit differential expression between HYPD and DDPD. This approach aids in understanding the up-regulation or down-regulation of the identified compounds(Xiong et al., 2020). Additionally, we have increased the number of decimals for M/z values in the table.

10.I don’t understand the order of the compounds in table 1. The authors should use the retention time or m/z.

As mentioned above, in order to better determine the differentially expressed metabolites between HYPD and DDPD, we utilized VIP values for evaluation. Therefore, in Table 1, we arranged the selected differential metabolites in descending order based on their VIP values. This was done to highlight the differences between DDPD and HYPD metabolites in untargeted metabolomics results. As for the sorting of compounds, we also took into consideration the findings of Liu et al. (2022).

11.Row 187, explain what PC (16:0/0:0) means. Or the other PCs. The same for MGDG, HODE and HOTrE.

We thank the reviewer for raising this concern. Phosphatidylcholine (PC) is an important class of phospholipids that widely exist in the cell membrane of organisms(Tavasoli et al., 2022). It is one of the main components of the cell membrane and plays an essential role in cell structure and function. PC consists of two fatty acid chains, one glycerol backbone, and one nitrogenous choline group(Kaoutari et al., 2021). The two fatty acid chains can be different types of fatty acids, and based on the carbon number and degree of unsaturation of these fatty acid chains, various PC molecules can be formed. Therefore, PC (16:0/0:0) represents a molecule that has only one 16-carbon saturated fatty acid chain attached to the glycerol backbone without any additional fatty acid chains. PC (15:0/0:0), PC (18:2/0:0), and PC (18:1/0:0) are different PC molecules that have different fatty acid chain structures. PC (15:0/0:0) contains one 15-carbon saturated fatty acid chain and one nitrogenous choline group, with the other position unoccupied by any fatty acid chain. PC (18:2/0:0) contains one 18-carbon diunsaturated fatty acid chain and one nitrogenous choline group, with the other position unoccupied by any fatty acid chain. PC (18:1/0:0) contains one 18-carbon monounsaturated fatty acid chain and one nitrogenous choline group, with the other position unoccupied by any fatty acid chain. In addition, Phosphatidylethanolamine (PE) consists of one glycerol backbone, one nitrogenous ethanolamine group, and two fatty acid chains. Like PC, PE is one of the main components of the cell membrane and participates in regulating cell shape and fluidity. Therefore, PE (18:2/0:0) indicates that this molecule consists of one 18-carbon diunsaturated fatty acid chain and one nitrogenous ethanolamine group, with the other position unoccupied by any fatty acid chain. PE (18:1(9Z)/0:0) contains one 18-carbon monounsaturated fatty acid chain (with cis double bond at position 9) and one nitrogenous ethanolamine group, with the other position unoccupied by any fatty acid chain. Monogalactosyldiacylglycerol (MGDG) is a type of phospholipid molecule that consists of one galactose group and two fatty acid chains. Hydroxyoctadecadienoic acid (HODE) is a derivative of a twenty-carbon diunsaturated fatty acid. Hydroxyoctadecatrienoic acid (HOTrE) is a derivative of a twenty-carbon triunsaturated fatty acid. To facilitate readers' better understanding of the names of these fatty acids in the table, we have added footnotes below the table for PC, PE, MGDG, HODE, and HOTrE in the revised manuscript.

12.Row 191, the authors presented 11(R)-HEDE. Why not the S isomer? Or the racemic? On what data do they base the assessment of the optic isomers? Please check all the other ones.

We thank the reviewer for this comment. Hydroxyeicosadienoic acid (HEDE) is a twenty-carbon polyunsaturated fatty acid that is metabolized from arachidonic acid by cyclooxygenase and peroxidase enzymes. 11-HEDE consists of two optical isomers, 11(R)-HEDE and 11(S)-HEDE. The optical isomers exhibit differences in mass-to-charge ratio (m/z) and relative abundance of fragment ions, which can be qualitatively observed by comparing the retention times of the isomers in a chromatographic system using reference standards. Untargeted metabolomics employs high-performance liquid chromatography as the separation system and tandem mass spectrometry with high resolution as the detection system for metabolite analysis. In this approach, metabolites are ionized in the ion source for primary mass spectrometry scanning, and specific ions enter the collision chamber for secondary fragmentation, generating daughter ions for secondary mass spectrometry scanning. Based on data-dependent acquisition, both primary and secondary spectra are obtained. Finally, the primary and secondary spectra are matched against an in-house standard compound database to identify primary and secondary metabolites. Therefore, this may also explain the detection of optical isomers in untargeted metabolomics. It is worth noting that in a previous study, similar findings were reported using untargeted metabolomic techniques, detecting compounds such as (10S,11R)-Pterosin C 4-glucoside and (R)-2-Benzylsuccinate in Tibetan tea(Liu et al., 2022).

13.The presence of some compounds looks strange in my view. For example, nitrophenol and 2-naphthylamine. Please comment on this.

We thank the reviewer for this question. Untargeted metabolomics is a systematic method for analyzing all small molecule metabolites in biological samples. It utilizes Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS), which employs LC as the separation system and tandem mass spectrometry with high resolution as the detection system for metabolite analysis. This technique offers high sensitivity, wide analytical range, and good repeatability. Untargeted metabolomics primarily relies on LC-MS/MS without pre-selecting or screening specific metabolites, but rather aims to explore all possible metabolites present in a sample to obtain comprehensive and integrated metabolic information.

In our results, we were also surprised to observe the presence of 4-nitrophenol. However, through further literature searching, we learned that 4-nitrophenol can enter plant tissues through various pathways, including soil pollution, atmospheric deposition, and water source contamination. Firstly, plants may absorb 4-nitrophenol from the soil through their roots. Some studies have shown that plants can uptake 4-nitrophenol into their roots and transport it to other parts of the plant via root cell transporters. Additionally, 4-nitrophenol can be absorbed by plant leaves in its gaseous or dissolved form in the atmosphere(Trang et al., 2022). Once inside the plant, 4-nitrophenol may undergo transformation, metabolism, or accumulation. Plants utilize various metabolic pathways to glucosylate and acetylate 4-nitrophenol, converting it into more stable and less toxic metabolites to reduce its harmful effects. Previous research on cell suspension cultures of glycine and triticum aestivum, as well as excised leaves of soybean, revealed that over 85% of 4-nitrophenol was primarily transformed into more polar and soluble conjugates, such as 1-(O-β-D-pyranoglucosyl)-4-nitrobenzene and 1-(6′-acetyl-O-β-D-pyranoglucosyl)-4-nitrobenzene (Schmidt et al, 1993). Another study also indicated the presence of 4-nitrophenol in the cuticles of tomato fruits and Ficus leaves(Riederer & Schönherr, 1986). Therefore, it is possible for 4-nitrophenol to exist in plants.

14.In 4.1. section the authors should add the botanical analyses that certified that the fruirs are exactly from the declared species and that the plant material is homogen. They should add the time of harvest.

Citrus is one of the most important fruits globally and also one of the most significant tree crops worldwide. Our research team has been dedicated to citrus research for over thirty years, and the locations involved in this experiment are advantageous geographical areas for this variety. Moreover, in our previous studies, we have also conducted research on these citrus species(Guo et al., 2019;Guo et al., 2018). Additionally, for this study, citrus fruits were collected from June to July, and citrus fruits with a diameter of 2.0-3.0 cm were selected as experimental materials. We have included this important information in the revised manuscript (Materials and Methods, Page13, Line 413-415).

15.Row 369, please detail on the tissues of the plant that were used. Exocarp, albedo, flavedo, seeds? The authors should present if the plant material was cut in pieces or grinded up.

We apologize for not providing detailed information. In this study, we dried and ground the fallen and mature citrus fruits, sieved them through a 60-mesh screen, and then carried out subsequent operations. We have also included this detail in the revised manuscript (Materials and Methods, Page14, Line 428-429).

16.Row 403, add details on database search and identification protocol.

Thank you. We revised it accordingly in the Materials and Methods (Page14, Line 461-468).

17.The quality of all the figure should be improved.

Thank you for your feedback. We have now implemented the use of a clear TIFF format in the revised manuscript.

18.The discussion section needs improvement. It is just a description of the results, and not a critical analysis of them. Please detail the weak points of the study, highlight possible shortcomings or sources of error. Describe how the results could be used in future studies. The discussion section presents in a synthetic way all the results produced. How are the results correlated? Chemical composition and biological effects?

We think these revisions would be important and hopefully help to strengthen this otherwise impressive paper. Imbalance between the cellular and tissue oxidative and antioxidant systems can lead to excessive production of reactive oxygen species (ROS) and related reactive oxygen species, resulting in oxidative stress. Polyphenols are natural compounds present in plants and possess various biological activities. Phenolic compounds and flavonoids can interact with ROS, thereby terminating the chain reaction before cell viability is severely affected. In fact, the mechanisms involved in the antioxidant capacity of polyphenols include inhibition of enzymes involved in their generation, scavenging of ROS, upregulation or protection of antioxidant defenses to suppress ROS formation. In our study, we found that citrus physiological premature fruit drop has abundant phytochemicals and remarkable in vitro antioxidant activity. Interestingly, we also found that Citrus aurantium L. 'Daidai' physiological premature fruit drop (DDPD) and Citrus aurantium 'Changshan-huyou' physiological premature fruit drop (HYPD) exhibit different antilipidemic effects against HepG2 and 3T3-L1 cells. However, based on the results of untargeted metabolomics analysis, we believe that this may be closely related to the differential components between DDPD and HYPD.

As the reviewer pointed out, we have now made modifications to the discussion section in the revised manuscript. Our current research on citrus fruit drop is limited to in vitro experiments and lacks results from in vivo experiments as well as exploration of its main active components. In the future, we will further combine in vitro and in vivo experiments to verify the therapeutic potential of citrus physiological premature fruit drop for obesity and elucidate the mechanism underlying its physiological effects.

19.The conclusions are very general. Please be specific and exact. For example, “stronger antioxidant activity than mature fruits” should be replace with something like “with x % higher” or any other clear quantitative measure.

We thank the reviewer for this critique. We have made revisions to the conclusions section in the revised manuscript.

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I would like to thank the authors for their favorable response to my comments. Only the clarity of figure 3 and 4  still needs improvement which may be attained during the proof reading and production stage.

Author Response

Dear Reviewer,

Thank you very much for your continued support and for providing us with valuable feedback. We truly appreciate the time and effort you have dedicated to reviewing our manuscript titled "Untargeted metabolomic analyses of citrus physiological premature fruit drop and antilipidemic effect on oleic acid-induced steatosis in HepG2 and 3T3-L1 cells".

We are pleased to inform you that we have carefully addressed your concern regarding the clarity of figures 3 and 4 in the revised version of our paper. We have taken great care to ensure that the new images are much clearer and easier to interpret. We believe these improvements will significantly enhance the overall quality of the manuscript.

Your dedication to providing thoughtful suggestions despite your busy schedule is truly commendable. We are grateful for your expertise and guidance throughout this process. Your input has been instrumental in refining our work, and we sincerely thank you for your valuable contributions.

Once again, we would like to express our heartfelt appreciation for your time and commitment.

Yours sincerely,

Yang Shan, Academician of Chinese Academy of Engineering

Chinese Academy of Engineering

Hunan Academy of Agricultural Sciences

Reviewer 2 Report

Comments and Suggestions for Authors

The authors made important changes to their paper and improved its quality significantly. I think that some of the explanations that they provided in the response to the first review could be incorporated in the paper. In this way the reader will understand better the paper.

Comments on the Quality of English Language

needs correction

Author Response

Dear Reviewer,

We appreciate your valuable time and effort in reviewing our revised manuscript titled "Untargeted metabolomic analyses of citrus physiological premature fruit drop and antilipidemic effect on oleic acid-induced steatosis in HepG2 and 3T3-L1 cells". We are pleased to hear that the improvements we made were well-received. We have incorporated some of the explanations provided in our response to the initial review into the Discussion of the paper, as per your suggestion. We believe that this will enhance the reader's understanding of the research.

We truly appreciate your valuable feedback and the effort you have put into providing further suggestions despite your busy schedule. Your input has been immensely helpful in refining our work.

Thank you once again for your continued support and guidance.

Yours sincerely,

Yang Shan, Academician of Chinese Academy of Engineering

Chinese Academy of Engineering

Hunan Academy of Agricultural Sciences