Association between dietary flavonol intake and mortality risk in the U.S. adults from NHANES database

Using updated National Health and Nutrition Examination Survey (NHANES) follow-up data, and a large nationwide representative sample of adult U.S. citizens, the aim of this study was to explore the relationship between dietary flavonol intake, all-cause and cause-specific mortality risks. In this prospective cohort study based on NHANES (2007–2008, 2009–2010, and 2017–2018), a total of 11,679 participants aged 20 years and above were evaluated. The amount and type of food taken during a 24-h dietary recall were used to estimate dietary flavonol intake, which includes total flavonol, isorhamnetin, kaempferol, myricetin, and quercetin. Each analysis of the weighted data was dealt with in accordance with the NHANES reporting requirements' intricate stratification design. The Cox proportional risk regression model or Fine and Gray competing risks regression model were applied to evaluate all-cause and cause-specific mortality risks, respectively. The follow-up period was calculated using the time interval between the baseline and the death date or December 31, 2019 (whichever occurs first). Each data analysis was performed between October 1, 2023, and October 22, 2023. Dietary flavonol intake included total flavonol, isorhamnetin, kaempferol, myricetin, and quercetin. Up to December 31, 2019, National Death Index (NDI) mortality data were used to calculate mortality from all causes as well as cause-specific causes. A total of 11,679 individuals, which represents 44,189,487 U.S. non-hospitalized citizens, were included in the study; of these participants, 49.78% were male (n = 5816), 50.22% were female (n = 5, 863); 47.56% were Non-Hispanic White (n = 5554), 18.91% were Non-Hispanic Black (n = 2209), 16.23% were Mexican American (n = 1895), and 17.30% were other ethnicity (n = 2021); The mean [SE] age of the sample was 46.93 [0.36] years, with a median follow-up of 7.80 years (interquartile range, 7.55–8.07 years). After adjusting covariates, Cox proportional hazards models and fine and gray competing risks regression models for specific-cause mortality demonstrated that total flavonol intake was associated with all-cause (HR 0.64, 95% CI 0.54–0.75), cancer-specific (HR 0.45, 95% CI 0.28–0.70) and CVD-specific (HR 0.67, 95% CI 0.47–0.96) mortality risks; isorhamnetin intake was associated with all-cause (HR 0.72, 95% CI 0.60–0.86), and cancer-specific (HR 0.62, 95% CI 0.46–0.83) mortality risks; kaempferol intake was associated with all-cause (HR 0.74, 95% CI 0.63–0.86), and cancer-specific (HR 0.62, 95% CI 0.40–0.97) mortality risks; myricetin intake was associated with all-cause (HR 0.77, 95% CI 0.67–0.88), AD-specific (HR 0.34, 95% CI 0.14–0.85), and CVD-specific (HR 0.61, 95% CI 0.47–0.80) mortality risks; quercetin intake was associated with all-cause (HR 0.66, 95% CI 0.54–0.81), cancer-specific (HR 0.54, 95% CI 0.35–0.84), and CVD-specific (HR 0.61, 95% CI 0.40–0.93) mortality risks; there was no correlation observed between dietary flavonol intake and DM-specific mortality. According to the current study, all-cause, AD, cancer, and CVD mortality risks declined with increased dietary flavonoid intake in the U.S. adults. This finding may be related to the anti-tumor, anti-inflammatory, and anti-oxidative stress properties of flavonol.


Different flavonol intake level, survival status and cause of death in participants
Table 3 presented the general characteristics of flavonol intake in participants by survival status or cause of death.As the intake of total flavonol increases, there is a decreasing trend in all-cause mortality, AD-specific mortality, cancer-specific mortality, and CVD-specific mortality (all P values for trend < 0.050).When isorhamnetin intake increases, there is a declining tendency in all-cause mortality, AD mortality, cancer mortality, CVD mortality, DM mortality, and other cause mortality (all P values for trend < 0.050).As the intake of kaempferol increases, there is a decreasing trend in all-cause mortality, AD-specific mortality, cancer-specific mortality, CVD-specific mortality, and other cause mortality (all P for trend < 0.050).As the intake of myricetin increases, there is a decreasing trend in AD mortality (P for trend = 0.003).With increasing quercetin intake quartiles, there is a trend toward lowering all-cause mortality, cancer mortality, and other cause mortality (all P for trend < 0.050).

Sensitivity analysis
In our sensitivity analysis, we applied the final model (Cox proportional risk regression model and Fine and Gray competing risk regression model) including squared age in order to adjust the potential non-linear association between age and mortality.The observed results were consistent with those shown in the main analysis, which indicated our results were relatively robust (Table 5).

Discussion
Established the up-to-date NHANES follow-up data and NDI mortality data, we evaluated a nationally representative sample of 11,679 U.S. citizens aged 20 years old and above, representing 44,189,187 non-institutionalized U.S. residents, and we observed that flavonol intake was significantly related to lower all-cause mortality Table 6.Subgroup analysis stratified by race/ethnicity, using Cox proportional hazards models for all-cause mortality by dietary flavonol intake.HR hazard ratio, CI confidence interval.*P < 0.05; **P < 0.01; ***P < 0.001.a Adjusted for age, sex, educational attainment, marital status, poverty income ratio, alcohol consumption, BMI, disease history (DM, hypertension, hyperlipidemia, congestive heart failure, coronary heart disease, angina, heart attack, stroke).

Flavonol types
Non-Hispanic White Non-Hispanic Black Mexican American Other HR (95% CI) a HR (95% CI) a HR (95% CI) a HR (95% CI) a Total flavonols (mg/day) Q1 [0, www.nature.com/scientificreports/risk.What's more, intakes of total flavonol, isorhamnetin, kaempferol and quercetin were associated with lower cancer-specific mortality risk.Meanwhile, intakes of total flavonol, quercetin and myricetin were associated with reduced CVD-specific mortality risk.We also observed that myricetin intake was related to decreased mortality risk for AD-specific causes.As a result, consumption of flavonoids is protective against all cause, cancer, and CVD-mortality in particular.Subgroup analysis based on age further revealed that flavonoid intake had a more significant protective effect on all-cause mortality reduction in those aged ≥ 40 years compared to participants aged < 40 years.Compared to 2020, the global burden of cancer is estimated to increase by 47% in 2040, from 19.3 million cases to 28.4 million cases.Approximately one-third of cancer cases in affluent nations are linked to physical activity, nutrition, and food.It has been suggested that consuming an appropriate amount of fruits and vegetables high in flavonoids helps prevent cancer 17 .This study suggests an inverse association between flavonol intake and cancer mortality, which is consistent with the conclusions of previous epidemiological studies by Bondonno et al. 18 and Zhou et al. 19 .The previous meta-analysis also highlighted the negative relationship between flavonoid intake and the incidence rate of some cancers, including breast cancer 20 , prostate cancer 21 , lung cancer, gastric cancer and colorectal cancer 22 , and cancers related to smoking 22 .There is evidence that flavonol inhibit to the development and spread of cancer in vitro.For example, by focusing on the phosphoinositide 3-kinase / protein kinase B signaling pathway, kaempferol can suppress epithelial mesenchymal transition and cause apoptosis and cell cycle arrest in the G2 / M phase 23 .By targeting cell cycle proteins, quercetin causes cell cycle arrest in the G1 phase 24 , which prevents cells from proliferating.Furthermore, a high quercetin content can prevent the cell cycle from moving from G0/G1 to G2/M 25 .Quercetin has an impact on the cell cycle, but it can also cause apoptosis by signaling the pro-apoptotic PI3K/Akt and mitogen-activated protein kinase pathways 26,27 .Similar to other flavonol, however, it should be noted that flavonoids can exhibit toxic effects at high doses 28,29 .After the phenolic ring containing flavonoids is oxidized by oxidative enzymes, cytotoxic phenoxyl radicals are generated; unsaturated lipids, nucleic acids, ascorbate, nicotinamide, adenine dinucleotide, and glutathione are all oxidized by CO, which produces reactive oxygen species and is harmful to mitochondria.In some circumstances, quercetin and other flavonoids have been shown to cause a notable frequency of sister chromatid swaps and micronuclei as well as to suppress cell growth by their pro-oxidant properties 30 .Thus, it is necessary to look at how dietary supplements containing flavonoids affect cancer survival.
It is noteworthy that, following numerous confounding factor adjustments, we discovered that the highest flavonoid intake was linked to a lower risk of CVD death in comparison to the lowest consumption.This outcome is in line with the findings of a previous meta-analysis performed by Huxley et al., which revealed that there may be a negative correlation between the death rate from coronary heart disease and flavonol intake 31 .In a meta-analysis including sixteen studies, Wang et al. found that a 20 mg/d increase in flavonol consumption was linked to a 14% reduction in the risk of stroke (RR, 0.86; 95% CI 0.77-0.96) 32.The possible mechanisms by which flavonol reduce the risk of CVD may involve multiple pathways, which have been reported to be generally associated with their Vasodilatory, anti-inflammatory, and antioxidant properties 33,34 .Using flavonol as an example, a large body of research indicates that quercetin and other related flavonol may provide protection against the most prevalent CVD.By preventing one or more processes linked to the development of the disease, such as oxidative stress, endothelial dysfunction, and inflammation, flavonol protects against atherosclerosis 3 .By reducing endothelial dysfunction, hypertension, and atherosclerosis, flavonol can protect coronary arteries 3 .The majority of acute coronary events are caused by myocardial ischemia following the rupture of atherosclerotic plaque.On the other hand, quercetin can stabilize atherosclerotic plaques by decreasing matrix metalloproteinase expression 35 .Flavonoids may affect stroke at different stages.Flavonol can decrease excitatory toxicity, regulate oxidative stress, stop platelet aggregation and thrombosis, and enhance cerebral blood flow during the acute phase 36 .In the intermediate stage, flavonol can protect the integrity of endothelial cells and reduce inflammatory responses 37,38 .Flavonol may obstruct ischemia-induced cell death pathways such as necrosis and apoptosis in later stages [39][40][41] .
DM remains one of the major healthcare challenges in the world.Although there is a growing understanding of the pathophysiology of diabetes, currently available treatment methods can only provide a temporary hypoglycemic effect, and cannot completely prevent the development of these abnormalities 42 .Previous literature has reported that flavonol has the potential of anti-diabetes.For example, Yang et al. reported that kaempferol can regulate lipid metabolism, improve insulin resistance, improve insulin signal transduction, and restore the imbalance between autophagy and apoptosis to protect β cells 43 .Kalai et al. reviewed that isorhamnetin can lower the impact of diabetes related diseases by reducing glucose level, improving oxidation status, reducing inflammation, and regulating lipid metabolism and adipocyte differentiation 44 .Li et al. reported that long-term intake of myricetin can inhibit apoptosis of pancreatic islets β cells and regulation of glucose levels, and myricetin can induce glucose dependent insulin secretion 45 .According to Eid et al., quercetin regulates systemic glucose homeostasis by interacting with numerous molecular targets in the liver, skeletal muscle, adipose tissue, small intestine, and pancreas 46 .The anti -diabetes mechanism of quercetin involves inhibition of intestinal glucose absorption, insulin secretion, insulin sensitizing activity, and improvement of glucose utilization in peripheral tissues.However, our results did not indicate significant correlations between flavonol intake and DM-specific mortality (all P values > 0.05).Whether the flavonol intake can prevent the progress of diabetes and protect diabetic patients still needs to be further explored by longitudinal studies in the population.
Alzheimer's disease (AD) is one of the most prevalent types of age-related neurodegenerative illness.There is an urgent need for effective treatment and prevention strategies since the present therapy options are not able to appreciably modify the development of AD.Myricetin was related to a lower risk of AD-specific mortality in this investigation.Myricetin has been shown to be helpful in treating Alzheimer's disease (AD) in animal trials.Ramezani et al. showed that intraperitoneal injection of myricetin dramatically raised the number of hippocampal CA3 pyramidal neurons in Alzheimer's disease rats, improving learning and memory problems 47 .www.nature.com/scientificreports/Myricetin treatment dramatically reversed the novel effects of polyamines in the AD mouse model, which included downregulating brain iron and inhibiting acetylcholinesterase (AChE).Furthermore, myricetin administration improves the activity of antioxidant enzymes and decreases oxidative damage in mice 45 .In summary, our research indicates that consuming more myricetin may help stop or slow the progression of AD.Previous studies did not take into account the competing risks of death when calculating specific causes of death.In our approach, we tackled this methodological challenge by employing a multiple confounder-adjusted competing risks model.This allowed us to elucidate the significant association between dietary flavonol intake and all-cause and specific-cause mortality risks.Nevertheless, the study still includes the following limitations: first, more than half of the sample size lacks data on dietary flavonol intake in the NHANES database, and the remaining 11,679 participants may not be nationally representative.Second, only an estimate of the flavonol dose was made at baseline, therefore it may not precisely reflect the flavonol intake over the course of the investigation.Third, the NHANES collected the representative participants in the North America, the western dietary pattern is most prevalent, which has an impact on the dietary composition of flavonol and other nutrients.Due to the limitation of database, we were unable to give the main food sources for dietary flavonol intake.Further, subgroup analysis showed that flavonol intake had a significant protective effect on mortality in the Non-Hispanic White population, suggesting that dietary patterns of different race/ethnicity may play an important role.Fourth, since the adjusted confounding factors in the model do not include total energy intake, micronutrient/antioxidant supplement intake, vitamin intake, and other dietary factors like lycopene or green tea catechins, it may be impossible to exclude the influence of these factors on the observed effect of flavonol intake.

Conclusion
Through comprehensive updating NHANES records, this study concluded that dietary flavonol intake was significantly linked with overall, AD, cancer, and CVD-specific mortality risks.The outcome of our research elucidated the relationship between flavonol intake, all-cause, and cause-specific mortality risks in a sample representing the entire nation of non-hospitalized citizens in the United States, presenting evidence for flavonol intake as an independent, practical, quantitative, and reliable predictor of disease survival status, this means that it is suitable for the health and risk alert management of AD, CVD and cancer patients.Our findings have practical significance for public health, because flavonol can be supplemented by making daily dietary modifications and eating habits better.

Study population
Dietary Flavonol intake data in this study were collected from three cycles of the nationwide representative NHANES, corresponding to years 2007-2008, 2009-2010, and 2017-2018.The National Center for Health Statistics, which examined the health and nutritional status of U.S. citizens using a nationally representative sample, sponsored and supervised NHANES 22 .The three cycles NHANES provided a total of 111,066 participants.84,841 participants were removed because they did not have dietary flavonol intake values from FNDDS, the percentage of missing values for dietary flavonol intake were 76.4%.Additionally, 14,546 individuals were excluded because they were who had no data on marital status, educational attainment, poverty income ratio, alcohol consumption, and body mass index (BMI), and disease history (Fig. 1).Finally, the present analyses were based on a total of 11,679 individuals who were aged ≥ 20 y and had completed a battery of questionnaires, in-person assessments, and laboratory tests were mandated for participants, either at the mobile examination center (MEC) or at home.The National Center for Health Statistics Research Ethics Review Board approved the NHANES programs, and all study participants gave written informed consent and followed the guidelines of the Helsinki's Declaration 48 .Relevant ethical certifications can be found in Supplement A.

Statistical analysis
Three NHANES cycles (2007-2008, 2009-2010, and 2017-2018) were integrated into one dataset, and all analyses were carried out using the design that the NHANES Analytics and Reporting Guide supplied.The intake of dietary flavonol including total flavonol, isorhamnetin, kaempferol, myricetin, and quercetin, was classified by quartiles, with quartile 1 (Q1) indicating the lowest intake, and quartile 4 (Q4) indicating the highest intake.The basic characteristics of each participant and their dietary flavonol intake distribution were described using numbers and weighted percentages for included categorical variables.These comprised the following: age, sex, race/ethnicity, marital status, educational attainment, poverty income ratio, alcohol consumption, and BMI.A chi-square test was applied for categorical variables.Then, we described the number and percentage of overall deaths and specific-cause deaths, corresponding to different intake levels of flavonol.And trend chi-square test was applied to determine the relationship between dietary flavonol intake and death outcomes in participants.To determine basic characteristics (sociodemographic variables and disease history) related to survival endpoints, we performed the Cox regression analysis adjusted age and sex to estimate the hazard ratio (HR) and 95% confidence interval (CI) for survival.Further, covariates significantly correlated with mortality and dietary flavonol intake were added to the Cox regression model, which calculated the HR and population attributed risk of dietary flavonol intake on mortality.We applied the Fine and Gray competing risks regression model to estimate specific-cause (e.g., AD, cancer, CVD, DM) mortality risks, given that deaths from other causes can be considered the competing risk event for one specific cause of death 53 , and this method has been used by previous studies with high quality 54,55 .Sensitivity analyses was perform by adjusting for age and squared age to evaluate the potential non-linear association between age and mortality.The Variance Inflation Factor (VIF) was used to examine the covariate effect, and all VIFs were less than 3.00.The P-values on both sides are less than 0.05, which is considered statistically significant.R software (version 4.2.1, http:// www.R-proje ct.org) was applied to analyze all the data, with packages "nhanesR", "reshape2 ", "survey", "do", "dplyr" and others.

Table 1 .
Statistical description of dietary flavonol intakes.

Table 2 .
Sociodemographic, and Disease history characteristics of participants with dietary flavonol intake.HS high school, BMI body mass index.a All proportions,are weighted estimates of the U.S. population characteristics, taking into account the complex sampling design of the NHANES.

cause mortality by basic characteristics of sociodemographic variables and disease history We
applied Cox regression analysis to explore the relationship between basic characteristics (including sociodemographic variables, and disease history) and all-cause mortality in U.S. participants, and the detailed outcome was presented in Table4.It is important to notice that HRs increased significantly every ten years of age.