Hydroxyhydroquinone and Quassinoids as Promising Compounds with Hypoglycemic Activity through Redox Balance

Round 1

Reviewer 1 Report

The manuscript entitled “Hydroxyhydroquinone and quassinoids as promising compounds with hypoglycemic activity through redox balance”, describes the potential of hydroxyhydroquinone (synthesized from 1,4-hydroquinone) and hydroethanolic extracts obtained from P. crenata and R. cuspidata as possible glycemic controlling agents for insulin sensitization was evaluated. Nevertheless, there are some concerns about the methodology used and the overall discussion. For example:

·      The manuscript discusses the evaluation of quassinoids from Picrasma crenata and flavonoids from Rourea cuspidata as potential agents for glycemic control and insulin sensitization. Nevertheless,it is noteworthy that the procedure for isolating these compounds is inadequately explained. Therefore, it is imperative to provide a thorough account of the isolation method or consider reorienting the manuscript's focus.

·       The total flow rate of 1.0 mL*min^-1 is excessively high for accurate analysis in the mass spectrometer.

·       The inclusion of MS/MS analysis is essential for the identification of compounds.

·        Due to the coelution of certain compounds, it is imperative to initially enhance the separation and subsequently employ a base peak chromatogram to estimate the concentration of these compounds.

·      To prevent misidentifications of compounds within the R. cuspidata extract, it is essential to utilize HPLC coupled with mass spectrometry and reference standards. For instance, signal 291.0863 might correspond to epicatechin, while signal 577.1341 may correspond to another procyanidin type A dimer.

To my view, the manuscript in its present form is not suitable for publication to Compounds except if the major revisions are made.

Author Response

Response to reviewers (compounds-2551044)

Author's Reply to the Review Report (Reviewer 1)

Thank you for valuable comments, below please find the responses.

The manuscript entitled “Hydroxyhydroquinone and quassinoids as promising compounds with hypoglycemic activity through redox balance”, describes the potential of hydroxyhydroquinone (synthesized from 1,4-hydroquinone) and hydroethanolic extracts obtained from P. crenata and R. cuspidata as possible glycemic controlling agents for insulin sensitization was evaluated. Nevertheless, there are some concerns about the methodology used and the overall discussion. For example:

•     The manuscript discusses the evaluation of quassinoids from Picrasma crenataand flavonoids from Rourea cuspidata as potential agents for glycemic control and insulin sensitization. Nevertheless, it is noteworthy that the procedure for isolating these compounds is inadequately explained. Therefore, it is imperative to provide a thorough account of the isolation method or consider reorienting the manuscript's focus.

We have emphasised the focus of the paper to be clearer. The isolation of specific compound was never our goal. We show synthesized HHQ, a molecular motif of quassinoids and flavonoids that can reduce ROS and increase glucose uptake in insulin resistant cells. 

We added “Hydroxyhydroquinone or 1,2,4-trihydroxy benzene (HHQ) is a simple aromatic molecule with redox potential, which can bind or release electrons according to the environment and metabolic demand. Likewise, auto-oxidized flavonoids are capable of producing oxygen radicals presenting lower oxidation potentials than those that did not undergo auto-oxidation. Its low redox potential and it oxidate homologous hydroxybenzoquinone makes a good flavonoid analog to test verifying phenolic activities to improve cell insulin sensitivity”.

      The method description was improved.

• The total flow rate of 1.0 mL*min^-1is excessively high for accurate analysis in the mass spectrometer.

      We have added to Supplementary Material showing results from direct infusion. We see consistent data. We have added in text - section 2.2.2. – the following: “A report of mass spectrum through direct infusion of P. crenata using electrospray source in positive mode can be visualized in supplementary material (Figure S4).”

      Our spectrometer coupled with electrospray source is compatible with flow rates of 1 mL min^-1 without compromising signal resolution for low molar mass molecules, with an injection of 20 L of a diluted solution of the mixture (0.01% m/v). These settings allowed chromatograms with a good signal/noise ratio without signal saturation. The mobile phase (H₂O/MeOH without ionizing agent) is completely evaporated at source settings at 200°C and N₂ flow at 10 L min^-1 and capillary pressure at 5 bar. Furthermore, the spectrometer is configured to have optimized signal gain to improve signal-to-noise ratio.

•      The inclusion of MS/MS analysis is essential for the identification of compounds.

      We have complied a direct infusion analysis. It was performed using electrospray source in positive mode and the mass spectrum. Results are attached in supplementary material.

• Due to the coelution of certain compounds, it is imperative to initially enhance the separation and subsequently employ a base peak chromatogram to estimate the concentration of these compounds.

Our emphasis has been to identify compounds rather than their isolation. The separation and analysis methodology for the P. crenata extract was replicated from a methodology validated by Cardoso, et al, 2009 (https://doi.org/10.1080/10826070802548663) for the sole purpose of quantification. A quantitative investigation would require the use of authentic standards, which makes implementation unlikely. Furthermore, the objective of this work is not centred on a specific analytical chemistry investigation, but on the application of a concentrate of quassinoids already published as biochemical agents acting in the metabolic pathway of glycol corticosteroids.

•     To prevent misidentifications of compounds within the R. cuspidata extract, it is essential to utilize HPLC coupled with mass spectrometry and reference standards. For instance, signal 291.0863 might correspond to epicatechin, while signal 577.1341 may correspond to another procyanidin type A dimer.

      LCMS analysis for R. cuspidata extract was previously carried out and published by Paim, et al, 2021 (DOI: 10.1002/pca.3087) in our laboratory, where authentic flavonoid standards were used. The infusion analysis presented in Figure 4 was performed to confirm the presence of the already identified flavonoids.

To my view, the manuscript in its present form is not suitable for publication to Compounds except if the major revisions are made.

Reviewer 2 Report

This manuscript presents an interesting study investigating the efficacy of hydroxyhydroquinone (HHQ), quassinoids from Picrasma crenata, and flavonoids from Rourea cuspidata as potential agents for insulin sensitization. The chosen HepG2 insulin-resistant cell model adds relevance to the investigation. However, despite the study’s potential, there are several areas that require significant improvement and clarification. These areas include, but are not limited to, the language used, the presentation of methods, and the clarity of the results and discussion sections. In particular, the manuscript's presentation and discussion of results are quite confusing. It would benefit from a clearer organization to enhance the reader's understanding of the study's significance. Detailed comments are provided below to guide the authors in strengthening their manuscript.

Abstract:

-        The abstract could benefit from a clearer and more concise presentation of the main results. I recommend improving this part.

-        The sentence "HHQ was synthetized and extracts from P.crenata and R.cuspidata were tested in hepatic cell line; which were evaluated through cytotoxic activity; levels of ROS; ATP; mitochondrial membrane potential and expression of FoxO1 protein in a high glucose environment" is quite complex and could be split into two sentences for clarity.

-        Line 13: Use “,” instead of “;”. The use of semicolons in the abstract is inconsistent and sometimes confusing. Consider revising the punctuation to improve readability.

-        I would suggest adding some more keywords in the manuscript. They will help increase the visibility and accessibility of the research.

Introduction and Methodoly:

-        Check for consistency in the use of abbreviations and ensure that they are defined when first used in the text.

-        Line 60: The sentence "Hydroxyhydroquinone or 1,2,4-trihydroxy benzene (HHQ) is a simple aromatic molecule with redox potential like flavonoids and its redox properties." is somewhat confusing. Consider breaking it into two separate sentences to clearly describe the redox potential of HHQ and how it relates to flavonoids.

-        Line 84: The extended name should be written in line 80.

-        Line 85: “In the presence of insulin, hydroxyhydroquinone and quassinoids compounds were able to decrease expression of FOXO1, most likely inhibiting gluconeogenesis and ultimately reducing glucose production”. What is the reference for this sentence? Is it just the anticipation of your results? It is completely misleading.

-        It is important to include the name of the manufacturer of the materials used in the process, to ensure transparency and reproducibility of the study.

-        At what stage were the cells at when the experiments were performed?

Results and Discussion:

-        The abstract mentions that HHQ and quassinoids downregulate the expression of FoxO1. Can the authors provide more detailed results or quantitative data to support this statement?

-        Line 334: The transition between discussing the pharmacological properties of Connaraceae plants and the development of type II diabetes could be smoother. Perhaps a sentence could be added to bridge the two topics. In the last sentence, it is stated that "Oxidative stress can be modulated by flavonoid ingestion." It would be beneficial to provide some examples or evidence from the study to support this statement.

-        Why did you show MTT results about incubation with resveratrol, rapamycin, Myo inositol and Chiro inositol. This could be completely misleading, as the focus of the manuscript is the evaluation of the efficacy of other compounds.

-        Line 449: Please remove the italics for this sentence.

Conclusion:

-        The manuscript’s conclusion should provide a clear and concise summary of the study's main findings, their implications, and potential future research directions. In its current form, the conclusion is a bit scattered and could benefit from a more structured presentation.

-        While the authors discuss the promising effects of hydroxyhydroquinone and quassinoids, it would be helpful to include a summary of the specific results that led to these conclusions. This would provide a clearer link between the study’s findings and the broader implications discussed in the conclusion.

The manuscript would benefit significantly from a thorough review and improvement of the English language used. I would strongly recommend having the manuscript reviewed and edited by a native English speaker or a professional scientific editor to enhance the clarity and overall quality of the writing.

Author Response

Response to reviewers (compounds-2551044)

Author's Reply to the Review Report (Reviewer 2)

Thank you for valuable comments, below please find the responses. The document was checked by a native English-speaking colleague (co-author Dr. A. Joseph Rastegar). We also send the manuscript to the English MDPI editing service.

This manuscript presents an interesting study investigating the efficacy of hydroxyhydroquinone (HHQ), quassinoids from Picrasma crenata, and flavonoids from Rourea cuspidata as potential agents for insulin sensitization. The chosen HepG2 insulin-resistant cell model adds relevance to the investigation. However, despite the study’s potential, there are several areas that require significant improvement and clarification. These areas include, but are not limited to, the language used, the presentation of methods, and the clarity of the results and discussion sections. In particular, the manuscript's presentation and discussion of results are quite confusing. It would benefit from a clearer organization to enhance the reader's understanding of the study's significance. Detailed comments are provided below to guide the authors in strengthening their manuscript.

Abstract:

-        The abstract could benefit from a clearer and more concise presentation of the main results. I recommend improving this part.

The abstract has been improved and updated. Please see new text.

-        The sentence "HHQ was synthetized and extracts from P.crenata and R.cuspidata were tested in hepatic cell line; which were evaluated through cytotoxic activity; levels of ROS; ATP; mitochondrial membrane potential and expression of FoxO1 protein in a high glucose environment" is quite complex and could be split into two sentences for clarity

-        Line 13: Use “,” instead of “;”. The use of semicolons in the abstract is inconsistent and sometimes confusing. Consider revising the punctuation to improve readability.

Semi colons are no longer used, and sentences are split.

-        I would suggest adding some more keywords in the manuscript. They will help increase the visibility and accessibility of the research.

We have added 8 more keywords.

Introduction and Methodoly:

-        Check for consistency in the use of abbreviations and ensure that they are defined when first used in the text.

We have checked.

-        Line 60: The sentence "Hydroxyhydroquinone or 1,2,4-trihydroxy benzene (HHQ) is a simple aromatic molecule with redox potential like flavonoids and its redox properties." is somewhat confusing. Consider breaking it into two separate sentences to clearly describe the redox potential of HHQ and how it relates to flavonoids.

The sentence was reformulated: “Hydroxyhydroquinone or 1,2,4-trihydroxy benzene (HHQ) is a simple aromatic molecule with redox potential, which can bind or release electrons according to the environment and metabolic demand. Likewise, auto-oxidized flavonoids are capable of producing oxygen radicals presenting lower oxidation potentials than those that did not undergo auto-oxidation”.

-        Line 84: The extended name should be written in line 80.

Sentence was corrected.

-        Line 85: “In the presence of insulin, hydroxyhydroquinone and quassinoids compounds were able to decrease expression of FOXO1, most likely inhibiting gluconeogenesis and ultimately reducing glucose production”. What is the reference for this sentence? Is it just the anticipation of your results? It is completely misleading.

The sentence anticipated results for the FOX protein and was out of place. We relocate part of the sentence to the conclusion section.

-        It is important to include the name of the manufacturer of the materials used in the process, to ensure transparency and reproducibility of the study.

We have included all manufactures.

At what stage were the cells at when the experiments were performed?

The cells were between the third and the fifth passages. We have included the statement in the methods.

Results and Discussion:

-        The abstract mentions that HHQ and quassinoids downregulate the expression of FoxO1. Can the authors provide more detailed results or quantitative data to support this statement?

As shown in Figure 12B, we have quantified Fox01 through densitometric analysis taken from western blot images. The Fox01 bands were normalized to b-actin (control marker) using Image J software. We also performed indirect immunofluorescence using anti-FOXO1 antibody, as shown in the green plot of Figure 12C, representing a qualitative expression of FOXO1 in HepG2 and HepG2/IRM cells.

-        Line 334: The transition between discussing the pharmacological properties of Connaraceae plants and the development of type II diabetes could be smoother. Perhaps a sentence could be added to bridge the two topics. In the last sentence, it is stated that "Oxidative stress can be modulated by flavonoid ingestion." It would be beneficial to provide some examples or evidence from the study to support this statement.

Added

A study from Paim et al. (2022) [19] reveals that catechin, quercetin and proanthocyanidins comprise the highest concentration of phenolic compounds in plants of the genus Con-naraceae. The pharmacological properties of these plants for the treatment of diseases are associated with their diferent species. Rourea cuspidata Benth. ex Baker is used for type II diabetes, Rourea induta Planch. is traditionally used in rheumatic disease and Connarus angustifolius (Radlk.) G. Schellenb. for the treatment of inflammation.

-        Why did you show MTT results about incubation with resveratrol, rapamycin, Myo inositol and Chiro inositol. This could be completely misleading, as the focus of the manuscript is the evaluation of the efficacy of other compounds.

We have initially tested all this compounds and included some well-known molecules as an internal control of experiments. The reason is because the literature points a range of IC50 and mechanisms for these compounds (for example resveratrol is an antioxidant, rapamycin can induce insulin sensitivity). We have taken these compounds out of figure 6 and reformulated the text. Thank you.

-        Line 449: Please remove the italics for this sentence.

 italic removed.

Conclusion:

-        The manuscript’s conclusion should provide a clear and concise summary of the study's main findings, their implications, and potential future research directions. In its current form, the conclusion is a bit scattered and could benefit from a more structured presentation.

-        While the authors discuss the promising effects of hydroxyhydroquinone and quassinoids, it would be helpful to include a summary of the specific results that led to these conclusions. This would provide a clearer link between the study’s findings and the broader implications discussed in the conclusion.

Reformulated all the conclusion section, highlighting the results and further research directions.

Reviewer 3 Report

This is a very interesting research work on the effect of flavonoid-like hydroxyhydroquinoneand quassinoid extracts on glycemic control and insulin sensitization. These compounds (and others) were tested on in a high-glucose environment by monitoring ROS activity, the concentration of adenosine triphosphate, and the measurement of mitochondrial membrane potential. The expression of FOXO1 protein, was also investigated.

The paper is very well written, easy to read and to follow.

The results are robust and thoroughly explained.

The abstract is well written providing the framework of the topic, mentioning the methods and describing the main results.

The introduction contains information relevant to the subject researched, provides enough information to understand the subject.

A clear hypothesis is suggested by the authors and the underlying methods are adjusted to prove (or disprove) this hypothesis.

The justification for the current experiments is well argued in the context of the topic and the research methodology is presented with precision.

The discussion of results is clear and draws several answers regarding the metabolic route for the biosynthesis of the compouds produced, the metabolic mechanism of oxidative stress, production of ROS. lipid peroxidation. The role of oxidative stress in insulin resistance has also been addressed, as well as other metabolic pathways to explain the pathologies addressed (mostly diabetes) in the paper and the impact of the studied compounds in the contribution to assisting in the treatment of insulin resistance.

The conclusions reflect the main findings of the research, as thus are adequate.  

Thus, I acknowledge the effort of the authors, and the interesting results obtained, that are valid to the scientific community.

Therefore, I believe that it can be published in the present form.   

Round 2

Reviewer 2 Report

The manuscript has been sufficiently improved to warrant publication in Compounds.