Dietary Flavonoids: Cardioprotective Potential with Antioxidant Effects and Their Pharmacokinetic, Toxicological and Therapeutic Concerns

Flavonoids comprise a large group of structurally diverse polyphenolic compounds of plant origin and are abundantly found in human diet such as fruits, vegetables, grains, tea, dairy products, red wine, etc. Major classes of flavonoids include flavonols, flavones, flavanones, flavanols, anthocyanidins, isoflavones, and chalcones. Owing to their potential health benefits and medicinal significance, flavonoids are now considered as an indispensable component in a variety of medicinal, pharmaceutical, nutraceutical, and cosmetic preparations. Moreover, flavonoids play a significant role in preventing cardiovascular diseases (CVDs), which could be mainly due to their antioxidant, antiatherogenic, and antithrombotic effects. Epidemiological and in vitro/in vivo evidence of antioxidant effects supports the cardioprotective function of dietary flavonoids. Further, the inhibition of LDL oxidation and platelet aggregation following regular consumption of food containing flavonoids and moderate consumption of red wine might protect against atherosclerosis and thrombosis. One study suggests that daily intake of 100 mg of flavonoids through the diet may reduce the risk of developing morbidity and mortality due to coronary heart disease (CHD) by approximately 10%. This review summarizes dietary flavonoids with their sources and potential health implications in CVDs including various redox-active cardioprotective (molecular) mechanisms with antioxidant effects. Pharmacokinetic (oral bioavailability, drug metabolism), toxicological, and therapeutic aspects of dietary flavonoids are also addressed herein with future directions for the discovery and development of useful drug candidates/therapeutic molecules.


Introduction
The introduction should briefly place the study in a broad context and highlight mitochondrial cellular oxidative stress. It has been proven that oxidised low-density lipoprotein (ox-LDL) increases the development of reactive oxygen species [9] in human umbilical vein endothelial cells (HUVECs). Angiotensin II and uremic toxin indoxylsulfate-induced endothelial cell dysfunction are two other recognised causes of ROS noticed in CVDs [10].
It has been well established via previous reports that sugars are involved in the development of atherosclerosis, hypertension, peripheral vascular disease, coronary artery disease, cardiomyopathy, heart failure and cardiac arrhythmias and that these effects of added sugars are mediated through ROS as glucose can produce ROS via various pathways including the sorbitol pathway, insulin pathway, NADPH-oxidase (Nox). Noxsignalling is crucial for normal physiology, but overstimulated Nox enzymes contribute to oxidative stress and cardiovascular disease [11]. In AT-II-induced hypertension, NOX-2 activation induces Sirt3 S-glutathionylation which causes acetylation of vascular SOD2 and reduces SOD2 activity, which further results in increased mitochondrial superoxide levels and lessened endothelial nitric oxide bioavailability which acts as an antioxidant in-vivo [11,12].
Diets low in saturated fat and high in fruits, vegetables, and essential fatty acids, as well as moderate wine intake, appear to protect against the production and progression of CVDs, according to epidemiological evidence.Long term metabolic studies have shown that the fatty acid composition of the diet, rather than the overall amount of fat consumed, predicts serum cholesterol levels. Saturated fatty acids (SFA) and transfatty acids are the ones associated with elevated cardiovascular risk however monounsaturated fatty acids (MUFA, omega-9) and polyunsaturated fatty acids (PUFA, omega-3, omega-6) explicit decreased risk of coronary heart disease (CHD) [13]. The activity of enzymes involved in the desaturation of fatty acids in the body is highly influenced by dietary fat quality. Plant sterols and stanols (saturated form of sterols) are natural elements of plants structurally related to cholesterol. Plant stanols lessen cholesterol absorption in the GIT thereby dipping plasma LDL concentrations. These stanols are found abundantly in vegetable oils, olive oil, fruits and nuts. Recent progressions in food technology have perceived the emergence of nutrition products such as margarine, milk, yoghurt, and cereal products being supplemented with plant sterols/stanols and encouraged as a food that can help lower serum cholesterol [14]. It has been found via clinical studies that serum LDL cholesterol was significantly dropped when stanols were added to milk (15.9%) and yoghurt (8.6%), but significantly less when added to bread (6.5%) and cereal (5.4%). Nonetheless, routine consumption of phytosterols has emerged as an effective strategy in the management of hypercholesterolemic patients in the clinical situation. Alternatively, red yeast rice (Monascuspurpureus) is natural compound capable of reducing cholesterol levels. This fermented rice holds plentiful monacolins that are naturally occurring HMG-CoA reductase inhibitors [15]. effective, and efficient strategy to prevent CVD worldwide. The INTERSALT study (an international study of electrolyte excretion and BP) confirmed a direct association between salt intake and the increase in BP with age [16].

Dietary Occurrence
Flavonoids are secondary metabolites located in the vacuoles of the plants.  [18]. Flavonols, such as quercetin, kaempferol and myricetin are one of the most common flavonoids found in fruits and vegetables, for example, apples, grapes, berries, tomatoes, onions, lettuce, etc. The chemical structure of flavonols is characterized to have a ketone group, and a hydroxyl group located in the position 3 of the C ring, that can have different glycosylation patterns and for this reason are the largest subgroups present in plants and foods [19].
On the other hand, the most well-known compounds in flavanones group are hesperidin, naringenin and eriodyctiol, regularly found in the white part of the peel of citrus fruits such as lemon, orange, and grapefruit. Structurally, those compounds are very similar to flavonols, the only difference is the saturation of C ring in the 2 and 3 position [19].
Isoflavonoids are less distributed on plants, and are usually present in lentils, beans, soybean, and other leguminous plants. The most important bioactive compounds on this group are genistein and daidzein, well known as a phytoestrogen due to their osteogenicactivity [18].
Neoflavonoids are a less studied group, their structure is characterized to have a 4-phenylchromen backbone with no hydroxyl group substitution at position 2. The hydroxyl group is bound to position 3 of the C ring [18]. One of the neoflavone is calophyllolide from Calophylluminophyllum seeds, found in other plants and flowers [20]. Flavanols also known as catechins, are abundantly distributed in berries, bananas, peaches, and apples.
Anthocyanins are a flavonoids class widely studied, their notable blue, black, red, and pink colours depend on the pH as well as by the methylation or acylation in the hydroxyl groups on A and B rings. This characteristic produced high interest in the food industry in a variety of applications. The well-known anthocyanins are cyanidin, delphinidin, malvidin, pelargonidin and peonidin. Those compounds are present in strawberries, raspberries, blueberries, blackberries, blue corn, black beans, among others (Table 1) [18].

Health benefits, Medicinal Significance and Nutraceutical Importance
Flavonoid-rich foods are widely studied and considering as potent bioactive compounds with different biological activities, participating in different important signalling pathways related to chronic disease [23]. Herbal supplements enriched with flavonoidsare frequentlyreported for their ameliorative effects in the management of metabolic syndrome including CVDs and diabetes mellitus. Anthocyanins, like cyanidin and delphinidin 3-glucoside, have shown to improve insulin resistance, insulin production and hepatic glucose uptake during type 2 diabetes mellitus [24]. Many flavonoids, specifically flavanols, are well-known for their antihypertensive effect and endothelialprotectionby lowering triglycerides and detrimentallipid accumulation. Several flavanoid molecules have been established for their wide range of therapeutic benefits in CVDs including endothelial dysfunction, coronary artery disease, cardiac fibrosis, myocardial infarction, ischemic reperfusion injury etc. [9,25].
A study suggests that regular consumption of 100 mg of total flavonoids in a day may reduce the risk of developing morbidity as well as fatality due to CVDs by approximately 10% [26]. Due to the presence of multiple hydroxyl groups (-OH) in the flavanoid structure, they exert as strong antioxidant and neutralizes the oxidative insult during various pathological events [18]. Flavanoids also often reported as strong inhibitor of DNA damage due to oxidative stress. Nevertheless, flavonoids have also been explored for their positive impact in neurological health and found to be effective in neural regeneration and counter inflammation in the nerve cells. A study indicated that [6]-Epigallocatechingallate, a flavonoid mainly found in green tea, can produce microglial activation and protects against inflammation in Alzheimer's disease [27].
Now-a-daysflavanoids are increasingly being recognized in the field of nutraceuticals for management of chronic life style related disorders and maintenance of healthy aging.
Several herbal beverages enriched with high content of flavanoids are commercially available as anti-aging, antidiabetic and antiobesity and blood pressure lowering purposes. For example, hibiscus tea, blue motcha tea, green tea, red tea, rose wine, kiwi wine, red wine are the most popular beverages commercially available and widely acclaimed for their scientifically proven beneficial health effects.

Antioxidant Potential of Dietary Flavonoids in OS-induced CVDs
Cardiovascular system is the one, which being most prevalent to be affected by the oxidative stress triggered by spontaneously generated ROS due to the intake of high calorie diet, drugs and other xenobiotics. Mostly the high calorie diet intake longer period of time alone can lead to the depletion of myocardial antioxidantstatus and also allows developing chronic abnormalities like endothelial dysfunction, ischemia and cardiac hypertrophy [28]. Flavonoids consumption have been proven to exhibitnoticeablepositiveinfluencein preventing damages produced by ROS and other free radicals in the human body. The beneficial effects of flavonoids have been mostly linked to their strong antioxidant activity. The basic antioxidant mechanism of flavonoids consists in the oxidation of flavonoids by free radicals, resulting in a more stable, less-reactive radical [17]. The high reactivity of the hydroxyl group of the flavonoids produces inactivation of the free radicals. Some of the flavonoids can directly scavenge superoxide, whereas other flavonoids can scavenge the highly reactive oxygen-derived radical like peroxynitrite ions [29]. The preventive action of flavonoids on cardiovascular diseases has been one of the most studied topics. It is well known that the antioxidant activity of these compounds is responsible for diminution of the oxidative damages of cellular components and induction of cardiomyocytes apoptosis [16,25]. Moreover, other mechanism action of flavonoids is the vasodilation by maintaining the action of

Dietary Flavonoids and Their Health Implications in CVDs
Flavonoids are naturally occurring organic compound groups generated by plants as secondary metabolites. In a metanalysis of prospective cohort studies, regular diets containing flavonoids were accompanying with a lesser risk of CVD mortality.

Cardioprotective Mechanisms of Dietary Flavonoids
Over the decade growing interest of scientific research regarding flavonoid con sumption to prevent CVDs and to improve vascular health has been noticed. Several signalling pathways [9,24]. Some important molecular mechanisms of the cardiovascular protective function of flavonoids are described below ( Table 2).

Intracellular Antioxidant Signalling Pathways
Unlike the in-vitro environment, antioxidative mechanisms of flavanoids in the in-vivo system often do not work only on the principle of scavenging free radicals.
Rather flavanoids have been found to activate intracellular antioxidant signalling pathways to accelerate the production of endogenous antioxidants like GSH, SOD, and catalase etc. [87].

Counter Inflammatory Pathways
Inflammation is thought to be one of the most aggravating factors in the progression of a variety of CVDs, from endothelial dysfunction to myocardial apoptosis [90].

Mitochondrial and Intracellular Pathways
Mitochondria plays vital role in the normal functioning of cardiomyocytes and endothelial cells. Synthesis of ATP by catabolism of carbon rich sources via oxidative phosphorylation is one of the major roles of mitochondria. Integrity of inner mitochondrial membrane is very much essential to have the normal physiological and biophysical functioning [93]. Mitochondrial damage during oxidative insult like accumulation of cardiotoxins or due to ischemia/reperfusion is considered as a key event leading to cardiomyocytes dysfunction and apoptosis [94]. In this regard, protective potential of various flavonoids on mitochondrial functions have been widely investigated. The mechanism of action of certain flavonoids on mitochondrial targets may be another reason for the cardioprotective effect, which is enabled by maintaining  [96,97]. Other study suggested that the dietary flavanoid consumption also acts as cardioprotective agents by activation of Ca +2 channels and modulation of mitochondrial Ca 2+ uptake [94].
Oxidative phosphorylation and maintenance of respiratory chain or electron transport chain are the vital functions of mitochondria. However, due to oxidative insult in the cardiac tissue hampers the complex formation (Complex I) and subsequently release cytochrome C [94,96]. Notably anthocyanin flavanoidslike cyanidin 3-O-glucoside and delphidin 3-O-glucoside have been found to reduce oxidative stress in cardiac cells by restoration of mitochondrial bioenergetics and safeguard the preservation of normal functioning of complex I [98]. Flavonoids have also been found to suppress the generated ROS due to mitochondrial respiration by directly inhibiting enzymes and chelating the trace elements involved in ROS generation [94]. Evidently flavanoids prototypes like quercetin, kaempferol, and epicatechin etc. has been found to inhibit H2O2 production in isolated rat heart mitochondria [99].

Bioavailability and Biotransformations of Dietary Flavanoids
Although flavonoids have shown countless health benefits, however their low oral bioavailability has been a major concern in the drug development.

Toxicities and Interactions with Drugs/Foods/Herbs
In contrast to the beneficial effects of flavanoids, the toxic effects and interactions with drugs/ foods/ herbs and other phytochemicals have been less explored. isoforms of CYP450 enzyme in the gut and liver and also found to modify the action of xenobiotic efflux in the gut [111,112]. This phenomenon often found to increase the bioavailability of many drugs, which is of course beneficial for the drugs with low bioavailability or metabolic stability. However, these pharmacokinetic alterations turns negatively for the drugs with extremely narrow therapeutic index like digoxin, lisinopril, captopril etc. [111]. intake. On the contrary, higher doses in food supplements the same can become pro-oxidants and generates free radicals rather than acting as antioxidants [110]. Hence, it is very important to have a better understanding of the timing and amount of intake of dietary flavanoids in order to maximize the benefits while minimizing the risks. Some important flavonoid-drug intercations are depicted in Table 3.

Strategies to Overcome Pharmacokinetic and Toxicological Limitations
The delivery of phytochemicals like flavonoids is challenging due to poor solubility, radical scavenging activity and promote bioavailability [114]. The delivery system is capable of increasing the antioxidant activity of flavonoids by preventing degradation of the formulation due to encapsulation and maintaining the drug concentration over time which in turn increases the antioxidant/radical scavenging activity of the active compound compared to the unloaded one. Furthermore, these also help in compounding sustained and controlled release formulations which can be used for flavonoid targeted therapies [115]. In comparison to the conventional formulation micro or nano-emulsion increase the penetration rate through biological membranes and also enhance their ADME phase thereby decreasing associated toxicities [116]. The use of biopolymers in formulations used for CVDs treatment adds an advantage because of its favourable properties such as biodegradability, good biocompatibility, and attractive biomimetic characteristics [117]. Structural modification of the parent flavanoid compounds also has been proven as one of the successful strategy to overcome poor solubility and GI absorption. Glycosylation and glucuronide conjugation are the useful tailoring reactions which may significantly change the physicochemical properties of hydrophobic flavonoids. Introduction of new polar groups or masking the selective functional groups in the structural skeleton, which is popularly known as pro-drug approach become useful to improve the pharmacokinetic profile of various dietary flavanoids [118]. It is often observed that co-administration of food and flavanoids together serves better absorption flavanoids from the gut. Hence, the complex carrier formation approach like cyclodextrin complex, lipid/carbohydrate-flavanoid conjugate is some of the approaches to overcome pharmacokinetic limitations [104,112]. Formulation of nanoparticles or nanocrystals is the most common approach to enhance the absorption and bioavailability of flavanoids and found remarkably effective in the cancer chemoprevention [119,120].
However, all these strategies to improve the pharmacokinetic profile of dietary flavanoids are exclusively depends on the area of their application and most of them are still under experimental investigational phases which need more in-depth studies to make any conclusive statement.