Allium Flavonols: Health Benefits, Molecular Targets, and Bioavailability

Allium species are revered worldwide as vegetables, condiments, and spices as well as the therapeutic agents in traditional medicine. The bioactive compounds in alliums mainly include organosulfur compounds, polyphenols, dietary fibers, and saponins. Flavonoids, particularly flavonols from alliums, have been demonstrated to have the antioxidant, anticancer, hypolipidemic, anti-diabetic, cardioprotective, neuroprotective, and antimicrobial activities. However, flavonols are mostly characterized from onions and have not been comprehensively reviewed across different species. This article therefore focuses on flavonol profiles from different Allium species, their health effects, underlying molecular mechanisms, and bioavailability. Intriguingly, the functional health effects of flavonols were mainly ascribed to their antioxidant and anti-inflammatory activities involving a cascade of multiple signaling pathways. Although the Allium-derived flavonols offer tremendous potential in preventing chronic disease risks, in-depth studies are needed to translate their clinical application.


Introduction
Chronic diseases pose a huge burden on global health care system and economy accounting for nearly seventy percent of pre-mature mortality en masse [1]. According to the World Health Organization (WHO), poor dietary habits which include consumption of foods with low-nutrient density and high in fat, sugar, and salt as well as overall calories, are one of the major contributors to the leading causes of chronic illness and related deaths [1,2]. We emphasize that the dietary co-administration of the antioxidant-rich foods along with clinical treatments may ameliorate chronic illnesses mediated by heightened free radicals in the body. It has particularly been observed that people with chronic illnesses are more vulnerable to clinical complications and deaths by novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) infections [3][4][5]. The on-going coronavirus disease (CoVID-19) pandemic has forced people across the globe to rethink their nutritional habits, switching to antioxidant-rich foods to avoid chronic illness with stronger immunity [6,7]. Antioxidant-rich foods reportedly enhance the T-cell subsets, interleukin production, and natural killer cells necessary to ward off influenza-like infections [7,8]. Regular consumption of Allium herbs is traditionally considered beneficial for human health owing to their rich contents of antioxidant compounds. The genus Allium (Amaryllidaceae) is one of the largest monocot genera comprising approximately 915 species including culinary herbs like garlic, onion, shallot, leek, chives, and scallions [9]. The main bioactive constituents in alliums include organosulfur compounds (OSCs) and polyphenols [10]. However, the complex chemistry of OSCs, due to their highly-volatile and thermally-unstable nature, likely impairs their health benefits [10][11][12][13]. In this context, allium polyphenols, with relatively higher stability than OSCs and associated antioxidant functions have emerged as more viable bioactive constituents in of Allium-derived polyphenols, in particular, flavonols, are attributed to their antioxidant activity which governs their functional properties including anti-inflammatory, antimicrobial, antiglycemic, and anticancer effects [14][15][16]. Though various Allium species are rich source of dietary flavonols, most studies have focused on the onion. The present review highlights the current knowledge about well-characterized flavonols from different Allium spp., their established health effects, and the associated subtle mechanisms based on their antioxidant properties as well as bioavailability.

Flavonoids in Allium: Structural Properties
The major polyphenolic compounds in alliums include flavonoids, phenolic acids, and lignans (http://phenol-explorer.eu/) [17] (Figure 1). Flavonoids are the largest class of polyphenols followed by phenolic acids in alliums, while lignans are a minor polyphenol class [17]. Flavonoids have a characteristic 15-carbon (C6-C3-C6) skeleton, which consists of two phenyl rings (A and B) and a heterocyclic ring (C) [18,19]. They are classified into different subclasses (flavonols, flavanols, flavanones, flavones, anthocyanins, and isoflavones) based on the degree of unsaturation and oxidation of the C-ring. Among different subclasses, further division is based on the number and nature of substituent groups attached to their heterocyclic rings [18,19]. Flavonols and anthocyanins are the main subclasses of flavonoids present in alliums, though the latter have been found only in red onions [20,21]. Notably, the flavonol content in different Allium spp. ranges from 7 to 1917 mg/kg fresh weight (Table 1). Herein, we intended to give a comprehensive detail of the flavonol constituents and related bioactivities from alliums.  (A. tuberosum) Leaves 160 mg/kg D.W. [28] Welsh onion A. fistulosum Leaves 2329 mg/kg D.W. [28] Yellow flowered garlic A. flavum subsp.

Flavonols
Flavonols, the most abundant class of flavonoids in alliums [22][23][24], contain a 3-hydroxyflavone backbone (IUPAC name: 3-hydroxy-2-phenylchromen-4-one) [18,19]. Their diversity arises from the different positions of the phenolic -OH groups. Quercetin, kaempferol, isorhamnetin, myricetin, fisetin, and morin are the major flavonol aglycone representatives in alliums (Figure 2). At least 52 different kinds of flavonols have been identified from different Allium spp. ( Table 2). Most of these flavonols exist as their glycosylated derivatives in nature, where sugars are attached through oxygen at 3, 4 , and/or 7-positions of the aglycones. Glucose is the most common sugar moiety; however, rhamnose, galactose, xylose, and glucuronic acid have also been identified (Table 2). Generally, flavonol glycosides have significantly lower antioxidant capacity than their respective aglycones [31]. Chemically, the antioxidant activity of flavonols is owed to their phenolic hydroxyl group via donation of hydrogen atom to free radicals [32]. The hydroxyl groups in B-ring (4 -position) and C-ring (3-position) have higher hydrogen donation ability than the A-ring hydroxyl groups [31]. The concentration (Table 1) and type ( Table 2) of flavonols vary widely among species, cultivars, and parts of the plant. In addition, post-harvest practices [33] and seasons [34] are also reported to affect the flavonol concentration in alliums. Major flavonols found in onion cultivars are the derivatives of quercetin while kaempferol and isorhamnetin derivatives are lesser abundant [22][23][24]. Quercetin 3,4 -glucoside (3,4 -Qdg) and quercetin 4 -glucoside (4 -Qmg) account for more than 90% of flavonols in onion [23,24]. In general, the levels of flavonols are higher in yellow onions than red onions [24]. Sweet onion contains two-three-fold higher isorhamnetin 4 -glucoside than red onion cultivars [35]. In chives, leeks, and scallions, kaempferol glycosides are the major flavonols [24,27,36]. Although several Allium flavonoids are characterized, the associated in vivo mechanisms are not fully understood owing to their cryptic catabolism following the dietary uptake. Below we discuss the health effects and associated functional mechanisms of Allium-derived flavonols based on the state-of-the-art literature and available metadata information. different positions of the phenolic -OH groups. Quercetin, kaempferol, isorhamnetin, myricetin, fisetin, and morin are the major flavonol aglycone representatives in alliums (Figure 2). At least 52 different kinds of flavonols have been identified from different Allium spp. ( Table 2). Most of these flavonols exist as their glycosylated derivatives in nature, where sugars are attached through oxygen at 3, 4′, and/or 7-positions of the aglycones. Glucose is the most common sugar moiety; however, rhamnose, galactose, xylose, and glucuronic acid have also been identified (Table 2). Generally, flavonol glycosides have significantly lower antioxidant capacity than their respective aglycones [31]. Chemically, the antioxidant activity of flavonols is owed to their phenolic hydroxyl group via donation of hydrogen atom to free radicals [32]. The hydroxyl groups in B-ring (4′-position) and Cring (3-position) have higher hydrogen donation ability than the A-ring hydroxyl groups [31]. The concentration (Table 1) and type ( Table 2) of flavonols vary widely among species, cultivars, and parts of the plant. In addition, post-harvest practices [33] and seasons [34] are also reported to affect the flavonol concentration in alliums. Major flavonols found in onion cultivars are the derivatives of quercetin while kaempferol and isorhamnetin derivatives are lesser abundant [22][23][24]. Quercetin 3,4′glucoside (3,4′-Qdg) and quercetin 4′-glucoside (4′-Qmg) account for more than 90% of flavonols in onion [23,24]. In general, the levels of flavonols are higher in yellow onions than red onions [24]. Sweet onion contains two-three-fold higher isorhamnetin 4′-glucoside than red onion cultivars [35]. In chives, leeks, and scallions, kaempferol glycosides are the major flavonols [24,27,36]. Although several Allium flavonoids are characterized, the associated in vivo mechanisms are not fully understood owing to their cryptic catabolism following the dietary uptake. Below we discuss the health effects and associated functional mechanisms of Allium-derived flavonols based on the stateof-the-art literature and available metadata information.
Another seminal study by Lee et al. [54] reported that onion-derived flavonols trigger the "mitochondria mediated and caspase-dependent apoptosis" in AGS human gastric cancer cells through the inhibition of PI3K/Akt signaling pathways. Besides suppressing the Akt phosphorylation, the flavonols upregulate the p53 expression and subsequent Bax induction, resulting in reduced MMP (∆Ψm) and cyt c release, which is directly linked with the activation of caspase associated molecular cascade [54]. The authors also reported that the flavonol extracts also suppress the expression of mitochondria localized anti-apoptotic Bcl-2, a key regulator of apoptosis [54]. The anti-proliferative activities of Chinese onion (A. chinense), onion (A. cepa), and Welsh onion (A. fistulosum) extracts rich in quercetin glucosides (quercetin-3,4 -di-O-glucoside, 3,4 -Qdg and quercetin-4 -O-glucoside, 4 -Qmg) are also reported against liver, colon, and pancreatic cancer cell lines [55]. The authors observed a notably higher efficacy of 4 -Qmg toward the growth inhibition of cancer cells, as compared with 3,4 -Qdg. More recently, the subcritical water extracts of A. hookeri roots (30.6 mg of quercetin equivalent/g) are demonstrated to have a dose-dependent antiproliferative effects against fibrosarcoma and breast cancer cell lines [56]. During carcinogenesis, host immune system is often subverted by inflammation which in part due to oxidative stress [57]. The anti-inflammatory and immunomodulatory effects of flavonol-rich (quercetin and quercetin-4 -O-glucoside) red onion scale extract on prostate cancer rat model are observed by the suppressed expressions of pro-inflammatory molecules including interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha (TNF-α) in prostatic tissues [14]. Furthermore, the attenuation of lipopolysaccharide (LPS)-induced oxidative as well as inflammatory stress in colon cancer cells (HT-29) has been reported by downregulating TNF-α expression and upregulating the expression of heme oxygenase-1 (HO-1) and glutathione S-transferase (GSTs) detoxification genes (i.e., GSTM1, GSTT1, and GSTP1) after onion peel extract (OPE) treatments [50]. Oxidative stress also acts a major stimulus of angiogenesis (the formation of new blood vessels from existing capillary networks) during tumor progression [58]. Seyfi et al. [59] demonstrated significant anti-angiogenic effects of flavonoid-rich A. ascalonicum (shallot) fraction in vitro, ex vivo, and in vivo models; however, the underlying mechanism is not investigated.

Anti-Obesity and Hypolipidemic Effects
Obesity is the underlying cause of several chronic diseases and is characterized by the excessive accumulation of adipose tissues (body fat) when energy intake surpasses the energy expenditure [60]. Obesity is a complex health issue that arises from a combination of causes and individual factors such as behavior and genetics. Behaviors can include physical activity, dietary patterns, medication use, and exposures to various environmental factors. Additional contributing factors include the food and physical activity environment, education and skills, and food marketing as well as promotion. Allium derived flavonol-rich extracts inhibit adipogenesis and intracellular lipid accumulation in cultured adipocytes and diet-induced obese animal models (Table 3). Several mechanistic studies suggested that quercetin-rich OPE induces lipolysis through the downregulated expression of transcriptional factors, including peroxisome proliferator-activated receptor-gamma (PPAR-γ) and CCAAT-enhancer binding protein alpha (C/EBPα) [61][62][63]. In addition, the genes associated with lipid metabolism including lipoprotein lipase (LPL), adipocyte fatty acid-binding protein (AFABP) and carnitine palmitoyltransferase 1α (CPT-1α) are also affected following OPE exposure [62,63]. Recent studies also have shown that autophagy plays an important role in adipogenesis and lipid metabolism. Allium-derived flavonols positively regulate autophagy via activation of AMP-activated protein kinase (AMPK) in cellular and animal models [64,65]. AMPK also plays an important role in the development and maintenance of brown and beige adipose tissue [66]. Recently, flavonol rich (quercetin and isoquercetin) OPE demonstrated browning effect on white adipose tissue through an AMPK-dependent pathway in mice adipose tissue and in cultured adipocytes [64]. The AMPK activation regulates the expression of brown adipocyte-specific genes such as uncoupling protein-1 (UCP-1), peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α) and cell death-inducing DNA fragmentation factor-alpha-like effector A (CIDEA) [64].
In vitro: 3T3-L1, 1-4 µg/mL for 24 h Antiobesity -Reduces lipid accumulation -Reduces adipogenesis -Induces lipolysis A. cepa (peel) Quercetin and isoquercetin In vitro: 3T3-L1, 50-150 µg/mL for 11 d (on day 5, 7, and 9) In vivo: HFD-fed C57BL/6 mice, 0.5% in diet for 8 wk Antiobesity -Induces adipocyte browning -Reduces adipogenesis -Reduces lipogensis       The high lipid uptake into adipocyte in obese conditions often increases the mitochondrial substrate load, which subsequently increases electron transport chain activity and ROS-mediated oxidative stress. These enhanced ROS levels have been associated with increased insulin resistance [92]. Hence, it has also been argued that the anti-obesity effects of onion flavonols may be linked with reduced insulin resistance [93] and elevated blood antioxidant parameters [70,71,94] following the dietary interventions. Reportedly, quercetin and its glycoside derivatives isolated from onion waste improve lipid metabolism in HFD-fed rats through promoting the enzymatic activity of intestinal microbiota and the antioxidant capacity of blood [71]. In a follow-up study, the authors suggested a higher efficacy of 4 -Qmg in lowering plasma cholesterol and triglycerides than the 3,4 -Qdg in high-fat diet (HFD)-fed rats [94].
Some randomized controlled trials (RCTs) have also been investigated toward probing the effects of quercetin-rich Allium extracts on obesity in human subjects. In a study by Kim and Yim [70], OPE administration (100 mg/d, 50 mg bis a die) for 12 weeks significantly reduces waist and hip circumference, and the effects are ascribed to the reduced oxidative stress in obese women. However, the follow-up study showed no influence on the inflammatory mediators among the obese women [95].

Anti-Diabetic Effects
Type 2 diabetes is a worldwide epidemic and characterized by the elevated blood sugar levels due to impaired insulin action and/or secretion [96]. When diabetes is left unmanaged or untreated, increased blood glucose can damage the heart, blood vessels, eyes, kidneys, and nerves, leading to disability and premature death [96][97][98][99]. Several pre-clinical studies reported antidiabetic effects of flavonol-rich extracts from different Allium spp. in vitro [72,75] and in vivo [36,72,73] (Table 3). The ethanolic extract of onion skin (6.04 g quercetin/100 g dried weight of onion skin) is reported to lower postprandial blood glucose response in diabetic rats and the effects are ascribed to the inhibition of carbohydrate digestive enzymes including α-amylase, α-glucosidase, and sucrase [72]. Oxidative stress and inflammation are inter-linked and play a key role in the progression of type II diabetes [99][100][101][102]. Thus, the inhibition of oxidative stress might be an effective strategy to delay/prevent diabetes-related complications [103][104][105][106]. Jung et al. [73] reported that the supplementation of quercetin-rich OPE suppressed the biomarkers of serum oxidative stress (superoxide dismutase (SOD) activity and malondialdehyde (MDA) formation) and hepatic inflammation (TNF-α and IL-6), as well as improved the lipid profiles, levels of insulin receptors, and insulin-regulated glucose transporter type 4 (GLUT4) in HFD/streptozotocin (STZ)-induced diabetic rats. The antidiabetic mechanisms of flavonol may also be attributed to the insulin signal transduction through modulating the increased expression and phosphorylation of insulin receptors, insulin receptor substrate, and glucose transporter (GLUT) proteins [100]. Previously, Schulze et al. [75] also demonstrated the ability of onion extracts containing mainly the quercetin and its glucoside derivatives to inhibit intestinal "sodium-glucose linked transporter 1" (SGLT1) in vitro. Notwithstanding, the same study failed to elicit any hypoglycemic effects in vivo in normoglycemic mice and human volunteers, which might be attributed to some variations in dose efficacy or compound bioavailability.
Kaempferol is another important class of Allium flavonol which displayed promising antidiabetic potential [107][108][109][110]. A kaempferol-3-O-β-d-6(P-coumaroyl) glucopyranoside from onion displayed blood glucose lowering ability in alloxan-induced diabetic rats and it was comparably higher to a standard drug [74]. The authors suggested that the antidiabetic effect might be due to the beneficial effects of kaempferol glycoside on lipid metabolism and hepatic enzymes. Recently, kaempferol glycoside-enriched butyl alcohol fraction of A. tuberosum is also reported to reduce blood glucose levels via improving serum lipid profile and antioxidant parameters in alloxan-induced diabetic rats [36]. The antidiabetic effects of Allium flavonols are thought to be mediated by reducing blood glucose, serum lipids, oxidative stress, and lipid peroxidation, as well as increasing antioxidant enzyme activity and insulin secretion [36,[72][73][74]76]. Nevertheless, the exact mechanism of hypoglycemic effects of Allium-derived flavonols is yet to be elucidated.

Cardio-Protective Effects
Cardiovascular disease (CVD) is the leading cause of deaths worldwide. A number of recent meta-analyses of prospective cohort studies have indicated that individuals with the highest flavonoid intakes have lower relative risk of mortality from CVD and all-causes compared with that of individuals with the lowest intakes [111][112][113]. Several studies evidenced the beneficial effects of Allium flavonols on the cardiovascular system via their regulatory effects on platelet aggregation [83,84,96], atherogenesis [78], thrombotic activities [79], endothelial function [81], hypertension [77,82], lipid metabolism [77], and oxidative stress [42] (Table 3). Ingestion of quercetin rich onion soup (69 mg total quercetin) inhibits platelet aggregation and essential components of the collagen-stimulated platelet activation pathway in human and may reduce the risk of thrombosis, a common pathology of CVD [114]. Dietary supplementation of onion powder containing high quercetin derivatives (50% 4 Qmg, 30% quercetin, and 20% 3,4 Qdg) attenuates the risk of atherosclerosis via lowering the atherogenic index and the incremental elastic modulus in rat models [78]. The oral supplementation of quercetin rich OPE (2 and 10 mg) demonstrates anti-thrombotic effects in a rat model [79]. The underlying mechanism of OPE-mediated anti-thrombosis involves the downregulated expression of thrombin-induced tissue factor, which is partly driven by the inactivation of mitogen-activated protein kinase (MAPK) signaling pathway, as observed by the reduced phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) [79]. In another in vitro study by Ro et al. [83], quercetin-rich OPE inhibits collagen-induced rat platelet aggregation in a dose-dependent manner with IC 50 value of 80.0 µg/mL. The anti-platelet aggregation effects of OPE has been ascribed to the reduced levels of intracellular Ca 2+ and thromboxane A2 (TXA2) via regulation of cyclooxygenase-1 (COX-1), and TXA2 synthase (TXAS) activities. In addition, OPE also elevates cAMP levels, which might be related to the regulation of protein kinase activities [83]. Olayeriju et al. [42] showed that the ethyl acetate extract of red onion tunic affects hemodynamic parameters in rats. The active flavonols in the extract are identified as quercetin, quercitrin, isoquercitrin, rutin, and kaempferol, which might potentially reduce systolic and diastolic pressure, mean arterial blood pressure, pulse rate, and heart rate via modulating the oxidative stress [42].
Several human intervention studies have also demonstrated the efficacy of flavonol-rich onion in the reduction of CVD risks. Supplementary intake of onion extracts was found to lower blood pressure in smokers [77] and overweight-to-obese patients with pre-(hypertension) [82]. Endothelial function is another important predictor of cardiovascular events [80]. Chronic onion extract (rich in quercetin) intake ameliorates endothelial dysfunction through improving postprandial flow-mediated dilation (FMD) in healthy men [80]. Consumption of OPE (100 mg quercetin/d) for 12 weeks by healthy overweight and obese individuals is reported to improve endothelial function by increasing the FMD and circulating endothelial progenitor cell (EPC) counts [81]. However, not all flavonol-rich onion fractions demonstrate cardioprotective effects. Recently, Brüll et al. [115] observed no influence of onion skin extract (54 mg quercetin per capsule) on blood pressure, heart rate, or any biomarker of endothelial function in overweight-to-obese adults with hypertension.

Neuroprotective Effects
Neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, frontotemporal dementia, Huntington disease, and amyotrophic lateral sclerosis are a growing burden worldwide due to their high prevalence yet poor treatment [116]. Oxidative stress and inflammation are the major contributors to the neurodegenerative disease [117][118][119]. The quercetin-rich A. cepa extract improves motor coordination and memory functions in aluminium chloride (AlCl 3 )-induced neurotoxicity in mice by ameliorating the tested oxidative stress biomarkers in serum including lipid peroxidation, glutathione levels, and catalase activity [89]. In addition, acetylcholinesterase (AChE) activity, a possible marker of low-grade systemic neuro-inflammation [120][121][122], is also significantly reduced in AlCl 3 exposed animals receiving onion extract [89]. Previously, the kinetics studies of quercetin suggested its competitive type of inhibition against AchE [123]. Another study reported that the A. cepa outer scale extract (rich in quercetin) had positive effects on ischemia-reperfusion cerebral injury in a mice model by inhibiting lipid peroxidation and increasing antioxidant enzymes as well as antioxidants [91]. In addition, several in vitro studies investigated the mechanisms of neuroprotective effects of Allium-derived flavonols. Yang et al. [87] reported that onion derived quercetin protected hippocampal neuronal cells (HT22) against glutamate-mediated oxidative stress by reducing intracellular ROS production, Ca 2+ influx, maintaining MMP, and downregulating several apoptosis-related biochemical markers. The onion flavonoids quench ROS in hypoxia-induced oxidative stress in neuronal cells via inactivation of protein kinase C-(PKC-) and p38 mitogen-activated protein kinase (p38MAPK), induced by the phosphorylation of ERK1/2 [88]. The flavonoids from A. victorialis var. platyphyllum leaves and onion peel [124] were reported to exert neuroprotective effects in activated glial cells by inhibiting NO production, a pro-inflammatory mediator. These effects are ascribed to the strong antioxidant potential of flavonoids and their capacity to scavenge ROS [124]. A 3,4 -Qdg isolated from onion bulb protects human striatal precursor cells (HSPs) under nutrient deprived condition by reducing apoptosis and improving adhesion capacities via the expression of some adhesion molecules, such as pan-cadherin and focal adhesion kinase [90]. These studies indicate the potential application of Allium-derived flavonols in providing neuroprotective effects.

Antimicrobial Effects
In an antibiotic crisis world, flavonol-rich Allium extracts could act as a promising source of antimicrobial or antivirulence agents. Flavonol compounds exhibit antimicrobial activity against a broad range of antibiotic resistant pathogens in vitro. Recently, Sharma et al. [125] demonstrated antimicrobial activity of methanolic extract and isolated quercetin from onion against several multi-drug resistant (MDR) bacteria through in vitro and in silico experiments. The extract displayed higher zone of inhibition as compared with the isolated quercetin, which may be attributed to the synergistic effects of other co-existing bioactive compounds. The molecular docking analysis revealed that quercetin might interfere with the metabolism of bacterial proteins (β-lactamase, gyrase A, 2-trans-enoyl-acyl carrier protein reductase-inhA and topoisomerase IV) [125]. Snoussi et al. [46] demonstrated the in vitro antimicrobial potential of methanolic extracts of different plant parts from A. roseum var. odoratissimum against bacteria and yeast isolated from contaminated meat and sea foods. The authors attributed the observed antimicrobial effects of Allium extracts to the apigenin and kaempferol derivatives.
No matter how effective a new antimicrobial agent may be, pathogens will eventually develop resistance over time. Therefore, recently scientists lauded for studies on "antivirulence drugs" instead of "antimicrobial drug" to cope up with antibiotic resistance [126]. An antivirulence agent is not bactericidal per se but it attenuates the detrimental phenotypes of a pathogen and therefore, generally used as adjuncts to potentiate the existing antibiotic therapy [127]. Recently, Mahomoodally et al. [128] demonstrated that the polyphenol-rich extracts of onion and garlic augmented the efficacy of streptomycin and chloramphenicol against standard and clinical bacterial strains. The ability of 4 -Qmg from onion peel to inhibit biofilm formation regulated by quorum sensing (QS)-mechanisms and hence the virulence factors of Chromobacterium violaceum and Pseudomonas aeruginosa, has also been reported [129]. They established that 4 -Qmg treatment prevented the production of virulence factors (violacein, elastase, and pyocyanin) and biofilm formation in the test pathogens by antagonizing the virulence factor regulator (Vfr) and QS signal receptor (LasR) [129]. Another recent study witnessed the anti-QS activity of quercetin aglycone and 3-Qmg from onion on C. violaceum, P. aeruginosa, and Serratia marcescens by inhibiting violacein production and swarming ability [130]. The above discussion imparts that the use of Allium-derived flavonols could be a sustainable solution to mitigate issues relating to the resistant microbial infections. However, we emphasize further in vivo studies and clinical trials prior to any formal promotion of flavonols toward disease management involving MDR pathogens.

Other Health Benefits
Immunomodulatory effects of flavonoid-rich fraction of A. ascalonicum (shallot) extract is reported via inducing delayed-type hypersensitivity and TH1 cytokine-IFNγ expression in a mouse model [131]. A. mongolicum extract (100 and 200 mg/kg) containing flavonols (quercetin, kaempferol, and their respective glucosides) has been reported to induce the recovery of fecal excretion and water content while enhancing colon thickness and number of goblet cells in a loperamide (LOP)-induced constipated mouse model [132]. The anti-constipation effects are mediated by the downregulation of aquaglyceroporin-3 (AQP3) expression and up-regulation and activation of G protein alpha (Gα) and phosphoinositide 3-kinases (PI3K) [132]. Cho et al. [133] reported the protective effects of onion extract (quercetin; 17.1 µM) against radiation-induced cyto-and geno-toxicity in human lymphocytes pertaining to its antioxidant and DNA repair properties.
The A. tuberosum extracts containing glycosides of quercetin, kaempferol, and isorhamnetin demonstrated antiviral effects in ovo against an avian influenza (low-pathogenic) virus [40]. The antiviral efficacy of flavonols is attributed to their ability to interact with various molecular targets [134][135][136]. Quercetin derivatives are reported to target cap-binding site of PB2 of influenza viral RNA polymerase [134]. Schwarz et al. [136] found that kaempferol derivatives block virus ion channels in SARS coronavirus. A recent molecular docking analysis revealed that fisetin, kaempferol, and quercetin target SARS-CoV2-S spike protein of CoVID-19 [135]. These studies indicate that Allium-derived flavonols may present a promising and relevant therapeutic option for the management of CoVID-19 infection.

Molecular Mechanisms Underlying the Physiological Effects of Flavonols
Oxidative stress as denoted by the elevated levels of reactive oxygen species (ROS) has pleiotropic effects on the development of chronic diseases [137][138][139][140]. The inextricable correlations between the dietary intake of flavonols and their functional effects against certain chronic disorders are evident. Mechanistically, the health benefits of the Allium flavonols are mainly attributed to their modulatory effects on oxidative stress and inflammation (Table 3). During a chronic disease condition, these compounds can regulate the cellular ROS levels by various mechanisms involving direct scavenging and detoxification as well as engagement of redox signaling pathways (Figure 3). We present here a few examples of molecular targets of Allium-derived flavonols.
Antioxidants 2020, 9, x FOR PEER REVIEW 5 of 36 activity of isorhamnetin to the increased Nrf2 activity and its target gene expression of HO-1 and GCL via the phosphorylation of ERK1/2, PKCδ, and AMPK. Nuclear factor kappa-B (NF-κB) is a family of transcription factors that plays a pivotal role in multiple aspects of innate and adaptive immune functions. Normally, NF-κB exists in the cytoplasm as an inactive complex in physical association with inhibitory proteins called inhibitors of κB (IκBs) [154]. Upon activation, NF-kB triggers inflammatory response by increasing the expression of proinflammatory cytokines (TNF-α, IL6, IL1-β), chemokines (monocyte chemoattractant protein-1, MCP-1), and adhesion molecules as well as by regulating the cell proliferation, apoptosis, morphogenesis, and differentiation [154,155]. The transcription of NF-κB-dependent genes also influences the levels of ROS and vice versa [156]. Therefore, a tightly regulated NF-κB signaling is essential to prevent any exacerbated inflammatory responses during a chronic illness [157]. Previous reports found that quercetin [158][159][160], myricetin [161], fisetin [162], and isorhamnetin [163] have suppressed the inflammatory responses via the inhibition of NF-κB signaling in animal models and human with chronic diseases. These studies suggest that the transcriptional specificity of NF-κB may be shaped by the crosstalk with other signaling pathways (p38, MAPK and ERK) and transcription factors (Nrf2 and p53). Furthermore, some enzymes including inducible nitric oxide synthase (iNOS), xanthine oxidase, cyclooxygenase-2 (COX-2), arachidonate 12-lipoxygenase, arachidonate 5-lipoxygenase, and cytochrome p450 enzymes that promote the production ROS are also regulated by NF-κB signaling [156].
p53, a transcription factor that regulates cell proliferation, senescence, DNA repair, and cell death and its activity is modulated by the degree of oxidative stress imposed [164]. The low levels of oxidative stress inhibit p53 expression, which promotes cell survival and repair by direct scavenging of free radicals through the expression of antioxidant genes [165]. Reciprocally, the acute/high stress promotes p53 expression and initiates DNA fragmentation to induce apoptosis via caspase cascade Nuclear factor kappa-B (NF-κB) is a family of transcription factors that plays a pivotal role in multiple aspects of innate and adaptive immune functions. Normally, NF-κB exists in the cytoplasm as an inactive complex in physical association with inhibitory proteins called inhibitors of κB (IκBs) [154]. Upon activation, NF-kB triggers inflammatory response by increasing the expression of pro-inflammatory cytokines (TNF-α, IL6, IL1-β), chemokines (monocyte chemoattractant protein-1, MCP-1), and adhesion molecules as well as by regulating the cell proliferation, apoptosis, morphogenesis, and differentiation [154,155]. The transcription of NF-κB-dependent genes also influences the levels of ROS and vice versa [156]. Therefore, a tightly regulated NF-κB signaling is essential to prevent any exacerbated inflammatory responses during a chronic illness [157]. Previous reports found that quercetin [158][159][160], myricetin [161], fisetin [162], and isorhamnetin [163] have suppressed the inflammatory responses via the inhibition of NF-κB signaling in animal models and human with chronic diseases. These studies suggest that the transcriptional specificity of NF-κB may be shaped by the crosstalk with other signaling pathways (p38, MAPK and ERK) and transcription factors (Nrf2 and p53). Furthermore, some enzymes including inducible nitric oxide synthase (iNOS), xanthine oxidase, cyclooxygenase-2 (COX-2), arachidonate 12-lipoxygenase, arachidonate 5-lipoxygenase, and cytochrome p450 enzymes that promote the production ROS are also regulated by NF-κB signaling [156].
p53, a transcription factor that regulates cell proliferation, senescence, DNA repair, and cell death and its activity is modulated by the degree of oxidative stress imposed [164]. The low levels of oxidative stress inhibit p53 expression, which promotes cell survival and repair by direct scavenging of free radicals through the expression of antioxidant genes [165]. Reciprocally, the acute/high stress promotes p53 expression and initiates DNA fragmentation to induce apoptosis via caspase cascade signaling [165]. Several flavonols are reported to cause cell cycle arrest at G2/M phase and triggered apoptosis in different types of human cancer cells via the activation of p53 [54,166,167].
Mitochondria-mediated ROS production accounts for approximately 90% of the total cellular ROS [168]. Lagoa et al. [169] identified mitochondrial respiratory chain complex-I and cyt c as the major molecular targets of quercetin and kaempferol in alleviating oxidative stress. Another mechanism pertaining to the beneficial effect of flavonols is identified as mitochondrial uncoupling, a regulated proton leak mediated by uncoupling proteins (UCPs) [170], which occurs through aldehydic lipid-peroxidation intermediates such as 4-hydroxy-2-nonenal [85] and AMPK activation [64]. The mitochondrial uncoupling activity of flavonols is also reported to induce apoptosis in cancer cells by dissipating MMP (∆ψ m ) through the activation of caspase cascades [54]. Influx of Ca 2+ into the mitochondria is a critical for the availability of ATP since major enzymes in the tricarboxylic acid cycle are activated by Ca 2+ [171]. Quercetin [87] and kaempferol [172] are reported to modulate mitochondrial Ca 2+ accumulation and thereby reduce ROS emission.
Different protein kinase pathways including PKC [88], PI3K/Akt [54], MAPK [153], and AMPK [64,65] have been identified as the targets of allium derived flavonols for providing cell survival signaling. PI3K/Akt and MAPK (ERK, p38, and JNK) signaling pathways play critical roles in the regulation of cell proliferation, apoptosis, and autophagy [173] while AMPK is identified as a key regulator of energy homeostasis [174]. The AMPK activation is reported to be beneficial for both the prevention and treatment of a wide variety of metabolism related chronic diseases via its regulatory effects on fatty acid, cholesterol, carbohydrate, and amino acid metabolism as well as autophagy, mitochondrial function (biogenesis, fission and mitophagy) and cell growth [174]. Flavonols are reported to mediate cell cycle arrest at G2/M phage, apoptosis, and autophagy in different types of cancer cells via suppressing the expression of (PI3K)/Akt [54] and MAPKs [153] as well as increasing the expression of AMPK [64,65] signaling pathways.
Although the precise molecular details of the beneficial effects of Allium-derived flavonols not fully elucidated, these studies suggest that some cryptic mechanisms of action are subtly involved.

Bioavailability of Allium-Derived Flavonols
Bioavailability refers to the amount of a compound/nutrient that enters systemic circulation and reaches the intended biological tissues [175,176]. Absorption, metabolism, and excretion are the main indices of bioavailability of an ingested compound [177,178]. The molecular actions of Allium flavonols are largely dependent on their bioavailability at the target tissue of humans. However, the bioavailability studies often involve the use of flavonoid-rich alliums rather than a pure isolated compound, where the possibility of the effects induced by other phytochemicals could not be excluded. Quercetin and its glucosides contribute to the major dietary flavonol intake in humans and have been the most extensively studied compounds for their bioavailability [177,179,180]. However, it should be noted that the complete bioavaialbility of quercetin in the body is not clearly understood. After quercetin, kaempferol contributes significantly to the flavonol intake in humans [181]. Nevertheless, there is a lack of scientific data on the bioavailability of Allium-derived kaempferol, isorhamnetin, myricetin, fisetin, and morin in humans. A simplified scheme of dynamics of flavonol/flavonol glycosides inside the body from oral intake to excretion is shown in Figure 4.
The site and manner in which flavonols are absorbed depend on their chemical structure [180,182,183]. Crespy et al. [183] reported gastric absorption of quercetin, but not its glycosides in a rat model, indicating a limited contribution of stomach in the bioavailabilty of flavonols. The pioneering study by Hollman et al. [184] indicated that 52% absorption of onion-derived quercetin glucosides whereas, 24% of quercetin aglycone given as a pure compound was absorbed in the small intestine of healthy ileostomy volunteers. The greater absorption of quercetin glucoside is attributed to its water-soluble nature and is mediated by sodium-dependent glucose transporter 1 (SGLT1) [184]. In contrast, higher quercetin aglycone (dried shallot skin) absorption is reported when quercetin aglycone was consumed as an integral food component as compared with its glucosides (shallot flesh) in humans [185]. This may be ascribed to the fact that quercetin aglycone is relatively lipophilic and thereby can easily enter intestinal enterocytes because of passive transport, whereas its respective glycosides must be hydrolyzed by phlorizin lactase or β-glucosidase located at the intestinal brush border membrane prior to absorption by an enterocyte [186,187]. Hollman et al. [188] pointed out that sugar moiety is the main determinant for the absorption of quercetin in humans; glucosides of quercetin being more efficiently absorbed than the rutinoside. However, the position of sugar moiety had no effects on their absorption when the same levels of pure 3-Qmg and 4'-Qmg were fed in humans [189]. Apart from glycosylation pattern, dietary source [188,190] and components of food matrix [191] also affect the oral bioavailability of flavonols. For example, higher absorption of quercetin from onions was reported as compared with apples in ileostomy patients [188] and healthy volunteers [190]. In an animal study using a porcine model, the fat content in the diet positively influences the bioavailability of 3-Qmg [191]. Dietary fat-dependent improvements in quercetin bioavailability, likely by enhancing its micellarization at the small intestine have also been reported in humans [192].  The site and manner in which flavonols are absorbed depend on their chemical structure [180,182,183]. Crespy et al. [183] reported gastric absorption of quercetin, but not its glycosides in a rat model, indicating a limited contribution of stomach in the bioavailabilty of flavonols. The pioneering study by Hollman et al. [184] indicated that 52% absorption of onion-derived quercetin glucosides whereas, 24% of quercetin aglycone given as a pure compound was absorbed in the small intestine of healthy ileostomy volunteers. The greater absorption of quercetin glucoside is attributed to its water-soluble nature and is mediated by sodium-dependent glucose transporter 1 (SGLT1) [184]. In contrast, higher quercetin aglycone (dried shallot skin) absorption is reported when quercetin aglycone was consumed as an integral food component as compared with its glucosides (shallot flesh) in humans [185]. This may be ascribed to the fact that quercetin aglycone is relatively lipophilic and thereby can easily enter intestinal enterocytes because of passive transport, whereas its respective glycosides must be hydrolyzed by phlorizin lactase or β-glucosidase located at the intestinal brush border membrane prior to absorption by an enterocyte [186,187]. Hollman et al. [188] pointed out that sugar moiety is the main determinant for the absorption of quercetin in humans; glucosides of quercetin being more efficiently absorbed than the rutinoside. However, the position of sugar moiety had no effects on their absorption when the same levels of pure 3-Qmg and 4'-Qmg were fed in humans [189]. Apart from glycosylation pattern, dietary source [188,190] and components of food matrix [191] also affect the oral bioavailability of flavonols. For example, higher absorption of quercetin from onions was reported as compared with apples in ileostomy patients [188] and healthy volunteers [190]. In an animal study using a porcine model, the fat content in the diet positively influences the bioavailability of 3-Qmg [191]. Dietary fat-dependent improvements in quercetin bioavailability, likely by enhancing its micellarization at the small intestine have also been reported Once absorbed, all the flavonols are metabolized via three types of conjugation in the liver: Methylation, sulfation, and glucuronidation by catechol-O-methyl-transferases (COMTs) sulfotransferases (SULTs), and uridine 5'-diphospho-glucuronosyl transferases (UGTs), respectively, prior to reaching systemic circulation [177,186,193]. It is noteworthy to mention that only 5-10% of the total dietary polyphenols is absorbed in the small intestine, while the remaining 90-95% together with their conjugates (excreted through bile) reaches the colon and undergoes microbial metabolism, leading to the production of low molecular weight phenolic acids and aromatic compounds as well as CO 2 [194][195][196][197]. The simpler phenolic compounds can be re-absorbed in the small intestine, then subjected to conjugation again in liver, reach the target tissues, and ultimately excreted via urine and feces as their respective conjugates, and via breath as CO 2 [194][195][196][197].

Stability During Domestic and Technological Processing
Domestic and technological processing may bring a significant variability in allium flavonol contents. The kind and position of the sugar moiety in flavonol glycosides often determine their fate during these processing practices [208,209]. Data in the literature about the effects of the processing techniques on the flavonol content are restricted to onions only. For instance, Rodrigues et al. [210] reported that maceration of chopped red onion slightly reduced the levels of 3,4 -Qdg and 4 -Qmg during the first 5 h when kept at room temperature under continuous light exposure. However, Makris and Rossiter [211] observed no marked changes in 3,4 -Qdg and 4 -Qmg following 60 min of maceration of chopped onion, indicating the fact that longer time is needed for the enzyme action. Cooking significantly alters the flavonol content due to thermal degradation and transformation; however, changes vary with the type of culinary treatment and the length of exposure [209][210][211][212]. The losses of onion flavonols during different cooking treatments are supposedly affected in the following order: Boiling > microwave roasting > oven roasting > frying [210]. Intriguingly, Lee et al. [212] reported a very different trend of flavonol losses following various cooking treatments: Frying > sautéing > boiling in water with 3% salt > boiling in water with 1% salt > steaming > microwaving. However, baking resulted in the gain of onion flavonols. The high loss of flavonols after boiling and frying may be attributed to a combined effect of thermal degradation and leaching into cooking water and oil [209,210,212]. Thermal treatments led to deglycosylation of quercetin glucosides (4 -Qmg and 3,4 -Qdg) to their corresponding aglycone [209,210]. The sugar moiety attached at 3-O-position (C-ring) is more susceptible to thermal degradation as compared with that attached at 4-O-position (B-ring) [209]. Garlic and onion are often marketed as dried powders for culinary uses due to their longer shelf-life. Freeze-drying is considered as the best method to produce high-quality food powders with maximal retention of bioactive compounds in the final products as compared to other drying techniques [213]. Pérez-Gregorio et al. [214] reported that freeze-drying increased the flavonol levels (4 -Qmg and 3,4 -Qdg) in onions by 32% and no significant changes were observed in the flavonols of freeze-dried onions during 6-months of storage.
Fermentation of Allium vegetables is an important part of Asian cuisine. Several studies have substantiated the fermentation mediated changes of Allium flavonols. Most notably, the red onion fermentation by Lactobacillus plantarum S1 leads to the hydrolysis of quercetin diglucoside to its monoglucoside and aglycone derivatives [215]. Yang et al. [216] demonstrated that the controlled fermentation of yellow onion using a β-glucosidase-rich crude extract from Aspergillus kawachii resulted in elevated quercetin aglycone levels owing to the enzymatic cleavage of 4 -Qmg and 3,4 -Qdg. The regioselective de-glycosylation of onion quercetin glucosides by Saccharomyces cerevisiae is also reported following the fermentation [217]. Similarly, Lee et al. [218] demonstrated that onion fermentation using Leuconostoc mesenteroides at varying salt concentrations resulted in higher relative abundance of quercetin and isorhamnetin glucosides, while their corresponding aglycones were gradually increased via β-glucosidase action. Recently, Kothari et al. [40] also observed higher relative abundance of flavonol glycosides following the controlled L. plantarum-mediated fermentation of A. tuberosum. Contrastingly, no flavonol glucosides but only the traces of free quercetin are detected in onion following fermentation, suggesting a rapid decomposition of flavonols into smaller molecules including acetate, butyrate, and carbon dioxide [219]. Therefore, further studies should focus on a tailored microbial fermentation of alliums for the controlled production of bioactive metabolites toward enhancing the nutritional and health properties.

Food Fortification
The increasing nutrition knowledge has improved the consumer perception to opt for flavonoid fortified food products. Several researchers attempted to incorporate flavonol-rich alliums into food products to augment the health quality of foods. For instance, the onion skin extract addition improves the polyphenolic content and antioxidant activity of bread [220,221] and bean paste [222] in a dose-dependent manner. A cereal bar containing A. fistulosum extract (containing ferulic acid and quercetin) displayed anti-obesity effects in a rat model [223]. Recently, a quercetin derivative isolated from onion/beef soup demonstrated autophagy in the colon cancer cell in vitro [224]. However, not all the Allium-fortified foods have enhanced nutritional values. The fortification sometimes reported to negatively influence protein digestibility due to the formation of flavonoid-protein insoluble complexes [220,222]. Therefore, while designing an Allium-rich food formulation, the food matrix components also need to be considered to ensure its end-product quality. In addition, the scale-up and technical constraints related to the flavonol fortifications in food must be thoroughly evaluated considering their varying solubility, unstable nature, and low bioavailability in food matrices. Furthermore, biofortified products should also be subjected to public awareness, consumer participation, and acceptance parameters.

Future Perspectives and Limitations
From the above literature review, it is evident that the consumption of Allium flavonols may engender a lower risk of chronic disease development through modulating the oxidative stress and related low-grade systematic inflammation. However, the epidemiological studies regarding the health effects of Allium flavonols are surprisingly limited pertaining to several challenges including (1) time-consuming and labor-intensive isolation of a single bioactive flavonol from its natural source, (2) elusive underlying mechanism of action, and (3) their low bioavailability. Most of the Allium-derived flavonol studies are based on using solvent extracts that have multiple bioactive constituents such as OSCs, saponins, and other polyphenols and thus their additive or synergistic actions cannot be dismissed. In most of the studies reporting the health effects and related bioactivities, the specific flavonol compounds have not been fully characterized using ultra-high performance liquid chromatography (UHPLC) or gas chromatography (GC) coupled to high resolution-mass spectrometry (HR-MS), and nuclear magnetic resonance (NMR) spectroscopy. Hence, we emphasize on both functional and chemical characterization of the flavonols from Allium species having substantiated pharmacological effects. In addition, most of the mechanistic evidence derived from culture-based and/or animal model studies using the native forms of flavonol either in isolation or combination. However, following the ingestion, flavonols undergo a cascade of biotransformation reactions depending on individual's genetics, dietary habits, and various environmental factors. The in vivo studies often use animal models of similar genetic makeup and involves normalization of diet as well as the environment. However, the humans are genetically diverse and exposed to numerous exogenous factors, and their diets often includes highly diverse nutritional components [225]. These variables make it very difficult to ascertain the true effect(s) of dietary Allium-derived flavonols on the human host. Therefore, mechanistic studies should be thrived in both animal models and humans with the help of integrated multi-omics approach. To address the low bioavailability issue, further research is needed toward developing micro-and nano-delivery systems to maximize the absorption of flavonols and thereby enhance their target specificity and therapeutic efficacy.

Conclusions
Long-term oxidative stress contributes to the development of several chronic conditions including age-related neurodegenerative and cardiovascular diseases, cancer, diabetes, obesity, and low-grade systematic inflammations, among many others. Disease prevention models practicing healthy lifestyle changes including dietary modifications are suggested to be one of the effective strategies to address these chronic disorders. Herein, we exclusively discuss the Allium-derived flavonols as natural antioxidants and their possible role toward the prevention of chronic diseases in humans through alleviating the oxidative stress and associated chronic inflammation. Nonetheless, the potential importance of other biologically active compounds, such as phenolic acids, anthocyanins, saponins, and OSCs of alliums, cannot be undermined. Currently, insufficient scientific evidence prevails to draw any conclusion on flavonol intake from alliums and their exclusive health benefits. Further mechanistic studies involving animal models and human volunteers are required to substantiate any potential health benefit claims. The poor bioavailability of flavonols and food matrix constituents should also be taken into consideration while designing any formulations. Finally, individual health conditions that could interfere the metabolism and, thus, the health effect should also be considered for the clinical use of Allium-derived flavonols.