FLAVONOIDS: BIOCHEMISTRY, BIOPHYSICS, MEDICINE - Tarakhovsky Yu. S. - 2013

PART 1. MAJOR FLAVONOIDS

1.4. Flavonols

Flavonoids are the most widespread Class of flavonoids in nature (Fig. 10). They are found in a variety of fruits and vegetables, with a typical daily intake ranging from 20 to 50 mg. Flavonol molecules occur as both aglycones and diverse glycoside forms, in which the sugar moiety is attached to an oxygen atom, predominantly at the 3, 7, 3', and 4' positions. Glycosides may contain not only glucose and mannose, but also rarer sugars such as allose, galacturonic acid, and apiose.

Fig. 10. Most significant flavonols.

1.4.1. Quercetin and rutin

Quercetin and its glycoside rutin are among the best-known and most thoroughly studied flavonols widely distributed throughout the plant kingdom. The name "quercetin" is derived from the Latin quercus (oak), referring to the tree in whose wood and bark this substance is present. Quercetin is found in the highest concentrations in tea (up to 2500 mg/kg of dry leaves). It is present in significantly lower amounts in apples, onions (especially red onions), red grapes, citrus fruits, tomatoes, and broccoli, among others.

Quercetin is believed to exert beneficial effects on METABOLISM by helping to prevent obesity. This mechanism is associated with the activation of apoptosis in preadipocytes—adipose tissue precursor Cells—thereby inhibiting fat accumulation in the body. The molecular mechanism underlying this phenomenon involves the enhanced phosphorylation of AMP-activated protein kinase and its substrate acetyl-CoA carboxylase, which disrupts The regulation of adipocyte proliferation.

It was also previously thought that quercetin could prevent The Development of diabetes; however, attempts to counteract the Insulin resistance that develops during obesity were unsuccessful. Consequently, the antidiabetic activity of quercetin has been called into question.

Both animal models and human studies have demonstrated that quercetin can exhibit anti-inflammatory properties and help prevent the progression of atherosclerosis. It may also inhibit tumor Cell proliferation, reduce the expression of cardiovascular disease risk factors (such as SAA and fibrinogen), and is considered a promising agent for suppressing atherosclerotic processes. Animal studies have shown that the quercetin glycoside quercetin-3-O-beta-D-glucuronide, derived from Polygonum perfoliatum L., possesses anti-inflammatory and antiviral activity against Influenza A virus. Animal experiments have further indicated that quercetin may be effective in treating allergic asthma. Emulsions containing quercetin are capable of suppressing inflammatory and allergic processes in the Airways of mice. Additionally, quercetin has been found to bind to serotonin receptors and competitively inhibit the effects of serotonin on inflammatory pathways.

Experiments involving human volunteers have shown that quercetin can improve the condition of patients suffering from inflammatory and oxidative stress, although it has no marked effect on healthy individuals. Metabolic transformations of quercetin in The Human Body can significantly influence its biological activity. For instance, quercetin has been identified as a potent inhibitor of leukotriene B4 production in leukocytes, which may account for its anti-inflammatory efficacy. This activity is determined by the presence of a double bond between carbons 2 and 3 in the C ring. Sulfation or methylation of the hydroxyl group at carbon 3 reduces this activity by more than 50%, whereas glycosylation of this group completely abolishes quercetin's activity.

Quercetin can exhibit anticarcinogenic activity by initiating apoptosis in human Lung Cancer cells through the Induction of the apoptotic Gene Bax. Suppression of human Bladder cancer cell growth has been observed due to an increase in the conductance of Ca²⁺-activated potassium channels (MaxiK channels), which leads to Plasma Membrane hyperpolarization and inhibits cell proliferation. The high toxicity of quercetin toward certain types of cancer cells manifests at various Stages of the Cell Cycle. Quercetin is capable of mimicking the 17-β-estradiol signal, inducing apoptosis in human Colorectal Cancer cells by upregulating the expression of the tumor suppressor protein PTEN. Quercetin can also influence the expression of the p53 tumor suppressor protein, thereby triggering tumor cell death. It has been shown that, via a p53-dependent mechanism, quercetin can halt Cell Division and initiate apoptotic processes by acting on Mitochondria. Cell cycle arrest occurs through the activation of the caspase signaling cascade and is mediated by mitochondrial signaling pathways. It is known that prostate cancer cells possess exceptionally high invasive activity, which promotes rapid tumor metastasis. The Serine protease uPA is involved in this process. Quercetin has been shown to suppress the expression of this protease, as well as A number of other Proteins participating in the activation of cancer metastasis. Furthermore, quercetin can inhibit a wide range of protein Kinases implicated in carcinogenesis. Numerous studies on the anticarcinogenic activity of quercetin, conducted mostly in vitro, indicate that it exhibits no toxic effects on healthy cells. For instance, studies on breast tumor cells have demonstrated that quercetin enhances the efficacy of the drug doxorubicin, whereas the Toxic effects of

doxorubicin on healthy cells are, conversely, mitigated in the presence of quercetin. To enhance the therapeutic efficacy of quercetin against tumors, it is desirable to use high concentrations of this agent; however, it has poor Water solubility. A significant increase in solubility can be achieved by utilizing polymeric nanoparticles composed of polyethylene glycol and modified caprolactone.

Despite numerous studies investigating the effects of quercetin in animals and human volunteers, large-scale clinical trials confirming the feasibility of using quercetin or its glycosides as pharmaceutical agents remain insufficient. The U.S. Food and Drug Administration (FDA) has not yet recognized any medicinal properties in quercetin.

1.4.2. Myricetin

Myricetin is present in red wine. It acts as an estrogen receptor agonist in breast cancer cells and inhibits tumor cell proliferation. Myricetin increases the bioavailability of tamoxifen, a pharmaceutical agent that also targets Hormone Receptors in breast cells. This provides at least a partial explanation for the ability of red wine to reduce the risk of breast cancer in women. Animal experiments have revealed that myricetin affects the bioavailability of certain medications; for example, it delays the elimination of the α- and β-blocker carvedilol, which is used in the Treatment of various cardiovascular diseases (such as myocardial ischemia and Hypertension). This effect is associated with the inhibition of specific cytochrome P450 isoforms responsible for xenobiotic excretion.

Myricetin not only lowers Blood glucose levels in diabetes but also exerts a protective effect on The Kidneys in rats with experimentally induced diabetic nephropathy. It prevents the progression of glomerulosclerosis and reduces urinary protein excretion. The red wine Phenolic Compounds piceatannol and myricetin are capable of inducing apoptosis in human leukemia and hepatoma cell cultures. Notably, a synergistic effect has been observed between these compounds. Due to its direct inhibitory action on several protein kinases, myricetin exerts a chemoprotective effect against ultraviolet-induced Skin cancer in mice. It also suppresses UV-induced angiogenesis, while preventing wrinkle formation and the degradation of skin Collagen.

Myricetin is a potent antioxidant. It is capable of inhibiting DNA damage and slows down The formation of hydroxyl radicals initiated by peroxynitrite. Myricetin prevents colorectal cancer induced by 1,2-dimethylhydrazine in experimental animals. This protective effect is attributed to myricetin's ability to shield cells from oxidative stress and DNA damage. The observed effect is explained not only by the Antioxidant Properties of the myricetin molecule itself, but also by its capacity to upregulate antioxidant Enzymes such as catalase and Glutathione peroxidase, as well as to stimulate hepatic protective Functions.

There is mounting evidence supporting the anticarcinogenic activity of myricetin. Specifically, it has been shown that myricetin can not only induce the death of Pancreatic Cancer cells in in vitro experiments, but also reduce tumor size and inhibit metastasis in animal models. This action is potentially linked to myricetin's ability to suppress The activity of PI3 protein kinases. Myricetin inhibits hepatoma cell proliferation and triggers cell cycle arrest at the G₂/M phase. Concurrently, an upregulation of the p53/p21 cascade proteins and a decrease in the activity of the B-type cyclin/Cdc2 complex are observed, which may account for the arrest of cell division.

Myricetin exerts beneficial effects on The Nervous system. It has been found to inhibit the phosphorylation of mitogen-activated protein kinases (MAPKs) and protects neural cells against toxins that disrupt mitochondrial Oxidative Phosphorylation. Myricetin acts as a natural regulator of metal-dependent amyloid protein aggregation, a process implicated in the Pathogenesis of Alzheimer's disease. Both myricetin and quercetin protect Brain Neurons against ischemic injuries resulting from glucose and oxygen deprivation. Under such conditions, free radicals typically accumulate and cellular Swelling occurs. These flavonoids counteract these pathological processes, likely by preventing an excessive rise in cytoplasmic calcium ion concentrations.

1.4.3. Morin

Morin is a yellow-colored substance. It is derived from the fruits of the osage orange (Maclura pomifera), a decorative plant native to the southern and middle latitudes of North America that is now cultivated across all continents. Additionally, morin is present in the fruits of the tropical plant old fustic (Maclura tinctoria), which were historically used for dyeing fabrics khaki, as well as in the leaves of guava (Psidium guajava), a fruit-bearing plant native to the tropical regions of Asia, Africa, and the Americas.

Research has shown that morin holds potential for the treatment of osteoarthritis due to its potent anti-inflammatory properties. Morin reduces nitric oxide production by inhibiting the expression of inducible nitric oxide synthase (iNOS) and prostaglandin synthase (COX-2). It prevents amyloid formation from islet amyloid polypeptide (IAPP) and disaggregates pre-formed amyloid fibrils in pancreatic islets associated with type 2 diabetes. Myricetin, kaempferol, and quercetin lack this specific activity. Morin functions as an inhibitor of Glycogen synthase kinase 3β (GSK3β), an enzyme responsible for the hyperphosphorylation of tau protein in the human brain. As a result of excessive phosphorylation, tau protein forms aggregates within neurons (neurofibrillary tangles), leading to neurodegenerative disorders known as tauopathies. These include Alzheimer's disease, progressive supranuclear palsy, and Pick's disease—conditions accompanied by severe cognitive decline, dementia, and motor impairments manifesting in middle and older age. The recently discovered ability of morin to counteract these processes in vitro warrants further investigation. Morin also exhibits neuroprotective activity in Parkinson's disease models. Its therapeutic effect is associated not only with a reduction in Lipid Peroxidation products in the brain, but also with the suppression of apoptotic pathways. Morin exerts a hepatoprotective effect in alcohol-intoxicated animals, attenuating the accumulation of lipid peroxidation products and restoring normal levels of bilirubin and various other markers of Liver pathology. Furthermore, morin improves renal function and uric acid excretion, as demonstrated in animals with experimental hyperuricemia and Renal Dysfunction. Morin protects cells against oxidative stress induced by gamma radiation. This is accompanied not only by a general decrease in peroxidation products, but also by the protection of Membrane Lipids and DNA from radiation-induced damage. Additionally, by inhibiting several protein kinases, morin mitigates cellular apoptosis associated with inflammatory responses.

1.4.4. Kaempferol

Kaempferol gets its name from Kaempferia galanga, a decorative flowering plant belonging to the ginger family (Zingiberaceae). It is widely distributed across numerous edible plants, including various alliums (such as leeks and onions), horseradish, tarragon, napa cabbage, Brussels sprouts, green cabbage, mustard, rutabaga, broccoli, cucumber, pumpkin, strawberry, lettuce, apples, olive oil, parsley, gooseberry, blackberry, raspberry, cranberry, lingonberry, tomato, potato, spinach, grapes, and many others. It is also present in a multitude of medicinal herbs. Significant amounts of kaempferol can be extracted from tea seeds, which contain several kaempferol glycosides. Kaempferol aglycone is obtained by the Enzymatic Cleavage of the glycosidic moiety from the molecule. Preclinical studies indicate that kaempferol possesses a broad spectrum of beneficial properties with promising future biomedical Applications.

Kaempferol induces apoptosis in Ovarian cancer cells through the activation of the p53 tumor suppressor protein and the pro-apoptotic proteins Bad and Bax. It also triggers apoptosis in rat pheochromocytoma cells by suppressing NADPH oxidase activity and inhibiting the NOX-JNK signaling pathway. Thanks to its antioxidant properties, kaempferol enhances the body's resistance to oxidative stress during carcinogenesis. Furthermore, kaempferol dampens the Immune Response of dendritic cells—leukocytes responsible for initiating immune reactions—thereby exhibiting immunosuppressive activity. Among the flavonoids tested, kaempferol showed the highest efficacy in reducing cytokine production by dendritic cells, concurrently suppressing thymocyte activation. These findings suggest that kaempferol could hold therapeutic potential for chronic inflammatory and autoimmune diseases.

The anti-inflammatory and anti-allergic effects of kaempferol may be attributed to its ability to suppress The production of chemokines by monocytes. Chemokines are protein chemoattractants for phagocytes that regulate immune and inflammatory responses to foreign Antigens, particularly bacterial lipopolysaccharides. Kaempferol also inhibits mitogen-activated protein kinases (MAPKs), which consequently leads to the downregulation of T-helper cell (Th1 and Th2) activity.

Kaempferol prevents the development of colitis in experimental animal models. In the intestine, this is accompanied by a reduction in the levels of NO and several pro-inflammatory cytokines. Kaempferol improves the barrier function of the intestinal epithelium by upregulating the expression of tight junction proteins and promoting the interaction of these proteins (ZO-1, ZO-2, occludin, and claudins) with Actin in the cellular Cytoskeleton. This reinforces epithelial barrier integrity, which is experimentally observed as an increase in electrical resistance.

Kaempferol-3-O-sophoroside, isolated from the leaves of mountain ginseng, exhibits antitumor, anti-allergic, and anti-inflammatory activities. These effects are mediated by its ability to inhibit the release of HMGB1—an immune system protein that regulates the levels of cytokines driving inflammatory processes.

Kaempferol helps prevent the progression of atherosclerosis. Animal studies have demonstrated that kaempferol reduces atherosclerotic plaque size and improves vasorelaxation. This protective effect is linked to its capacity to downregulate pro-inflammatory cytokines, specifically osteopontin and its associated differentiation pathway involving CD44.

Kaempferol is capable of protecting neurons in the subcortical nucleus known as the substantia nigra, the degeneration of which—induced experimentally by neurotoxins—leads to Parkinson's disease. Notably, kaempferol treatment restored dopamine concentrations to normal levels, increased the activity of superoxide dismutase and glutathione peroxidase, and decreased malondialdehyde, a toxic biomarker of lipid peroxidation. Consequently, the treated animals showed significant improvements in motor coordination.

Kaempferol protects pancreatic β-cells from chronic hyperglycemia both in vitro and directly within the pancreatic islets of experimental animals. This protective effect occurs at nanomolar concentrations, with an optimum at 10 nM. Treatment successfully restores the expression of anti-apoptotic proteins Akt and Bcl-2 to normal levels. Moreover, it normalizes intracellular ATP and cAMP levels, which are typically depleted under such pathological conditions. Thus, kaempferol emerges as a promising natural antidiabetic agent capable of safeguarding pancreatic β-cells, potentially halting the onset of type 2 diabetes. Animal studies have further shown that kaempferol can protect against obesity and Osteoporosis by regulating the Transcription of genes responsible for the differentiation of preadipocytes, the precursor cells of adipose tissue. Concurrently, a reduction in bone calcium loss—the primary driver of osteoporosis—was observed.

Kaempferol demonstrates antiviral activity against the Japanese encephalitis virus (Encephalitis B), a mosquito-borne pathogen prevalent in South Asia associated with high mortality rates. Kaempferol can bind to specific viral RNA sites, thereby halting the spread of infection.

Like many other polyphenolic compounds, kaempferol has poor water solubility, which hinders the achievement of therapeutically effective blood concentrations. To overcome this limitation, nanoparticle-based delivery systems have been proposed, significantly enhancing kaempferol's solubility in aqueous media and substantially boosting its antioxidant efficacy.

1.4.5. Fisetin

Fisetin is found in A wide variety of plants, imparting yellow or reddish-yellow hues to them. It is present in mangoes (Mangifera indica), strawberries, blueberries, as well as certain leguminous trees native to Mexico and the southern United States, such as acacia species (Acacia greggii, Acacia berlandieri), honey locust (Gleditsia triacanthos), and bastard teak (Butea monosperma). It also occurs in trees native to southern Asia (Vietnam, Pakistan, Thailand, Indonesia), the Alaska yellow cedar (Callitropsis nootkatensis) of the cypress family in North America, the quebracho tree of South America (Schinopsis lorentzii), and the Japanese wax tree (Rhus succedanea), which serves as a commercial source of fisetin for the pharmaceutical industry.

Fisetin has garnered considerable attention as an anticancer agent. For instance, it induces apoptosis in HeLa Cervical cancer cells by activating the caspase-8 and caspase-3 regulatory pathways. Research on breast cancer cells has shown that fisetin targets caspases-7, -8, and -9. Fisetin also activated the p53 protein, although apoptosis via this pathway was inhibited by the pan-caspase inhibitor z-VAD-fmk. In prostate cancer cell studies, TRAIL (tumor necrosis factor-related apoptosis-inducing Ligand) was identified as a target of fisetin, with its activation triggering tumor cell apoptosis.

When applied to lung cancer cells, fisetin modulates multiple signaling pathways leading to cell death, including the PI3K/Akt pathway and the rapamycin-sensitive mTOR signaling pathway. Similarly, in prostate cancer cells, fisetin suppressed the mTOR signaling pathway and inactivated the PI3K/Akt pathway. In melanoma cells, fisetin disrupted Wnt/β-catenin signaling and consequently downregulated the transcription factor Mitf, resulting in cell cycle arrest and halted proliferation. In bladder cancer cells, fisetin arrested cell division and initiated apoptosis by activating p53 and inhibiting the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling pathway, which controls the expression of genes governing apoptosis, immune responses, and The Cell cycle. The ability to inhibit NF-κB also allows fisetin to target chemoresistant pancreatic cancer cells. In leukemia cells, myricetin and fisetin proved most effective, acting as catalytic inhibitors of both topoisomerase I and II.

Fisetin exhibits anti-inflammatory and anti-allergic properties and holds potential for asthma treatment. As demonstrated in several independent studies, fisetin's effects stem from its ability to suppress NF-κB activity. Treatment with fisetin leads to a decrease in eosinophil infiltration in the Lungs, reduced mucus production, and lowered nitric oxide synthase activity, all of which point to the attenuation of pulmonary inflammation. Studies on mast cells—immune cells of Connective Tissue—revealed a reduction in their activation upon fisetin exposure, mediated by the suppression of NF-κB and MAPK (mitogen-activated protein kinase) pathways, which regulate cell division, differentiation, and apoptosis. Fisetin also interfered with cell-to-cell communication, inhibiting the capacity of HMC-1 cells to become activated upon interaction with the membranes of active T cells, thereby impeding immune response progression. Furthermore, fisetin reduces the production of inflammatory cytokines, including TNF-α, the monocyte chemoattractant MCP-1, and vascular endothelial growth factor (VEGF). It also decreases the phosphorylation of extracellular signal-regulated kinase (ERK). Consequently, animal experiments show a significant suppression of rheumatoid Arthritis progression.

In investigations of the neuroprotective effects of phenolic compounds in animal models of Parkinson's disease, fisetin proved ineffective. However, it was found to significantly enhance memory. Fisetin was shown to induce the phosphorylation of CREB (cAMP response element-binding protein), a transcription factor involved in embryonic development, growth control, and the maintenance of Cellular Homeostasis. This was accompanied by long-term potentiation in the mouse hippocampus; the animals demonstrated improved object recognition and enhanced long-term memory. Additionally, fisetin exhibits antidepressant-like activity, elevating serotonin and norepinephrine levels in the frontal cortex and hippocampus while suppressing monoamine oxidase activity.

Fisetin helps preserve cognitive function in Aging animals. This effect is achieved not only through its intrinsic antioxidant activity but also by increasing the concentration of glutathione, the primary intracellular antioxidant, within the brain. Moreover, fisetin protects mitochondria against oxidative stress, displays anti-inflammatory activity in microglial cells, and lowers lipid peroxidation products by suppressing 5-lipoxygenase, an enzyme capable of oxidizing polyunsaturated lipids. Acting as a potent antioxidant, fisetin safeguards the brain from the damaging effects of chemical oxidants.

1.4.6. Isorhamnetin

Isorhamnetin is found in only a select few plant species. Notably, it is abundant in sea buckthorn berries (Hippophae rhamnoides L.), which have been used for centuries to treat ischemic conditions and Circulatory Disorders. Sea buckthorn extracts can also act as immunomodulatory, anti-stress, anticancer, antiseptic, and wound-healing agents. Isorhamnetin is likewise present in onions (Allium cepa L.), grapes, and wine.

Other plant sources containing isorhamnetin include certain species of the goosefoot family (genus Chenopodium). It is found in water pepper (Persicaria hydropiper), a plant widely distributed across Europe and Asia and extensively used in traditional medicine as an anti-inflammatory, hemostatic, and wound-healing remedy. Additional sources include dusty miller (Senecio cineraria), glasswort (Salicornia herbacea), Peucedanum morissonii L. (native to the Gorno-Altay, Western Siberia, and Central Asia), and Mexican tarragon (Tagetes lucida), a spicy herb of the sunflower family native to Mexico. Mexican tarragon possesses psychedelic properties and induces hallucinogenic effects, though its infusions have historically been used in small doses as a sedative and laxative.

Isorhamnetin exhibits anticancer properties by inducing apoptosis in myeloid erythroleukemia cells, a process accompanied by DNA fragmentation and the cleavage of PARP, a protein essential for DNA Repair. Additionally, it increases the activity of caspases—proteases central to apoptosis. Isorhamnetin suppresses skin cancer induced by epidermal growth factor receptor signaling by inhibiting cyclooxygenase-2 (COX-2), the enzyme responsible for converting arachidonic acid into prostaglandin H2 endoperoxide, which in turn helps curb inflammatory responses. Furthermore, isorhamnetin inhibits mitosis by suppressing MAP and ERK kinases, with MEK and PI3K being its primary molecular targets. Isorhamnetin also mitigates the cytotoxic effects of colorectal carcinoma cells while inhibiting proliferation and promoting tumor cell apoptosis.

Isorhamnetin can protect healthy cells, such as cardiomyocytes, from hydrogen peroxide-induced damage by suppressing mitochondrial apoptotic pathways. This protective action is associated with a decrease in reactive oxygen species (ROS) and the subsequent inhibition of the MAPK-dependent apoptotic pathway, positioning isorhamnetin as a promising candidate for cardiomyopathy treatment. Isorhamnetin also shields cardiomyocytes from injury during experimental ischemia by reducing Lactate dehydrogenase activity and suppressing apoptosis. Its antioxidant potency is further reinforced by its ability to coordinately upregulate various Components of the cellular antioxidant defense system. Specifically, it enhances the expression of superoxide dismutase, catalase, glutathione reductase, and heme oxygenase-1, while suppressing myeloperoxidase—an intracellular generator of hypochlorous acid.

Isorhamnetin, along with isoquercitrin from water pepper (Persicaria hydropiper), can inhibit adipocyte differentiation by downregulating genes in the Wnt/β-catenin signaling pathway within the liver, offering a potential therapeutic strategy for obesity management.