Therapeutic bioactivities of flavonoids

Antioxidant activity

Antioxidant activity is the biological property most commonly associated with flavonoids. As antioxidants, flavonoids act as free radical scavengers that neutralize reactive oxygen species (ROS), which cause cellular damage and contribute to various diseases, such as cardiovascular disorders, neurodegenerative disorders and cancer [132]. Flavonoids exert their antioxidant effects through multiple mechanisms, including direct scavenging of ROS [133], activation of endogenous antioxidant enzymes [29], metal ion chelation [134], inhibition of xanthine oxidase and NADPH oxidase [135], suppression of nitric oxide-mediated oxidative stress [136] and enhancement of low-molecular-weight antioxidants [137]. The hydroxyl groups of flavonoids, particularly ortho-dihydroxyl groups on the B-ring, play a crucial role by donating hydrogen atoms and electrons to stabilize free radicals [138]. Structural features such as the number and position of hydroxyl groups, conjugation and planarity strongly influence antioxidant potency [139].

Several flavonoid activities result from a synergistic combination of radical scavenging and enzymatic modulation [140]. Flavonoids can activate key defence proteins, including NAD(P)H: quinone oxidoreductase, glutathione S-transferase and UDP-glucuronosyltransferase, which serve as the primary defence line against electrophilic toxins and oxidative stress [141]. For instance, onion extract and quercetin can increase intracellular glutathione (GSH) concentrations by approximately 50 % [142]. This occurs through the up-regulation of γ-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis. GSH acts as a major cellular antioxidant, maintaining redox equilibrium, while GSH peroxidase facilitates the clearance of various ROS and reactive nitrogen species (RNS) [143].

Flavonoids can chelate or bind metal ions to prevent metal-induced oxidation. The ability of flavonoids to form stable complexes with metal ions is dictated by specific structural features within the molecule. Primarily, there are three potential binding sites for metal ions: the 3',4'-dihydroxy (catechol) group on the B-ring and the 3-hydroxy or 5-hydroxy groups in conjunction with the 4-carbonyl group on the C-ring [144]. Through these hydroxyl and carbonyl groups, a wide array of plant-derived flavonoids can sequester transition metals to prevent metal-induced oxidation. For example, epigallocatechin-3-gallate (EGCG), the primary constituent of green tea, representing over 50 % of its catechins, can effectively inactivate Cu(II) by chelating Cu(I), subsequently forming peroxymonosulfate species with higher oxidative activity than Fe(II) [145]. However, the outcome of these interactions is highly dependent on the local environment and concentration. Research indicates that the shift between antioxidant and pro-oxidant behaviour is governed by the concentration of both the flavonoid and the metal ions involved [146]. In practical applications such as canned foods, quercetin has been shown to form a quercetin-Tin(II) complex that simultaneously reduces the antioxidant capacity of the flavonoid and the concentration of tin [147]. The studies have shown that while Fe and Zn possess high pro-oxidant effects, inducing 37 and 33 % haemolysis, respectively, they can be successfully chelated by quercetin, rutin and catechin. Notably, this protective chelation effect appears markedly less significant against Zn ions compared to Fe [146].

Flavonoids function as potent regulators of oxidative stress through the targeted inhibition of key oxidase enzymes, most notably xanthine oxidase (XO) and NADPH oxidase (NOX) [148]. Their inhibitory efficacy is strictly governed by specific structure-activity relationships (SAR), where molecular planarity and the strategic positioning of hydroxyl groups—particularly at the C-5 and C-7 positions of the flavonoid skeleton—are essential for high-affinity binding to enzyme active sites. By blocking these enzymatic pathways, flavonoids prevent the catalytic production of harmful reactive species, such as hydrogen peroxide and superoxide anions. Beyond direct competitive inhibition, flavonoids like quercetin also offer a secondary layer of protection by inducing cytoprotective enzymes such as heme oxygenase-1 (HO-1), thereby suppressing NOX-mediated oxidative damage [149]. This dual action, acting as both direct inhibitors of pro-oxidant enzymes and indirect stimulators of cellular defence systems, positions flavonoids as critical therapeutic candidates for mitigating the oxidative pathologies associated with chronic conditions like diabetes and inflammatory disorders [150].

Antidiabetic activity

Flavonoids exhibit potent anti-diabetic properties by modulating glucose homeostasis, enhancing insulin sensitivity and protecting pancreatic function through multiple molecular pathways (Table 2). Each class of flavonoids exhibits diverse antidiabetic properties, including enhancing insulin secretion and pancreatic β-cell viability under high-glucose or pro-inflammatory conditions. Therefore, they improve insulin-stimulated glucose uptake in target cells, protecting muscle cells from fatty acid-induced insulin resistance and reducing hyperglycemia while improving glucose tolerance in animal models of obesity and type 2 diabetes mellitus [151]. Table 2 summarizes studies on the antidiabetic effects of several flavonoids.

Mechanism of Action | Flavonoid
Inhibits α-glucosidase and α-amylase | Luteolin [152], chrysin, salvigenin [153], nepetin [154], epigallocatechin gallate [155], catechin [156], anthocyanin [157]
Increases insulin secretion | Luteolin [158], salvigenin [159], rutin [160], epicatechin [161], genistein [162]
Reduces fasting blood glucose and HbA1c | Luteolin [163], quercetin [164], rutin [165,166], fisetin [167], catechin [168], daidzin and glycitin [169], daidzein [170], biochanin [171], formononetin [172]
Strengthens glucose-stimulated insulin secretion (GSIS) | Apigenin [173], epicatechin [174]
Protects beta-cells | Apigenin [175], kaempferol [176], quercetin [177], isorhamnetin [178], morin [179], epigallocatechin gallate [180], anthocyanin [181], genistein [182], equol [183], formononetin [184]
Enhances GLUT4 translocation | Acacetin [185], isorhamnetin [186], equol [187], formononetin [188], naringenin [189]
AMPK activation | Nepetin [190], quercetin [191], anthocyanin [192]. daidzein [193], naringenin [194]
Improves insulin resistance | Isorhamnetin [195,196], epigallocatechin gallate [197], epicatechin [198], anthocyanin [199], biochanin a [200], hesperetin and hesperidin [201]

Anticancer activity

The anticancer activity of flavonoids is linked to the ability of this group of compounds to interfere with key processes in cancer development (Table 3) [202]. The mechanisms of action can be divided to [203-207]:

• Interference with signalling pathways, by disrupting signal cascades such as MAPK, PI3K/Akt that regulate cancer cell growth and proliferation.
• Suppression of inflammatory mediators, for example, by inhibiting NFκB, thereby reducing chronic inflammation that often promotes tumour progression.
• Structural inhibition, flavonoids specifically target processes that enable tumours to form new blood vessels (angiogenesis) and spread to distant organs (metastasis).
• Induction of programmed cell death, flavonoids can trigger apoptosis, a natural cellular self-destruction mechanism that is frequently dysregulated in cancer cells.

Mechanism of action | Flavonoid
Inhibits cell proliferation | Apigenin [208-210]
Reducing chemoresistance and drugs toxicity | Apigenin [211], scutellarin [212], salvigenin [213]
Induces apoptosis | Apigenin [214], acacetin [215], pectolinarigenin [216,217], luteolin [218], luteolin [219,220], nepetin [221], scutellarin [222], hispidulin [223], hesperetin cells [224]
Reduces cell proliferation by modulating the MAPK | Apigenin [225,226]
Regulates the production of pro-inflammatory cytokines (TNF-α, IL-6, IL-8, etc.) | Apigenin [227], hispidulin [228]
Suppresses cytoplasmic transcription factor (STAT3) | Apigenin [229], pectolinarigenin [230], luteolin [231], salvigenin [232]
Downregulates PI3K/AKT/mTOR pathway | Apigenin [233], pectolinarigenin [234], nepetin [235], scutellarin [236], hispidulin [237,238]
Inhibits cell proliferation, migration and invasion | Luteolin [239], nepetin [240], salvigenin [241], hesperetin [242,243]
Inhibitors of the transforming growth factor-β (TGF-β) | Hesperetin [244]

Anti-inflammatory

Flavonoids have been extensively researched as anti-inflammatory agents, which is linked to their planar structural conformation featuring an unsaturated bond at C2-C3, as well as the critical position of the hydroxyl groups for this property [245]. The flat shape of the molecule allows it to fit more effectively into the binding pockets of inflammatory enzymes like COX-2 or iNOS [246]. Research indicates that flavonoids downregulate pro-inflammatory cytokine expressions, including TNF-α, IL-1β, IL-6, IL-8 and MCP-1 in RAW macrophage cells, peripheral blood mononuclear cells and Jurkat T cells [247]. Flavonoids are also effective in decreasing pro-inflammatory enzymes such as iNOS, COX-2, glucuronidase and lysozyme, while enhancing antioxidant enzymes like GST, heme-oxygenase-1, SOD and CAT, which play vital roles in the progression of inflammatory diseases [248]. The presence of hydroxyl groups at the 3' and 4' positions of the B ring is responsible for the anti-inflammatory activity of the flavonoid group [249,250].

Apigenin, a flavone, has been reported to lower TNF-α-induced steady-state mRNA levels, thereby reducing the expression of ICAM-1, E-selectin and VCAM-1 in endothelial cells [251]. Flavonols like quercetin, morin, kaempferol and myricetin act as lipogenesis inhibitors and potentially inhibit arachidonic acid, phospholipase A2, cyclooxygenase and NOS, thus lowering the production of prostaglandins, leukotrienes and NO, which are key inflammatory compounds [252]. Catechin and quercetin were also reported to increase the production of IL-10, an anti-inflammatory compound, while simultaneously inhibiting IL-1β and TNF-α [251]. Quercetin blocks heat shock factor (HSF) activity required to induce the HSP70 protein, thereby reducing heat-induced damage [253]. Additionally, flavonoids can chelate iron and inhibit complement system activation to decrease inflammation [254].

Hispidulin increases dopamine in the prefrontal cortex of phencyclidine-induced rats and reverses "anti-social" behaviour in schizophrenia-1 mutant rats [255]. In epilepsy models, hispidulin reduces seizures with effects comparable to diazepam and alleviates motor disorders in hyper-dopamine-stressed rat models [256,257]. Its anti-seizure effects work by suppressing inflammatory processes and activating MAPK-A [258]. Acacetin has been reported to have anti-neuroinflammatory activity in Parkinson's disease models by protecting dopaminergic cells and inhibiting inflammatory factors like NO, prostaglandin E2 and TNF-α [259]. In dental inflammation, acacetin suppresses inflammation by regulating autophagy and GSK-3β signaling in human periodontal ligament cells [260]. Luteolin inhibits neuroinflammation and reduces endoplasmic reticulum stress markers in brain tissue [261]. In subarachnoid haemorrhage rat models, it improves oxidative damage by increasing Nrf expression and downregulating NLRP3 activation [262]. For intracerebral haemorrhage, it protects against microglia activation and infiltration and inhibits the TLR4/TRAF6/NF-κB signalling pathway [263].

Scutellarin protects against vascular inflammation induced by hyperglycaemia and inhibits testicular apoptosis and morphological disorders in diabetic rats [264]. Scutellarin protects against intervertebral disc degeneration (IVDD) by reducing ROS and mitochondrial damage [265]. In SARS-CoV-2 simulations, it interacts with the ACE2 receptor with a binding energy of -62.3415 kJ/mol at Glu495, Unk957 and Arg482 residues [266]. In stroke therapy, scutellarin aids in brain ischemia by activating JAK2/STAT3 signalling [267], and reduces brain tissue infarction in MCAO rat models [268]. Hesperidin and hesperetin prevent viral binding to ACE2, inhibit replication, and neutralize excessive pro-inflammatory reactions in SARS-CoV-2 infections [269]. Molecular docking shows hesperidin binding affinities for SARS-CoV-2 Mpro, SARS-CoV-2 PLpro, and spike glycoprotein are -24.27, -41.84 and -33.89 kJ mol-1, respectively [270]. It also has a high affinity for targets, preventing viral RNA synthesis (3CLpro and Helicase) and binds the spike-ACE2 interface via hydrogen bonding with the Tyr440 residue [271].

Cardiovascular protection

Cardiovascular disease (CVD) conditions can be categorized into atherosclerosis, hypertension, and cardiomyopathy [272]. In these cases, the cardiovascular protective effects of flavonoids have been reported across all three conditions [273]. The protective effect of flavonoids against cardiovascular disorders occurs through several specific mechanisms, primarily targeting hypertension, atherosclerosis and cardiomyopathy.

Hypertension is categorized into primary hypertension, which results from hyperlipidemia, insulin resistance and obesity, and secondary hypertension, which refers to high blood pressure caused by kidney failure, vascular disease (narrowing of blood vessels) and endocrine disorders [274]. Flavonoids lower the occurrence of hypertension through several key mechanisms.

First, flavonoids can act as vasorelaxants by directly modulating vascular smooth muscle and endothelial signalling pathways. Several flavones (acacetin, apigenin, luteolin, chrysin) and flavanones (hesperetin, pinocembrin) induce endothelium-dependent and -independent vasorelaxation, primarily via activation of Ca2+-activated potassium channels and enhancement of nitric oxide (NO) bioavailability [275-277]. Quercetin improves vascular function by restoring endothelial NO production and nitric oxide synthase (NOS) activity under hypertensive conditions [278]. Similarly, naringenin promotes vasorelaxation by activating K-Ca2+ channels in vascular myocytes and reduces blood pressure and cardiac hypertrophy through regulation of the AMPK/NOX2/MAPK signalling pathway [279]. In addition, hesperetin-7-O-β-glucuronide enhances NOS activity and attenuates oxidative stress in the aorta, further contributing to its vasorelaxant effects [280].

Second, flavonoids can suppress oxidative stress that induces endothelial dysfunction in the early event of atherosclerosis [281] and flavonoids improve endothelial function by directly enhancing NO-mediated vasoprotection and suppressing vascular inflammation [282]. They mitigate this process by preventing the reaction between nitric oxide (NO) and reactive oxygen species (ROS), thereby reducing the formation of the vasoconstrictive and cytotoxic species peroxynitrite (ONOO-) [283]. Compounds such as hesperidin and baicalin enhance endogenous antioxidant defences by increasing superoxide dismutase (SOD) and glutathione (GSH) activity while lowering malondialdehyde (MDA) levels [284,285]. In addition, luteolin attenuates vascular hypertension by inhibiting angiotensin II-induced vascular smooth muscle cell proliferation and migration through suppression of MAPK signalling and ROS generation [286]. Luteolin rapidly activates nitric oxide synthase (NOS), increasing NO production and inducing vasorelaxation in rat aortic rings and primary human aortic endothelial cells [287]. In parallel, quercetin metabolites inhibit endothelial adhesion molecule expression (VCAM-1 and ICAM-1), thereby reducing monocyte adhesion and preserving vascular homeostasis [288]. Flavonoids have been found to lower blood pressure by modulating calcium signalling in vascular smooth muscle. Epigallocatechin-3-gallate and hesperetin inhibit voltage-operated Ca2+ channels, reducing intracellular Ca2+ levels and ROS production [289], while genistein suppresses Ca2+-dependent proline-rich tyrosine kinase 2 signalling [290].

Third, they can modulate the overactivation of the renin-angiotensin-aldosterone system (RAAS), which is a major cause of hypertension. The inhibition of the RAAS system via suppression of angiotensin-converting enzyme (ACE) reduces the formation of the vasoconstrictor angiotensin II [291-293]. Flavonoids such as quercetin inhibit ACE, likely by chelating the active-site zinc ion, and significantly reduce ACE activity in vivo [294-296].

Fourth, the anti-inflammatory effects of flavonoids suppress persistent inflammation that leads to stroke and heart failure [297]. Flavonoids modulate the NF-κB and MAPK pathways, lowering the expression of TNF-α, IL-1β and COX-2 [298]. The gut microbiota ferment flavonoid glycosides into short-chain fatty acids (SCFAs) and phenolic metabolites that enter the circulation and modulate inflammation and vascular function [299,300]. For example, quercetin, metabolized by gut microbes to form 3-(3-hydroxyphenyl) propionic acid, inhibits monocyte adhesion induced by TNF-α [301].

The anti-atherosclerotic mechanisms of flavonoids related to vascular health include inhibition of low-density lipoprotein (LDL) oxidation, anti-platelet activity, reduction of atherosclerotic lesions, lowering of blood pressure and improvement of both endothelial and vascular smooth muscle function [302,303]. Flavonols and flavan-3-ols are the most extensively researched flavonoid groups regarding atherosclerotic effects because they are abundant in food and share structural similarities, specifically the presence of a hydroxyl group at C3, with differences occurring in the carbonyl group and the double bond in flavonols [304]. For example, quercetin and theaflavin significantly reduce atherosclerotic lesion size in ApoE-deficient mouse models [305], while glabridin reduces LDL oxidation [306]. In addition, myricetin protects against endothelial vascular cell damage and inhibits atherosclerotic plaque formation in ApoE-deficient mice [307]. Kaempferol has been shown to reduce the atherosclerotic lesion area, improve endothelium-dependent vasorelaxation, increase maximum relaxation values and decrease pro-inflammatory cytokines in ApoE-deficient mice [308].

Several studies indicate that the protective effects of flavonoids in cardiomyopathy occur through the modulation of autophagy, moving in the opposite direction of the triggers that cause cardiomyopathy [309]. In vivo research on hypertrophic cardiomyopathy (HC) rat models (induced by isoproterenol-ISO injection) showed that baicalein significantly attenuates HC conditions and restores cardiac function by increasing the expression of catalase and the mitophagy receptor FUNDC1 [310]. Diosmetin, a flavone, was reported to protect against HC under pressure overload via the p62/Keap1/Nrf2 signalling pathway in male C57BL/6 mice [311]. An in vitro study using rat cardiomyocyte cultures induced with polysaccharides showed that luteolin can decrease the expression of HC markers, such as atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), as well as reduce α-actinin and LC3 expression [312]. Furthermore, in vitro tests on H9c2 cells showed that treatment with puerarin, 24 hours before ISO induction, reduced hypertrophic and apoptotic markers, while the in vivo results in Sprague-Dawley rats showed improved left ventricular function and a decrease in HC markers and cardiomyocyte apoptosis following the administration of 100 mg per kg body weight of puerarin [313].

Isoflavones are a particularly interesting group of flavonoids regarding cardiovascular protection. As phytoestrogens, these soy-derived flavonoids improve cardiovascular risk through several mechanisms. Ecological research found that the high consumption of soy containing isoflavones in Asian countries lowers the risk of cardiovascular disease [314]. A study of 200 early-menopausal Caucasian women (average age 55) who were given 15 g of soy protein containing 66 mg of isoflavones for six months showed improvement in cardiovascular risk markers compared to those given soy protein without isoflavones [315]. Genistein directly affects NOS enzyme activity in vascular endothelial cells, leading to increased NO synthesis [316] and daidzein exhibits vasodilatory properties by stimulating prostaglandin production [317].

Anti-aging

Skin aging is triggered by intrinsic factors related to genetics and age and extrinsic factors related to environmental exposure; both lead to a decline in structural integrity and a loss of physiological skin function [318]. The mechanism of extrinsic skin aging involves the formation of ROS and oxidative stress caused by environmental factors like UV radiation, cigarette smoke and pollutants [319]. ROS and oxidative stress stimulate the upregulation of MMP (matrix metalloproteinases) and elastase (enzymes responsible for skin elasticity), resulting in the degradation of collagen and elastin in the skin matrix [320]. Tyrosinase is a critical enzyme in melanin biosynthesis across mammals, bacteria, plants and fungi. It is a copper-containing bifunctional enzyme that catalyses the hydroxylation of monophenols into O-diphenols and the oxidation of O-diphenols into O-quinones, which form melanin [321]. Excess melanin production leads to dermatological disorders such as age spots, melasma, freckles, lentigo, brown spots and even skin cancer [322].

Research has reported the ability of flavonoids to inhibit melanin formation reactions through targets such as microphthalmia-associated transcription factor (MITF), tyrosinase, tetratricopeptide repeat (TPR)-1 and TPR-2 [323]. Inhibiting tyrosinase by blocking its active site and preventing its natural substrate, tyrosine, from binding can suppress melanin production, making it a target for hyperpigmentation therapy [324]. Some flavonoids were reported to act as tyrosinase inhibitors. Multiflorin B, a flavone glycoside isolated from Rosa chinensis, exhibits tyrosinase inhibitory activity twice as strong as α-arbutin [325]. Tricin, an O-methylated flavonoid, shows higher effects than arbutin, with molecular docking showing hydrogen bonds with Asn80 and Arg267 near the catalytic core [326].

Four flavonoids isolated from Loranthus acutifolius show anti-tyrosinase and anti-melanin activities in an in vitro study using B16-F10 cells, with the IC50 value lower than 10 μM [327]. Apigenin possesses skin-whitening activity through its 7 and 4’ hydroxyl groups [328]. It forms hydrogen bonds with Met280 and hydrophobic interactions with Val248, Phe264 and Phe292 [329]. In vivo studies show it significantly inhibits hydroquinone-induced vitiligo by acting as an anti-inflammatory and modulating p38 MAPK [330]. Quercetin inhibits tyrosinase monophenolase and diphenolase activities [331]. Its catechol structure (3’,4’-dihydroxy group on the B-ring) chelates copper (Cu) at the tyrosinase active site, blocking the substrate L-DOPA. Quercetin-7-O-α-L-rhamnoside inhibits tyrosinase activity and melanogenesis in B16F10 melanoma cells stimulated by α-MSH plus IBMX. In addition, molecular docking simulations show the formation of hydrogen bonds between this flavonoid and the His85, His244, Thr261 and Gly281 residues of the tyrosinase [332].

Luteolin 5-O-β-D-glucopyranoside isolated from Cirsium japonicum var. maackii acts as a potent competitive inhibitor of L-tyrosine [333]. The 4’ hydroxyl group on the aglycone forms a strong hydrogen bond with the peroxide ion between two Cu ions at the active site, while the B-ring hydroxyls bind to Cys83. Luteolin was reported to inhibit cellular melanogenesis to an extent equivalent to arbutin in B16F10 murine melanoma cells that are stimulated by α-melanocyte-stimulating hormone (α-MSH) [334]. Pectolinarigenin has anti-melanogenesis activity by inhibiting the expression of MITF and tyrosinases (i.e. TRP-1 and TRP-2) [335]. Hesperetin has skin-protective activity through in vitro research using B16-F10 murine melanoma cells, where hesperetin stimulates melanogenesis via MAPK activation, phosphorylation of CREB and GSK-3β [336]. However, other studies indicate that hesperetin also acts as a competitive tyrosinase inhibitor, as demonstrated by inhibitory kinetics and molecular docking analyses predicting binding at Met280, His61, His85 and His259 [337].