G.B. Jadhav, C.D. Upasani , and R.A. Patil
Flavonoids are phenolic substances widely found in fruits and vegetables. Research in the field of flavonoids has increased since the discovery of the French paradox, i.e., the low cardiovascular mortality rate observed in Mediterranean populations in association with red wine consumption and a high saturated fat intake. Several other potential beneficial properties of flavonoids have since been ascertained. Flavonoids are most commonly known for their antioxidant activity. These effects are due to the physiological activity of flavonoids in the reduction of oxidative stress, inhibiting low-density lipoproteins (LDL) oxidation, platelet aggregation, acting as vasodilators in blood vessels, promoting fibrinolysis, acting as immunomodulators, anti-inflammatory agents and anti-tumour agent.The widespread distribution of flavonoids, their variety and their relatively low toxicity compared to other active plant compounds mean that many animals, including humans, ingest significant quantities in their diet. The aim of this review was to give an overview of the potential valuable working mechanisms of flavonoids followed by present knowledge on the Intake, absorption, conjugation, and toxicity of these substances.
Flavonoids belong to a group of natural substances with variable phenolic structures and are found in fruit, vegetables, grains, bark, roots, stems, flowers, tea, and wine1. These natural products were known for their beneficial effects on health long before flavonoids were isolated as the effective compounds. More than 4000 varieties of flavonoids have been identified, many of which are responsible for the attractive colors of flowers, fruit, and leaves2. Research on flavonoids received an added impulse with the discovery of the French paradox, ie, the low cardiovascular mortality rate observed in Mediterranean populations in association with red wine consumption and a high saturated fat intake. The flavonoids in red wine are responsible, at least in part, for this effect3. Furthermore, epidemiologic studies suggest a protective role of dietary flavonoids against coronary heart disease2. The association between flavonoid intake and the longterm effects on mortality was studied subsequently4 and it was suggested that flavonoid intake is inversely correlated with mortality due to coronary heart disease5. Until 50 years ago, information on the working mechanisms of flavonoids was scarce. However, it has been widely known for centuries that derivatives of plant origin possess a broad spectrum of biological activity6. In 1930 a new substance was isolated from oranges, which is believed to be a member of a new class of vitamins, and was designated as vitamin P. When it became clear that this substance was a flavonoid (rutin), a flurry of research began in an attempt to isolate the various individual flavonoids and to study the mechanism by which flavonoids act.
The flavones are characterized by a planar structure because of a double bond in the central aromatic ring. One of the best described flavonoids, quercetin, is a member of this group. Quercetin is found in abundance in onions, apples, broccoli, and berries. The second group is the flavanones, which are mainly found in citrus fruit. An example of a flavonoid of this group is narigin. Flavonoids belonging to the catechins are mainly found in green and black tea and in red wine2, whereas anthocyanins are found in strawberries and other berries, grapes, wine, and tea. An important effect of flavonoids is the scavenging of oxygen-derived free radicals. In vitro experimental systems also showed that flavonoids possess anti-inflammatory, antiallergic, antiviral, and anticarcinogenic properties1. The aim of this review was to give an overview of the potential valuable working mechanisms of flavonoids followed by present knowledge on the absorption, conjugation, and toxicity of these substances. Flavonoids can be divided into various classes on the basis of their molecular structure7. The molecular structure of each group of flavonoids is given in Figure 1.
Figure: 1 The molecular structure of each group of flavonoids.
The average daily flavonoid intake is estimated to be 23 mg/d12. Intakes of flavonoids exceed those of vitamin E and -carotene, whereas the average intake of vitamin C is 3 times higher than the intake of flavonoids. Flavonoid intakes seem to vary greatly between countries; the lowest intakes (2.6 mg/d) are in Finland and the highest intakes (68.2 mg/d) are in Japan (Friesenecker, 1994). Quercetin is the most important contributor to the estimated intake of flavonoids, mainly from the consumption of apples and onions13. A major problem in cohort studies of flavonoid intakes is that only a limited number of flavonoids can be measured in biological samples, and more importantly, only a relatively small number of fruit and vegetables are used to make an accurate estimation.
Data on the absorption, metabolism, and excretion of flavonoids in humans are contradictory and scarce 14-19. Some studies showed that the most intensely studied dietary flavonoid, quercetin, is absorbed in significant amounts14, 20. Naturally occurring flavones exist predominantly in a glycosylated form rather than in their aglycone form. The form of the flavonoid seems to influence the rate of absorption. Hollman and Katan18 suggested that the glycosylated forms of quercetin are absorbed more readily than are the aglycone forms; however, this has been questioned by other researchers19. The role of flavonoid glycosylation in facilitating absorption is questioned by the fact that catechin, which is not glycosylated in nature, is absorbed relatively efficiently21.
It is generally accepted that the conjugation pathway for flavonoids (catechins) begins with the conjugation of a glucuronide moiety in intestinal cells. The flavonoid is then bound to albumin and transported to the liver22,23. The liver can extend the conjugation of the flavonoid by adding a sulfate group, a methyl group, or both. The addition of these groups increases the circulatory elimination time and probably also decreases toxicity. There are several possible locations for the conjugates on the flavonoid skeleton. The type of conjugate and its location on the flavonoid skeleton probably determine the enzyme-inhibiting capacity, the antioxidant activity, or both of the flavonoid. Recent data suggest that the regular intake of flavonoids results in a more predominant formation of several conjugates, which probably results in greater activity. A detailed example is given in the study by Manach et al21, in which a high dose of quercetin was administered to a group of rats adjusted to flavonoid intake and to a nonadjusted group. Results of this study indicated that the conjugated compound isorhamnetin was formed in higher quantities in the adjusted group, which is important because it is known to be even more active than is the aglycone form of quercetin on xanthine oxidase inhibition24. Concentrations of individual flavonoids and their biologically active conjugates may not be high enough after occasional intake to explain the low mortality rates from cardiovascular disease in Mediterranean countries. However, because the half-lives of conjugated flavonoids are rather long (23–28 h)20, accumulation may occur with regular intakes, which may in turn result in sufficiently active flavonoid concentrations.
Formica and Regelson3 gave an interesting overview of the in vitro and vivo studies on quercetin. The early data on toxic side effects are mainly derived from in vitro studies. At a conference of the Federation of American Societies for Experimental Biology in 1984 on mutagenic food flavonoids, carcinogenicity was reported in just 1 of 17 feeding studies conducted in laboratory animals25, 26. Dunnick and Hailey27 reported that high doses of quercetin over several years might result in the formation of tumors in mice. However, in other long-term studies, no carcinogenicity was found28. In contrast with the potential mutagenic effects of flavonoids in earlier studies, several more recent reports indicate that flavonoids, including quercetin, seem to be antimutagenic in vivo 29, 30. A large clinical study by Knekt et al13, in which 9959 men and women were followed for 24 y, showed an inverse relation between the intake of flavonoids (eg, quercetin) and lung cancer. One possible explanation for these conflicting data is that flavonoids are toxic to cancer cells or to immortalized cells, but are not toxic or are less toxic to normal cells. If this is true, flavonoids might play a role in the prevention of cancer that is worthy of further investigation.
An overview of the hypothetical links between the working mechanisms and clinical effects of flavonoids is given in Figure 2.
Figure 2. Hypothesis of the links between the working mechanisms of flavonoids and their effects on disease.
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 damage8, 9. 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.
Flavonoids can prevent injury caused by free radicals in various ways. One way is the direct scavenging of free radicals. Flavonoids are oxidized by radicals, resulting in a more stable, less-reactive radical. In other words, flavonoids stabilize the reactive oxygen species by reacting with the reactive compound of the radical. Selected flavonoids can directly scavenge superoxides, whereas other flavonoids can scavenge the highly reactive oxygenderived radical called peroxynitrite. Epicatechin and rutin are also powerful radical scavengers10. The scavenging ability of rutin may be due to its inhibitory activity on the enzyme xanthine oxidase. By scavenging radicals, flavonoids can inhibit LDL oxidation in vitro11. This action protects the LDL particles and, theoretically, flavonoids may have preventive action against atherosclerosis.
Cyclooxygenase and lipoxygenase play an important role as inflammatory mediators. They are involved in the release of arachidonic acid, which is a starting point for a general inflammatory response. The exact mechanism by which flavonoids inhibit these enzymes is not clear. Quercetin, in particular, inhibits both cyclooxygenase and lipoxygenase activities, thus diminishing the formation of these inflammatory metabolites6, 41.
Antioxidant systems are frequently inadequate, and damage from reactive oxygen species is proposed to be involved in carcinogenesis31, 32. Reactive oxygen species can damage DNA, and division of cells with unrepaired or misrepaired damage leads to mutations. It has been stated that flavonoids, as antioxidants, can inhibit carcinogenesis33. Some flavonoids- such as fisetin, apigenin, and luteolin are stated to be potent inhibitors of cell proliferation34. Furthermore, it has been speculated that flavonoids can inhibit angiogenesis35. Angiogenesis is normally a strictly controlled process in the human body. The process of angiogenesis is regulated by a variety of endogenous angiogenic and angiostatic factors. It is switched on, for example, during wound healing. Pathologic, unregulated angiogenesis occurs in cancer36. Angiogenesis inhibitors can interfere with various steps in angiogenesis, such as the proliferation and migration of endothelial cells and lumen formation. Among the known angiogenesis inhibitors, flavonoids seem to play an important role37.
Platelet aggregation contributes to both the development of atherosclerosis and acute platelet thrombus formation, followed by embolization of stenosed arteries. Selected flavonoids, such as quercetin, kaempferol, and myricetin were shown to be effective inhibitors of platelet aggregation in dogs and monkeys38.
The antiviral activity of flavonoids was shown in a study by Wang et al39. Some of the viruses reported to be affected by flavonoids are herpes simplex virus, respiratory syncytial virus, parainfluenza virus, and adenovirus. Quercetin was reported to exhibit both antiinfective and antireplicative abilities. The interaction of flavonoids with the different stages in the replication cycle of viruses was previously described40. For example, some flavonoids work on the intracellular replication of viruses, whereas others inhibit the infectious properties of the viruses.
Some epidemiologic studies suggest a cardioprotective role of flavonoids against coronary heart disease. One large clinical study indicated that flavonoids may reduce mortality from coronary heart disease52. Various cohort studies indicated an inverse association between flavonoid intakes and coronary heart disease mortality . These studies are promising and indicate that flavonoids may be useful food compounds. Flavonoids have received much attention in the literature over the past 10 years and a variety of potential beneficial effects have been elucidated. However, most of the research involved in vitro studies; therefore, it is difficult to draw definite conclusions about the usefulness of flavonoids in the diet. The study of flavonoids is complex because of the heterogeneity of the different molecular structures and the scarcity of data on bioavailability. Furthermore, insufficient methods are available to measure oxidative damage in vivo and the measurement of objective endpoints remains difficult. There is a need to improve analytic techniques to allow collection of more data on absorption and excretion. Data on the long-term consequences of chronic flavonoid ingestion are especially scarce. In conclusion, the in vivo studies that have been performed do give a hopeful picture for the future. Currently, the intake of fruit, vegetables, and beverages (eg, tea and moderate amounts of red wine) containing flavonoids is recommended, although it is too early to make recommendations on daily flavonoid intakes.
The authors are grateful to Hon. Prashantdada Hiray, Hon. Apuravabhavu Hiray Management member and Principal for their continuous support and encouragement.
G.B. Jadhav, C.D. Upasani , and R.A. Patil
G. B. Jadhav
Department of Pharmacology, M. G. V’s Pharmacy College, Panchavati, Nashik-3, Maharashtra, India
C. D. Upasani
Departmeny of Pharmacology, Brahma Valley college of Pharmacy, Anjeneri, Nashik,Maharashtra, India.
R. A. Patil
Department of Pharmacology, M. G. V’s Pharmacy College, Panchavati, Nashik-3, Maharashtra, India