Flavones

Flavones are one of the important subgroups of flavonoids. Flavones are widely present in leaves, flowers and fruits as glucosides. Celery, parsley, red peppers, chamomile, mint and ginkgo biloba are among the major sources of flavones. Luteolin, apigenin and tangeritin belong to this subclass of flavonoids (Fig. 2). The peels of citrus fruits are rich in the polymethoxylated flavones, tageretin, nobiletin and sinensetin⁽ Reference Manach, Scalbert and Morand ^{16 )}. They have a double bond between positions 2 and 3 and a ketone in position 4 of the C ring. Most flavones of vegetables and fruits have a hydroxyl group in position 5 of the A ring, while hydroxylation in other positions, for the most part in position 7 of the A ring or 3′ and 4′ of the B ring, may vary according to the taxonomic classification of the particular vegetable or fruit.

Fig. 2. Flavonoid classes, subclasses and natural sources.

Flavonols

Flavonols are flavonoids with a ketone group. They are building blocks of proanthocyanins. Flavonols occur abundantly in a variety of fruits and vegetables. The most studied flavonols are kaempferol, quercetin, myricetin and fisetin (Fig. 2). Onions, kale, lettuce, tomatoes, apples, grapes and berries are rich sources of flavonols. Apart from fruits and vegetables, tea and red wine are also sources of flavonols. Intake of flavonols is found to be associated with a wide range of health benefits which includes antioxidant potential and reduced risk of vascular disease.

Compared with flavones, flavonols have a hydroxyl group in position 3 of the C ring, which may also be glycosylated. Like flavones, flavonols are very diverse in methylation and hydroxylation patterns as well and, considering the different glycosylation patterns, they are perhaps the most common and largest subgroup of flavonoids in fruits and vegetables. For example, quercetin is present in many plant foods⁽ Reference Iwashina ^{17 )}.

Flavanones

Flavanones are another important class which is generally present in all citrus fruits such as oranges, lemons and grapes. Hesperitin, naringenin and eriodictyol are examples of this class of flavonoids (Fig. 2). Flavonones are associated with a number of health benefits because of their free radical-scavenging properties. These compounds are responsible for the bitter taste of the juice and peel of citrus fruits. Citrus flavonoids exert interesting pharmacological effects as antioxidant, anti-inflammatory, blood lipid-lowering and cholesterol-lowering agents. Flavanones, also called dihydroflavones, have the C ring saturated; therefore, unlike flavones, the double bond between positions 2 and 3 is saturated and this is the only structural difference between the two subgroups of flavonoids. Over the past 15 years, the number of flavanones has significantly increased⁽ Reference Iwashina ^{17 )}.

Isoflavonoids

Isoflavonoids are a large and very distinctive subgroup of flavonoids. Isoflavonoids enjoy only a limited distribution in the plant kingdom and are predominantly found in soyabeans and other leguminous plants. Some isoflavonoids have also been reported to be present in microbes⁽ Reference Matthies, Clavel and Gütschow ^{18 )}. They are also found to play an important role as precursors for the development of phytoalexins during plant microbe interactions⁽ Reference Aoki, Akashi and Ayabe ^{19 ,} Reference Dixon and Ferreira ^{20 )}. Isoflavonoids exhibit tremendous potential to fight a number of diseases. Isoflavones such as genistein and daidzein are commonly regarded to be phyto-oestrogens because of their oestrogenic activity in certain animal models (Fig. 2). Szkudelska & Nogowski reviewed the effect of genistein inducing hormonal and metabolic changes, by virtue of which they can influence various disease pathways⁽ Reference Szkudelska and Nogowski ^{21 )}.

Neoflavonoids

Neoflavonoids are a class of polyphenolic compounds. While flavonoids have a 2-phenylchromen-4-one backbone, neoflavonoids have a 4-phenylchromen backbone with no hydroxyl group substitution at position 2. The first neoflavone isolated from natural sources in 1951 was calophyllolide from Calophyllum inophyllum seeds. It is also found in the bark and timber of the Sri Lankan endemic plant Mesua thwaitesii ⁽ Reference Linuma, Tanaka and Hamada ^{22 –} Reference Garazd, Garazd and Khilya ^{24 )}.

Flavanols, flavan-3-ols or catechins

Flavanonols, also called dihydroflavonols or catechins, are the 3-hydroxy derivatives of flavanones. They are a highly diversified and multisubstituted subgroup. Flavanols are also referred to flavan-3-ols as the hydroxyl group is always bound to position 3 of the C ring. Unlike many flavonoids, there is no double bond between positions 2 and 3. Flavanols are found abundantly in bananas, apples, blueberries, peaches and pears (Fig. 2).

Anthocyanins

Anthocyanins are pigments responsible for colours in plants, flowers and fruits. Cyanidin, delphinidin, malvidin, pelargonidin and peonidin are the most commonly studied anthocyanins (Fig. 2). They occur predominantly in the outer cell layers of various fruits such as cranberries, black currants, red grapes, merlot grapes, raspberries, strawberries, blueberries, bilberries and blackberries. Stability coupled with health benefits of these compounds facilitate them to be used in the food industry in a variety of applications⁽ Reference Giusti and Wrolstad ^{25 )}. The colour of the anthocyanin depends on the pH and also by methylation or acylation at the hydroxyl groups on the A and B rings⁽ Reference Iwashina ^{17 )}.

Chalcones

Chalcones are a subclass of flavonoids. They are characterised by the absence of ‘ring C’ of the basic flavonoid skeleton structure shown in Fig. 1. Hence, they can also be referred to as open-chain flavonoids. Major examples of chalcones include phloridzin, arbutin, phloretin and chalconaringenin. Chalcones occur in significant amounts in tomatoes, pears, strawberries, bearberries and certain wheat products. Chalcones and their derivatives have garnered considerable attention because of numerous nutritional and biological benefits. Table 1 describes the food sources of all dietary flavonoids discussed throughout the article for their bioactivity and research trends⁽ Reference Hertog, Hollman and Van De ^{26 –} Reference López-Lázaro ^{63 )}. The intake of flavonoids through food sources could be the simplest and safest way to combat diseases as well as modulate activities.

Table 1. Flavonoids, their classes and rich dietary sources

Anti-cholinesterase activity

Acetylcholinesterase (AChE) is a key enzyme in the central nervous system and inhibition of it leads to increases of neural acetylcholine levels which is one of the therapies for symptomatic relief of mild to moderate AD⁽ Reference Perry, Tomlinson and Blessed ^{64 )}. Hence the inhibition of cholinesterases is one of the central focus for drug development to combat AD. A number of flavonoids have been reported for their anti-cholinesterase activity. The in vitro inhibitory studies done on various flavonoids like quercetin, rutin, kaempferol 3-O-β-d-galactoside and macluraxanthone showed that quercetin and macluraxanthone possess a concentration-dependent inhibition ability against AChE and butyrylcholinsterase (BChE)⁽ Reference Khan, Orhan and Enol ^{34 )}. Macluraxanthone was found to be the most potent and specific inhibitor of both the enzymes with 50 % inhibitory concentration (IC₅₀) values of 8·47 and 29·8 µm, respectively. The enzyme kinetic studies revealed that quercetin inhibited both the enzymes in a competitive manner whereas macluraxanthone was non-competitive against AChE and competitive against BChE. To get insight of the intermolecular interactions, molecular docking studies of these two compounds were performed at active sites of both the enzymes. The docking studies showed that macluraxanthone binds much more tightly with both the enzymes than that of quercetin. Sheng et al.⁽ Reference Sheng, Lin and Zhang ^{65 )}, while designing, synthesising and performing the evaluation of flavonoid derivatives as potent AChE inhibitors, observed that most of the flavonoid derivatives have properties of inhibitory activities to AChE. The most potent inhibitor, isoflavone derivative 10d, inhibits AChE with an IC₅₀ of 4 nm, showing a high BChE:AChE inhibition ratio (4575-fold), superior to donepezil (IC₅₀ = 12 nm, 389-fold). Molecular docking studies were also performed to explore the detailed interaction with AChE.

Anti-inflammatory activity

COX is an endogenous enzyme which catalyses the conversion of arachidonic acid into prostaglandins and thromboxanes⁽ Reference Smith, DeWitt and Garavito ^{66 )}. The enzyme exists in two isoforms, COX-1 and COX-2. COX-1 is a constitutive enzyme and is responsible for the supply of prostaglandins which maintain the integrity of the gastric mucosa and provide adequate vascular homeostasis whereas COX-2 is an inducible enzyme and is expressed only after an inflammatory stimulus⁽ Reference Kurumbail, Stevens and Gierse ^{67 )}. The function of COX-2 is to synthesise prostaglandins for the induction of inflammation and pain⁽ Reference Kujubu, Fletcher and Varnum ^{68 )}. The studies done by using in silico methods on the binding modes of flavonoids with COX-2 explored that some flavonols and flavones containing a 2, 3-double bond may act as preferential inhibitors of COX-2⁽ Reference D'Mello, Gadhwal and Joshi ^{69 )}. These observations were found for the flavonol, flavone, and flavanone or isoflavone classes. This discovery led to the development of selective COX-2 inhibitors which are a class of compounds with good anti-inflammatory activity and reduced gastrointestinal side effects. The commercially available flavonoids like silbinin, galangin, scopoletin, hesperitin, genistein, daidzein, esculatin, taxifolin, naringenin and celecoxib were also evaluated for COX-inhibitory activity⁽ Reference Madeswaran, Umamaheswari and Asokkumar ^{70 )}. The selected flavonoids showed higher binding energy ranging between −8·77 to −6·24 kcal/mol (–36·69 to –26·11 kJ/mol) when compared with that of the standard (−8·30 kcal/mol; –34·73 kJ/mol) which led to the development of potent COX inhibitors for the treatment of inflammation. Madeswaran et al.⁽ Reference Madeswaran, Umamaheswari and Asokkumar ^{70 )} evaluated the COX-inhibitory activity of flavonoids using in silico docking studies. In this perspective, they used flavonoids like farobin-A, gericudranin-B, glaziovianin-A, rutin and xanthotoxin. Their docking results showed that all the selected flavonoids contributed better aldose reductase inhibitory activity because of their structural parameters. Hence, further deeper studies could develop potent aldose reductase inhibitors for the treatment of diabetes. Madeswaran et al.⁽ Reference Madeswaran, Umamaheswari and Asokkumar ^{71 )} also reported in silico docking studies of lipoxygenase-inhibitory activity of commercially available flavonoids. In this perspective, they selected flavonoids like aromadedrin, eriodictyol, fisetin, homoeriodictyol, pachypodol, rhamnetin, robinetin, tangeritin, theaflavin and azelastine for investigation. It was observed that all the selected flavonoids contributed to lipoxygenase-inhibitory activity because of their structural parameters and the whole analysis could lead to the further development of potent drugs for the treatment of inflammation. Wu et al.⁽ Reference Wu, Wu and Chung ^{72 )} worked on antiplatelet effects and selective binding of COX with flavonoids and lignans by using the molecular docking method. The flavonoids considered were ginkgetin, Taiwan-homo-flavone A, Taiwan-homo-flavone B and Taiwan-homo-flavone C and eight known lignans justicidin B, justicidin C, justicidin D, chinensinaphthol methyl ether, procumphthalide A, procumbenoside A and ciliatosides A and B from medicinal herbal plants, Cephalotaxus wilsoniana and Justicia species, respectively. Out of these flavonoids ginkgetin, Taiwan-homo-flavone C, justicidin B and justicidin D were found to be effective for antiplatelet effects.

Steroid-genesis modulators

Abyssinones and related flavonoids can be used as potential steroid-genesis modulators against three enzymes 3β-hydroxysteroid dehydrogenase (HSD), 17β-HSD and aromatase of the steroid-genesis pathway⁽ Reference Hatti, Diwakar and Rao ^{73 )}. The virtual screening experiment indicated higher affinity for flavonones than their respective chalcones. The flavonones possess consistent binding affinity to all the three enzymes used and are better steroidogenesis modulators in hormone-dependent cancer.

Xanthine oxidase modulators

XO catalyses the conversion of hypoxanthine to xanthine and subsequently xanthine to uric acid. The increase of uric acid level in blood serum, which is called hyperuricaemia, can lead to major complications such as gout and kidney stones⁽ Reference Borges, Fernandes and Roleira ^{74 ,} Reference Hille ^{75 )}. Alnajjar⁽ Reference Alnajjar ^{76 )} worked on natural flavonoids towards the discovery of a potential XO inhibitor. Licoisoflavone-A extracted from the roots of Glycyrrhiza glabra (liquorice) showed the most potent activity in the inhibition of XO. Umamaheswari et al.⁽ Reference Umamaheswari, Madeswaran and Kuppusamy ^{77 )} evaluated XO-inhibitory activity of flavonoids using in silico docking studies. The flavonoids butein, fisetin, diosmetin, tricetin, genistein, tricin, vitexycarpin, herbacetin, biochanin, rhamnetin, isorhamnetin, robinetin, peonidin and okanin were studied and it was found that all flavonoids exerted inhibition activity. The presence of a benzopyran ring in their basic nucleus would have contributed to its XO-inhibitory activity. This molecular docking analysis may further lead to the development of potent XO inhibitors for the prevention and treatment of gout and related inflammatory ailments. New drugs for the inhibition of the enzyme aldose reductase are in development and efforts are being made for their preclinical and clinical evaluation.

A novel approach emphasising the significance of natural products as a prime solution to unanswered questions like the treatment of the ‘silent killer’ ‘polycystic kidney disease’ (PKD) has been investigated⁽ Reference Shoba, Hari and Prabhavathi ^{78 )}. The key protein, namely cystic fibrosis transmembrane conductance regulator (which is responsible for PKD), and its mutated three-dimensional structure were subjected to molecular docking and in silico toxicity studies with flavonoids from vegetable sources. The outcome indicated the possible application of flavonoids from vegetable sources as potential and natural therapeutic agents to combat PKD.

Lin et al.⁽ Reference Lin, Xie and Wu ^{79 )} carried out in vitro kinetic studies of different flavonoids as inhibitors with various xanthine concentrations. In vitro studies and kinetic measurements of different flavonoids and various concentrations of xanthine were carried out⁽ Reference Lin, Xie and Wu ^{79 )}. Four potent XO inhibitors were found in 95 % ethanolic (v/v) gnaphalium affine extract. Among them, the flavone eupatilin exhibited the strongest inhibitory effect on XO compared with allopurinol, a known synthetic XO inhibitor. Apigenin, luteolin and 5-hydroxy-6, 7, 3′, 4′-tetramethoxyflavone also contributed to the inhibitory effect of gnaphalium affine extract on XO activity. This study provides a rational basis for the traditional use of gnaphalium affine against gout. The study on in vitro XO-inhibitory activity of the aglycone hesperetin and its glycosylated forms (hesperidin and G-hesperidin) and their effects on the plasma lipid profile and the oxidative–antioxidative system has been carried out in rats⁽ Reference De Souza, De Franco and De Araujo ^{80 )}. The concentrations of the major conjugated metabolites in rat plasma after oral administration of these compounds were also determined. It has been reported that hesperetin was found to have a stronger XO-inhibitor activity than the glycosylate derivatives.

Countering antibiotic resistance

β-Ketoacyl acyl carrier protein synthase III (KAS III), which initiates fatty acid synthesis in bacteria, is a key target enzyme to overcome the antibiotic resistance problem. Lee et al.⁽ Reference Lee, Jeong and Shin ^{81 )}, while working on the known flavonoid inhibitors of β-KAS III against the methicillin-resistant bacteria Staphylococcus aureus, found that flavonoids such as naringenin (5,7,4′-trihydroxyflavanone) and eriodictyol (5,7,3′,4′-tetrahydroxyflavanone) are potent antimicrobial inhibitors of Staphylococcus aureus KAS III. Ganugapati et al.⁽ Reference Ganugapati, Sirisha and Mukkavalli ^{82 )} worked on in silico modelling and docking studies of a superbug enzyme, namely New Delhi metallo-β-lactamase-1 (NDM-1), which is an enzyme found in Escherichia coli. It has been reported that this enzyme belongs to a B1 subclass of metallo β-lactamases and is known to induce resistance to standard intravenous antibiotics. Similar studies were carried out on inhibition of NDM-1 in superbugs by flavonoids using the technique of in silico molecular docking⁽ Reference Padmavathi, Prasanth Reddy and Rao ^{83 )}. At present, there are no effective antibiotics against the NDM-1-positive pathogen and therefore this study provides clues to investigate the molecular basis of extended antibiotic resistance of NDM-1 and then accelerate the search for new antibiotics against the NDM-1-positive strain in clinical studies. Quercetin and its analogue penta-O-ethylquercetin were found to be potential inhibitors of NDM-1.

Disease-combating activity

Ganugapati et al.⁽ Reference Ganugapati, Mukkavalli and Sahithi ^{84 )} studied green tea flavonoids as insulin mimetics. Diabetes mellitus is a metabolism disorder where glucose, a principal source of energy, cannot enter the cells due to deficiency of insulin. The study suggested that epicatechin acts as an insulin receptor activator and reduces the harmful effects of diabetes. Lu & Chong⁽ Reference Lu and Chong ^{85 )} carried out the computational work to predict the binding modes of flavonoid derivatives with the neuraminidase of the 2009 haemagglutinin 1 neuraminidase (H1N1) influenza virus. They employed molecular dynamics simulation techniques to optimise the 2009 H1N1 influenza neuraminidase X-ray crystal structure. All the twenty flavonoid derivatives were found to be satisfactory in binding and inhibiting the activity of the virus. These findings may help to develop a potential drug form of the flavonoid derivatives for the treatment of H1N1 influenza disease. Cardenas et al.⁽ Reference Cardenas, Arango and Nicholas ^{86 )} showed through a study on mice that apigenin, a dietary flavonoid, exerts immune-regulatory activity. The study carried out on NF-κB luciferase transgenic mice showed effective modulation of NF-κB with no effect on the rate of cell death, a decrease in lipopolysaccharide-induced apoptosis in lungs, and infiltration of inflammation, leading to re-establishment of normal lung architecture. These effects indicate the immune-regulatory roles of flavonoids. Kim et al.⁽ Reference Kim, Vance and Chun ^{87 )} reported that a flavonoid-rich diet is associated with a reduced risk of CVD. The study focused on individual as well as total flavonoid diet effects. Higher flavonoid intake was found to be associated with the improved CVD risk factors. Mulvihill et al.⁽ Reference Mulvihill, Burke and Huff ^{88 )} focused on the ability of citrus flavonoids to modulate lipid metabolism and other metabolic parameters related to the metabolic syndrome. This is one of the recent trends which have focused on citrus flavonoids as potential therapeutic agents for the treatment of metabolic dysregulation. The observational studies done by Hügel et al.⁽ Reference Hügel, Jackson and May ^{89 )} indicated that dietary flavonoids are associated with a decreased risk of hypertension and CVD. A diet rich in all flavonoid classes through herbs and beverages improves vascular health leading to a reduced risk of diseases. It has been observed that the consumption of them is associated with improvement in endothelial function via vascular endothelial nitric oxide synthase and protein kinase B (Akt) activation. The effect of regular quercitin intake on blood pressure in overweight and obese patients with pre-hypertension and stage I hypertension was studied in seventy patients. Ambulatory blood pressure and office blood pressure were measured. It was observed that the blood pressure level was reduced in patients with hypertension⁽ Reference Brüll, Burak and Stoffel-Wagner ^{90 )}.

Recently it has been reported that an apple of the type pelingo is rich in food components that can markedly inhibit in vitro tumorigenesis and the growth of human breast cancer cells⁽ Reference Schiavano, Santi and Brandi ^{91 )}. It was observed that pelingo juice induced cell accumulation in the G2/M phase of the cell cycle, autophagy, inhibition of extracellular signal-regulated kinases 1/2 (ERK1/2) activity and an increase in lipidated microtubule-associated protein-1 light chain-3β (LC3B). Hence it could be used as a source of bioactive compounds with potential chemopreventive activity. Through the review of randomised controlled trials, it has been observed that intake of purified and extract forms of anthocyanins leads to significant improvement in LDL-cholesterol with no adverse effects⁽ Reference Wallace, Slavin and Frankenfel ^{92 )}. An in vivo study model of rats was used to examine the effect of fenugreek seeds on renal pathology in alcoholics⁽ Reference Pribac, Sferdian and Neamţu ^{93 )}. The different concentrations of seeds and their exerted effects were checked through transmission electron microscopy. The results showed reduction in cell deterioration and improvement in renal morphology and function. A tannin-rich extract obtained from the pinhão (Araucaria angustifolia) seed was found to inhibit α-amylase⁽ Reference da Silva, Koehnlein and Bracht ^{94 )}. The same extract was also examined for inhibition of pancreatic lipase. An effective level of inhibition was observed for pancreatic lipase also. The extract also showed a significant reduction in TAG levels in mice. These results indicate that tannin can be used as a potential molecule for anti-obesity⁽ Reference Oliveira, Gonçalves and Inácio ^{95 )}. An extract of mixed polyphenolic compounds of grape seeds was found to comprise the ability to inhibit the aggregation and oligomerisation of β-amyloid in vitro and also improve the behavioural deficits in a mouse model of AD⁽ Reference Hayden, Yamin and Beroukhim ^{96 )}. Paris et al.⁽ Reference Paris, Mathura and Ghezala ^{97 )} worked on flavonoids which lower Alzheimer's amyloid protein (Aβ) production via a nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB)-dependent mechanism. It is well known that AD is due to the accumulation of Aβ peptides and the presence of neurofibrillary tangles in the brain^{( 98 ,} Reference Terry, Masliah and Salmon ^{99 )}. Aβ is believed to play an important role in AD and it has been shown that certain flavonoids such as genistein, quercetin, taxifolin, kaemferol, luteolin, apigenin, daidzein, aminogeneistein, and α- and β-napthofalvone can affect Aβ production. Recently, it was suggested that the Aβ-lowering properties of flavonoids are mediated by a direct inhibition of β active site cleavage enzyme-1 (BACE-1) activity, the rate-limiting enzyme responsible for the production of Aβ peptides⁽ Reference Paris, Mathura and Ghezala ^{97 )}. It has been reported that a strong correlation exists between the inhibition of NF-κB activation by flavonoids and their Aβ-lowering properties, suggesting that flavonoids inhibit Aβ production in whole cells via NF-κB-related mechanisms. As NF-κB has been shown to regulate BACE-1 expression, it has been concluded that NF-κB-lowering flavonoids inhibit BACE-1 transcription in human neuronal cells. Shimmyo et al.⁽ Reference Shimmyo, Kihara and Akaike ^{100 )}, while working on structure–activity relationships in cell-free, cell-based and in silico modes revealed novel pharmacophore features of flavonoids. Their results contributed to the development of new BACE-1 inhibitors by certain natural flavonoids (myricetin, quercetin, kaempherol, morin, apigenin) for the treatment of AD. Swaminathan et al.⁽ Reference Swaminathan, Chin and Sek ^{101 )} worked on a series of natural and synthetic flavones and flavonols to explore their activity against radio ligand binding at human cloned muscarinic receptors. It has been mentioned that muscarinic acetylcholine receptor-active compounds have potential to treat AD⁽ Reference Fisher ^{102 )}. Their findings indicated that there are several flavonoid compounds which possess competitive binding affinity, comparable with that of acetylcholine. Molecular modelling studies suggested that the compounds bind to the orthosteric site of the receptor, mainly through non-polar interactions. Further, it is mentioned that due to limitations in the docking and scoring functions used, no significant energy differences were observed for binding of the active compounds compared with the inactive compounds. These results give an indication of the potential of flavonoid compounds for the treatment of AD.

Flavonoid mechanisms

Almost every group of flavonoids has a capacity to act as antioxidants. It has been reported that the flavones and catechins seem to be the most powerful flavonoids for protecting the body against reactive oxygen species. Body cells and tissues are continuously threatened by the damage caused by free radicals and reactive oxygen species, which are produced during normal oxygen metabolism or are induced by exogenous damage⁽ Reference DeGroot ^{103 ,} Reference Grace ^{104 )}. The mechanisms and the sequence of events by which free radicals interfere with cellular functions are not fully understood, but one of the most important events seems to be lipid peroxidation, which results in cellular membrane damage. This cellular damage causes a shift in the net charge of the cell, changing the osmotic pressure, leading to swelling and eventually cell death. Free radicals can attract various inflammatory mediators, contributing to a general inflammatory response and tissue damage. To protect themselves from reactive oxygen species, living organisms have developed several effective mechanisms⁽ Reference Halliwell ^{105 )}. The antioxidant defence mechanisms of the body include not only the enzymes such as superoxide dismutase, catalase and glutathione peroxidase, but also non-enzymic counterparts such as glutathione, ascorbic acid and α-tocopherol. The increased production of reactive oxygen species during injury results in consumption and depletion of the endogenous scavenging compounds. Flavonoids may have an additive effect to the endogenous scavenging compounds⁽ Reference Nijveldt, Nood and Hoorn ^{106 )}. Codorniu-Hernández et al.⁽ Reference Codorniu-Hernández, Rolo-Naranjoa and Montero-Cabrera ^{107 )} carried out docking studies to understand flavonoid–protein interactions. The results indicated that hydrophilic amino acid residues demonstrate high-affinity interactions with flavonoid molecules, as was predicted by the theoretical affinity order. The docking modes among catechin molecules and four proteins (human serum albumin, transthyretin, elastase and renin) are also supporting this information. The theoretical affinity order among flavonoids and amino acid residues seems to have great applications in the theoretical predictions of flavonoid–protein interactions as a high-quality approach to understand the biological activity of flavonoids.

Radical scavenging

Flavonoids can prevent injury caused by free radicals in various ways and one way is the direct scavenging of free radicals. Flavonoids are oxidised by radicals, resulting in a more stable, less-reactive radical. In other words, flavonoids stabilise the reactive oxygen species by reacting with the reactive compound of the radical. Because of the high reactivity of the hydroxyl group of the flavonoids, radicals are made inactive, as explained in the following equation as given by Korkina & Afanasev⁽ Reference Korkina and Afanasev ^{108 )}:

$${\rm Flavonoid}\;({\rm OH}) + {\rm r(O)} + {\rm RH},$$

where R is a free radical and O is an oxygen free radical. Hanasaki et al.⁽ Reference Hanasaki, Ogawa and Fukui ^{109 )} found that some of the flavonoids can directly scavenge superoxides, whereas other flavonoids can scavenge the highly reactive oxygen-derived radical called peroxynitrite. They found that flavonoids such as epicatechin and rutin are powerful radical scavengers and the scavenging ability of rutin may be due to its inhibitory activity on the enzyme XO. Kerry & Abbey⁽ Reference Kerry and Abbey ^{110 )} reported that by scavenging radicals, flavonoids can inhibit LDL oxidation in in vitro studies. They further mentioned that this action protects the LDL particles and, theoretically, flavonoids may have preventive action against atherosclerosis.

Xanthine oxidase inhibition

Sanhueza et al.⁽ Reference Sanhueza, Valdes and Campos ^{111 )} worked on changes in the xanthine dehydrogenase:XO ratio in the rat kidney subjected to ischaemia–reperfusion stress and also studied the preventive effect of some flavonoids. They mentioned that the XO pathway is an important route in the oxidative injury to tissues, especially after ischaemia–reperfusion. Xanthine dehydrogenase is the form of the enzyme present under physiological conditions, but its configuration is changed to XO during ischaemic conditions. XO is a source of oxygen free radicals. In the reperfusion phase (reoxygenation), XO reacts with molecular oxygen, thereby releasing superoxide free radicals. Two flavonoids, quercetin and silibin, were found to inhibit XO activity, thereby resulting in decreased oxidative injury⁽ Reference Shoskes ^{112 ,} Reference Chang, Lee and Lu ^{113 )}. Cos et al.⁽ Reference Cos, Ying and Calomme ^{114 )} worked on structure–function relations in which the flavonoid luteolin (tetrahydroxyflavone) was reported to be the most potent inhibitor of XO.

Anti-inflammation

Nijveldt et al.⁽ Reference Nijveldt, Nood and Hoorn ^{106 )} reported about how immobilisation of leucocytes in the blood vascular system can damage tissues through the release of oxidants and inflammators. They mentioned in their paper that the immobilisation and firm adhesion of leucocytes to the endothelial wall lead to the formation of oxygen-derived free radicals and also release of cytotoxic oxidants and inflammatory mediators. Under normal conditions, leucocytes move freely along the endothelial wall. However, during ischaemia and inflammation, various endothelium-derived mediators and complement factors may cause adhesion of the leucocytes to the endothelial wall, thereby immobilising them and stimulating degranulation of the neutrophil. As a result, oxidants and inflammatory mediators are released, resulting in injury to tissues. Friesenecker et al.⁽ Reference Friesenecker, Tsai and Allegra ^{115 )}, while working on the oral administration of a purified micronised flavonoid fraction, found that the flavonoids suppresses leucocyte adhesion in ischaemia–reperfusion injury in hamsters. The decrease in the number of immobilised leucocytes by flavonoids may be related to the decrease in total serum complement and is a protective mechanism against inflammation-like conditions associated with reperfusion injury⁽ Reference Friesenecker, Tsai and Intaglietta ^{116 )}. Some flavonoids have been shown to inhibit degranulation of neutrophils without affecting superoxide production⁽ Reference Ferrandiz, Gil and Sanz ^{117 )}.

Compared with the research work done on the antioxidant capacities of flavonoids, there has been relatively little research on other possible mechanisms. One such mechanism by which flavonoids act is through interaction with various enzyme systems. Furthermore, some effects may be a result of a combination of radical scavenging and an interaction with enzyme functions⁽ Reference Codorniu-Hernández, Rolo-Naranjoa and Montero-Cabrera ^{107 )}. Alcaraz & Ferrandiz⁽ Reference Alcaraz and Ferrandiz ^{118 )}, while working on anti-inflammatory activity and the inhibition of arachidonic acid metabolism by flavonoids, reported that flavonoid inhibit the metabolism of arachidonic acid through the enzyme pathway. This feature gives flavonoids anti-inflammatory and anti-thrombogenic properties. The release of arachidonic acid is a starting point for a general inflammatory response and neutrophils containing lipoxygenase create chemotactic compounds from arachidonic acid.

Combating neurodegenerative diseases

The recent studies on different plant metabolites have shown that flavonoids may perform a key role in enzyme and receptor systems of the brain, exerting significant effects on the central nervous system, like prevention of the neurodegeneration associated with AD and Parkinson's disease⁽ Reference Panche, Chandra and Diwan ^{15 ,} Reference Jager and Saaby ^{119 )}. Flavonoids are capable of inhibiting enzymes, as there exist strong reports about inhibitory enzymes such as aldose reductase, XO, phosphodiesterase, Ca²⁺ ATPase, lipoxygenase and COX in preventive neurodegenerative diseases.

Considerable work has been carried out to search suitable and new flavonoids for therapeutic use in AD by using the technique of molecular docking. Hu et al.⁽ Reference Hu, Yanhong and Jing ^{120 )} have designed a new series of flavonoids, evaluated them and discovered potent AChE inhibitors. Most of them showed more potent inhibitory activities against AChE than rivastigmine, an AD drug. Further, it was mentioned that the isoflavone skeleton would be a promising structural template for the development of novel AChE inhibitors. Khan⁽ Reference Khan, Orhan and Enol ^{34 )} has examined AChE- and BChE-inhibitory activities of four flavonoid derivatives –quercetin, rutin, kaempferol galactoside and macluraxanthone. Out of four flavonoids, macluraxanthone displayed a concentration-dependent inhibition of AChE and BChE. A number of flavonoids were studied to lower Alzheimer's Aβ production using molecular docking studies. It has been reported that there exists a strong correlation between flavonoids and inhibitions of NF-κB-related mechanisms. While doing work on the molecular docking of flavones as BACE-1 inhibitors, it has been found that the flavonoids potently inhibit BACE-1 activity through the interactions of flavonoids with the BACE-1 catalytic centre⁽ Reference Shimmyo, Kihara and Akaike ^{100 )}.