C-Ring Structure-Dependent Redox Properties of Flavonoids Regulate the Expression of Bioactivity

Round 1

Reviewer 1 Report

This paper  compared physicochemical properties and biological activity of three flavonoids ( (-)-Epicatechin (EC), taxifolin (Tax), and quercetin (Q)) with different C-ring structures, by looking at redox properties at different pHs mimicking plant vacuole or gut, as well as their impact on sympathetic hyperactivation in a mouse model after acute dosing. Although the data seems interesting, the links between structure /redox properties and impact of sympathetic activity are not mechanistically very clear.

Major Points to address:

• Using urine catecholamines , as well as muscle arterial blood flow as indexes of sympathetic nerve activation is a substantial limitation of the work. Wouldn´t direct electrophysiological recordings of nerve activity be a more robust measure? Evidence of increases in Hear Rate or Blood Pressure. Flavonoids have been shown to lower blood pressure in humans and there is not any evidence suggesting that it results in increases in heart rate or blood pressure acutely in the human literature.

         A more robust rationale for the use of these outcome measures to assess sympathetic activation is needed and /or adding this as a limitation.

           A discussion of how these data align with human data is important also.

• Stress is easily induced in rodents, so what steps have been taken to control for stress exposure during the animal studies, particularly to ensure that all were subjected to exactly the same levels of stress. This could have an impact on the catecholamine excretion.
• Justify why increases in blood flow are being interpreted in this context as evidence for activation of the sympathetic nervous system. Could this increases in blood flow be mediated by other mechanisms related to modulation of the endothelial products such as NO?
• Contextualize the limitations of using Computational analysis to estimate Reactivity of flavonoids . Also explain the rationale for performing molecular dynamic simulations on the parent compounds, if those are highly metabolized in the body and unlikely to drive the biological effects?
• The discussion would benefit from separation into a few sub-sections to facilitate readability, specially given that experiments are discussed separately.
• It would be good to put into context the dose of epicatechin that was given to rodents and how would that translate into the typical effective doses in humans of 1 mg/kg BW.
• It is not clear the mechanism by which differences in C-ring structure (affecting stability and redox properties) links to different abilities to activate the nervous system. It would be good to more clearly clarify how these 2 observations are clearly linked and elaborate a bit more on specific experiments needed to establish a causal relationship.
• Consider simplifying Figure 4, 5 and 6 as there are too many graphs with a lot of data in each one, making it hard to read. Also each graph within the Figure should have a label as A, B, C etc…

Minor:

• Line 60: clarify how many hours do flavonoids spend in the large intestine. Is it 90 hours?
• Line 271 :Should be read Quercetin and not Quercetine
• Most Figures have a label on the bottom right corner., e.g. Fig 1, Fig 2, that need to be removed.

 

Author Response

RESPONSE TO REVIEWER 1： We wish to express our appreciation to Reviewer 1 for their insightful comments, which have helped us improve our paper significantly.

 

Comment 1  Using urine catecholamines , as well as muscle arterial blood flow as indexes of sympathetic nerve activation is a substantial limitation of the work. Wouldn´t direct electrophysiological recordings of nerve activity be a more robust measure? Evidence of increases in Hear Rate or Blood Pressure. Flavonoids have been shown to lower blood pressure in humans and there is not any evidence suggesting that it results in increases in heart rate or blood pressure acutely in the human literature. A more robust rationale for the use of these outcome measures to assess sympathetic activation is needed and /or adding this as a limitation. A discussion of how these data align with human data is important also.

Comment 2  Justify why increases in blood flow are being interpreted in this context as evidence for activation of the sympathetic nervous system. Could this increases in blood flow be mediated by other mechanisms related to modulation of the endothelial products such as NO?

Comment 4 It would be good to put into context the dose of epicatechin that was given to rodents and how would that translate into the typical effective doses in humans of 1 mg/kg BW.

Response

We appreciate Reviewer 1's insightful comments. While several meta-analyses of numerous intervention trials have confirmed that a single intake of 200mg or more of flavanols, such as epicatechin, increases the Flow-Mediated Dilatation (FMD) value (DOI: 10.1039/c9fo01747j), reports on other flavonoids are extremely scarce. Similarly, while repeated intake of flavanol-containing foods is known to have a blood pressure-lowering effect (DOI: 10.3389/fnut.2022.969823), this is not clear for other flavonoids. Although a few percent of the flavanol epicatechin is absorbed, it is metabolized in intestinal epithelial cells and liver cells, converting to water-soluble conjugates, meaning almost no unchanged form is present in peripheral blood. Previous studies reported increased eNOS expression in HUVEC cells in vitro following flavanol supplementation (DOI: 10.1007/s00394-011-0172-9). However, the effective concentration required for this effect was over 1000 times higher than typical blood concentrations, making it an unrealistic condition. In our previous study, we replicated the immediate circulatory changes observed after flavanol intake in experimental animals. Flavanols transiently increased blood pressure, heart rate, and skeletal muscle blood flow. Furthermore, we demonstrated that this effect is canceled by adrenergic receptor antagonists, confirming it is due to enhanced sympathetic nervous system activity (DOI:10.1016/j.freeradbiomed.2016.09.008, 10.1080/10715762.2020.1759805). In addition, the results of the FMD intervention trial showed that the effective amount of flavanol is 200-600 mg/person, of which approximately 20% is epicatechin, so the dose of epicatechin used in this trial is consistent with the clinical dose. Furthermore, following repeated flavanol administration, blood pressure decreases over time in rodents (DOI: 10.1186/1476-511x-13-64), similar to humans. It has also been confirmed that when flavanols are repeatedly administered to humans, the rate of increase in FMD after the final dose is plateau than the rate of increase after the initial dose (DOI:10.1017/s0007114515002822). These phenomena resemble the changes in hemodynamics observed during exercise due to increased sympathetic nerve activity. Specifically, as shown in the reference figure, a single dose causes a transient increase in blood flow, leading to increased eNOS expression due to elevated shear stress. However, repeated administration is thought to induce further angiogenesis, resulting in decreased blood pressure and reduced shear stress (DOI: 10.3164/jcbn.22-50). Furthermore, it has been demonstrated that following a single oral administration of flavanol to mice, blood adrenaline and noradrenaline levels peak 2-4 hours later (DOI: 10.1371/journal.pone.0201203). Recent experiments have demonstrated that flavanol acts as a stressor in mammals, activating the locus coeruleus-noradrenergic network and hypothalamic paraventricular nucleus CRH neurons, thereby enhancing sympathetic activity as a stress response(DOI: https://doi.org/10.1016/j.crfs.2025.101195). As Reviewer1 pointed out, direct measurement of neural potentials is likely the most robust indicator for verifying sympathetic activity. However, since changes in flavanol's circulation dynamics occur via the central nervous system as part of this stress response, direct verification is considered technically extremely difficult. Therefore,we adopted changes in circulation dynamics and urinary catecholamines as surrogate markers for sympathetic activity in this study.　However, as it is still difficult to understand, we have added the sentences to L86-118 as suggested by Reviewer1.

 

 

 

 

Mechanism of circulatory dynamics alteration due to increased sympathetic nervous activity

Comment 3

Stress is easily induced in rodents, so what steps have been taken to control for stress exposure during the animal studies, particularly to ensure that all were subjected to exactly the same levels of stress. This could have an impact on the catecholamine excretion.

Response

As Reviewer1 pointed out, stress management was particularly crucial in this experiment, and we attempted to mitigate it using the method inserted in L163-177.

Comment 5

Contextualize the limitations of using Computational analysis to estimate Reactivity of flavonoids. Also explain the rationale for performing molecular dynamic simulations on the parent compounds, if those are highly metabolized in the body and unlikely to drive the biological effects?

Response

As mentioned above, flavonoids are poorly absorbed and their unchanged concentrations in the blood are extremely low. Therefore, it is believed that the various effects of flavonoids originate in the gastrointestinal tract. As mentioned in the main text, flavonoids are originally stored in plants at a weakly acidic pH, but it is well known that changes in pH accelerate their decomposition. Because flavonoids change structure depending on pH, this study examined the stability of flavonoids under neutral pH conditions, simulating the intestinal tract (Fig. 1-3), and compared their reactivity with O2- (Fig. 4). Because the behavior of the three flavonoids differed, molecular dynamics simulations were performed and revealed that flavanols were significantly more unstable than flavanonols and flavonols. We added a paragraph to L386-388 about why I did computational chemistry.

Comment 6

The discussion would benefit from separation into a few sub-sections to facilitate readability, specially given that experiments are discussed separately.

Response

We have divided the discussion into subsections as suggested by Reviewer 1.

Comment 7

It is not clear the mechanism by which differences in C-ring structure (affecting stability and redox properties) links to different abilities to activate the nervous system. It would be good to more clearly clarify how these 2 observations are clearly linked and elaborate a bit more on specific experiments needed to establish a causal relationship.

Response

As Reviewer 1 pointed out, the C-ring structure significantly influences stability and redox properties. At neutral pH, EC was unstable/pro-oxidant, Q was unstable/antioxidant, and Tax, possessing intermediate properties, was stable/pro-oxidant. Our previous studies confirmed that ROS generated by flavanols is involved in their effects, as the vasodilatory action was canceled when flavanols containing EC were co-administered with the ROS scavenger N-acetylcysteine(DOI: 10.1016/j.bcp.2023.115682). On the other hand, Transient Receptor Potential V1 (TRPV1) and TRPA1, which are predominantly expressed in the enteric nervous system, possess ROS-sensitive domains. It is known that ROS activates these channels, transmitting signals to the central nervous system to induce stress responses (DOI: 10.1093/bbb/zbad154). Therefore, when flavanol was co-administered with TRP channel inhibitors, the blood flow-increasing effect was canceled, suggesting that these channels are likely involved in the mechanism of action(DOI: 10.1016/j.bcp.2023.115682). Consequently, we are currently advancing experiments using knockout mice and expect to report our findings soon. To clarify this point, we have added the following text to L584-595.

Comment 8

Consider simplifying Figure 4, 5 and 6 as there are too many graphs with a lot of data in each one, making it hard to read. Also each graph within the Figure should have a label as A, B, C etc…

Response

The figures have been revised according to Reviewer 1's instructions and also revised results section .

 

Minor

・Line 60: clarify how many hours do flavonoids spend in the large intestine. Is it 90 hours?

- We revised to 24 hours.

・Line 271 :Should be read Quercetin and not Quercetine

- We corrected.

・Most Figures have a label on the bottom right corner., e.g. Fig 1, Fig 2, that need to be removed.

- We deleted.

Author Response File: Author Response.docx

Reviewer 2 Report

This reviewer does not have any major comment. However, reviewer wish to suggested several things to attract the readers and boost the search engine performance, such as general terminology, new reaction figure, more journal review and hypothesis in discussion.

The manuscript is written reasonably well. The topic of this manuscript seems to be read by variously disciplined people. Thus, the introduction/abstract should be a little bit generalized for attracting more readers’ attention and selected by any search engines more effectively.

Minor suggestions for introduction:

• Manuscript deals with specific three flavonols among more than ~8,000 flavonoid molecules. This reviewer feel introduction need to describe/review more about those specific three in terms of physiological roles, abundant foods and health-related matters to be attracted by many readers.
• Terminology: Specifically, epicatechin belongs to flavan-3-ols and taxifolin is often called dihydroquercetin (DHQ). It might be a good idea to use both terms (at least once with a parenthesis).
• The three flavonoids,epicatechin, taxifolin, and quercetin, can coexist in the same plant. It might be a good idea to indicate the metabolic relationship among these three. For example, quercetin is produced by flavonol synthase (FLS, EC 1.14.20.6) from Taxifolin (dihydroquecetin). Epicatechin is produced from taxifolin by consecutive activities from Dihydroflavonol 4-reductase (DFR. EC 1.1.1.219), Anthocyanin synthase (ANS, or flavonoid 3',5'-hydroxylase, EC 1.14.11.12) and Anthocyanidin Reductase (ANR, EC 1.3.1.77). Or include some pathway figure.

Minor suggestions for discussion:

• It might be a good idea to expand the authors’ findings to other pairs of flavonoids ( i.e. hypothesize/speculate). Do the same pairs of flavonoid with a different number of hydroxyl group in B-ring (with same C-ring), display similar behavior? For example, one hydroxyl group in B ring (4' position or para) pairs ((-)-epiafzelchin, dihydrokaempferol, kaempferol) or three hydroxyl group in B ring (3', 4', and 5' positions)pairs (epigallocatechin-3-gallate (EGCG), dihydromyricetin, myricetin).
• Author describes the interaction with O2•⁻ and it will be easier to follow the authors' description of chemistry if there are corresponding figures for the plausible figures.

Error

Line 60: qo hours

Line 471: These results indicate that Tax is stable against EC.

Line 104: Spelling error: queercetin

Line 425: Stability of two or three flavonoids?

Author Response

RESPONSE TO REVIEWER 2： We would like to express our gratitude to Reviewer 2 for their insightful comments, which have contributed to a substantial improvement in the quality of our paper.

Comment 1

Manuscript deals with specific three flavonols among more than ~8,000 flavonoid molecules. This reviewer feel introduction need to describe/review more about those specific three in terms of physiological roles, abundant foods and health-related matters to be attracted by many readers.

Comment 2

Terminology: Specifically, epicatechin belongs to flavan-3-ols and taxifolin is often called dihydroquercetin (DHQ). It might be a good idea to use both terms (at least once with a parenthesis).

Comment 3

The three flavonoids,epicatechin, taxifolin, and quercetin, can coexist in the same plant. It might be a good idea to indicate the metabolic relationship among these three. For example, quercetin is produced by flavonol synthase (FLS, EC 1.14.20.6) from Taxifolin (dihydroquecetin). Epicatechin is produced from taxifolin by consecutive activities from Dihydroflavonol 4-reductase (DFR. EC 1.1.1.219), Anthocyanin synthase (ANS, or flavonoid 3',5'-hydroxylase, EC 1.14.20.4) and Anthocyanidin Reductase (ANR, EC 1.3.1.77). Or include some pathway figure.

Response

Following reviewer 2's instructions, I added a note to them in L123-140.

Comment 4

It might be a good idea to expand the authors’ findings to other pairs of flavonoids ( i.e. hypothesize/speculate). Do the same pairs of flavonoid with a different number of hydroxyl group in B-ring (with same C-ring), display similar behavior? For example, one hydroxyl group in B ring (4' position or para) pairs ((-)-epiafzelchin, dihydrokaempferol, kaempferol) or three hydroxyl group in B ring (3', 4', and 5' positions)pairs (epigallocatechin-3-gallate (EGCG), dihydromyricetin, myricetin).

Response

As Reviewer 2 pointed out, differences in the structures of the B- and C-rings of flavonoids may significantly affect their redox properties or physiological effects. Many studies have previously investigated the relationship between the number of hydroxyl groups on the B-ring and antioxidant activity, with activity reported to depend on the number of hydroxyl groups. Meanwhile, it has been reported that the presence of a double bond between the 2nd and 3rd positions on the C ring promotes electron delocalization throughout the molecule, further stabilizing radicals and enhancing antioxidant activity. However, we have found that a hydroxyl group on the 3rd position enhances pro-oxidant activity. We are currently investigating the differences between EC and epigallocatechin, which have different B-ring structures, as well as the glycosides of pelargonidin, cyanidin, and delphinidin, and we hope to report on these findings soon. We have added the statements in L618-623 in response to Reviewer 2's comment.

Comment 5

Author describes the interaction with O2•− and it will be easier to follow the authors' description of chemistry if there are corresponding figures for the plausible figures.

Response

As reviewer 2 pointed out, I think it would be easier to understand if there was a diagram, so we added the reaction equation in the SFig.4 and added L488-490.

Minor

・Line 60: qo hours

-We revised to 24 hrs.

・Line 471: These results indicate that Tax is stable against EC.

-We revised to Tax is more stable than EC

・Line 104: Spelling error: quercetin

-We corrected.

・Line 425: Stability of two or three flavonoids?

-We revised to three flavonoids

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors responded to all my commments. Can be accepted in the present form

The authors responded to all my commments. Can be accepted in the present form