ABSTRACT: Resveratrol (3,5,4′-trihydroxystilbene) is a natural polyphenolic phytochemical with a variety of bioactivities associated with health promotion. Resveratrol is readily absorbed with the other absorbable digestion products of its main human dietary sources (peanuts, peanut butter, grapes, and red wine). The polyphenolic structure of resveratrol confers antioxidant activity and may reduce oxidant-induced apoptosis and low-density lipoprotein (LDL) oxidation. Resveratrol may be responsible, in part, for the correlation between increased wine consumption and decreased risk of coronary heart disease. The cardioprotective activity of resveratrol is associated with the inhibition of platelet aggregation and LDL oxidation and the promotion of artery vasorelaxation. As a chemoprevention agent, resveratrol has been shown to inhibit tumor initiation, promotion, and progression, as well as inhibit the growth of cancerous cells through increased apoptosis and/or cell cycle blockage. Inflammatory processes are associated in the pathogenesis of many chronic diseases including heart disease and cancer. Resveratrol has been shown to reduce inflammation via inhibition of prostaglandin production, cyclooxygenase-2 activity, and nuclear factor-кB activity. In addition, the estrogenic activity of resveratrol may help prevent post-menopausal bone loss. Modulation of cellular signal transduction pathways (such as mitogen-activated protein kinases) may explain, in part, the diverse bioactivities associated with resveratrol. Scientific information summarized in this review supports the many potential health benefits of resveratrol; however, further understanding of the bioavailability, metabolism, and cellular effects of resveratrol is necessary.
Interest in the potential health benefits associated with dietary consumption of resveratrol (3,5,4′-trihydrostilbene) has increased significantly in the past decade. Resveratrol is a member of the stilbene family, a group of compounds that consist of 2 aromatic rings joined by a methylene bridge. Over 30 stilbenes and stilbene glycosides have been identified in the plant kingdom (Soleas and others 1997). Resveratrol is the parent molecule of viniferins, a family of phytoalexin polymers that prevent the progression of fungal infections. Increased consumption of monomeric resveratrol and/or resveratrol-containing foods may be associated with improved health. These health benefits are related to a diverse range of biological activities. This review will discuss several aspects of resveratrol including bioavailability, antioxidant capacity, cardioprotection, anticancer activity, anti-inflammatory effects, estrogenic/anti-estrogenic properties, and modulation of cellular signal transduction pathways.
Resveratrol is found in at least 72 plant species (Dercks and Creasy 1989) and is formed via a condensation reaction between 3 molecules of malonyl CoA and 1 molecule of 4-coumaroyl CoA (Figure 1) (Soleas and others 1997). Resveratrol synthase facilitates this condensation reaction, which also produces 4 molecules of CO2. Resveratrol exists in 2 structural isomeric forms, cis and trans (Figure 2), with the trans form being more common and possessing greater biological activity. One of the richest sources of this compound is Polygonum cuspidatum, a weed that is used in traditional Chinese and Japanese medicines. Trees such as eucalyptus and spruce have also been found to contain resveratrol (Hillis and others 1974; Rolfs and Kindl 1984); however, its presence in edible plants is rare. The primary dietary sources in the human diet are peanuts, peanut butter, grapes, and wine.
The concentration of resveratrol present in peanuts is dependent upon many different factors. Cultivar, growing season, weather conditions, and peanut maturity have been shown to affect resveratrol concentrations (Sobolev and Cole 1999; Sanders and others 2000; Chen and others 2002). Resveratrol concentration appears to vary among the different components of a peanut, with higher concentrations found in the seed coat and shell than the embryos and kernels (Sanders and others 2000; Sobolev and Cole 1999). Processed peanuts and peanut products also display variability in resveratrol concentration. Boiled peanuts were found to contain the highest concentration, followed by peanut butter and roasted peanuts (Sobolev and Cole 1999). The higher concentration in boiled peanuts was thought to be due to the greater use of immature kernels, which contain greater amounts of resveratrol than mature kernels.
Resveratrol was first identified in grapevines (Vitis vinifera) in 1976 (Langcake and Pryce 1976). Leaf tissue was found to synthesize resveratrol in response to fungal infection or ultraviolet light exposure. Creasy and Coffee (1988) found resveratrol only in the skins of grapes, not the flesh, when its production was induced by ultraviolet light. Climate, wilting conditions, and amount of fungal (Botrytis cinerea) infection influence the concentration of resveratrol in grape skin. Grape variety also affects resveratrol concentration, with higher concentrations observed in red grape varieties compared to white grape varieties (Sieman and Creasy 1992). Due to its presence in grapes, it is no surprise that resveratrol is also found in wines. The resveratrol concentration in wine is variable, with grape variety and growing conditions having significant influence. Vinification, the conversion of fruit juice to wine through fermentation, is influential in resveratrol concentration. Mattivi and others (1995) followed the development of resveratrol through the vinification process and found that the resveratrol glucosides cis- and trans-resveratrol β-d-glycopyranoside (cis- and trans-piceid) (Figure 3) were extracted from the grape before cis- and trans-resveratrol. As fermentation continued the concentrations of the piceid isomers decreased while the resveratrol isomers increased, with trans-resveratrol having the highest final concentration. The decrease in piceid isomers was explained as the hydrolysis of these isomers into resveratrol isomers. While hydrolysis of piceid isomers did increase the concentration of resveratrol, the extraction of resveratrol from the grapes was driven by increased ethanol production. The extraction of trans-resveratrol from wine is at a maximum concentration after approximately 10 days of fermentation (Soleas and Goldberg 1995; Gambuti and others 2004).
In a comparison of wines from the north coast of California, red Pinot noir wines were found to contain the highest resveratrol concentrations, while white Bordeaux and Chardonnay wines contained the lowest concentrations (Lamuela-Raventós and Waterhouse 1993). Similar results were found by Sato and others (1997) in their comparison of wines made from grapes grown in Japan. The fermentation of grape flesh with the skin in red wine production allows red wines to have greater resveratrol concentrations than white wines, which are produced by fermentation of the flesh only.
Absorption Bioavailability and Metabolism of Resveratrol
The potential health benefits of resveratrol depend, in part, upon its absorption, bioavailability, and metabolism. Several in vitro and in vivo models have been utilized to characterize the absorption and bioavailability of resveratrol. Using the Caco-2 human intestinal cell model, Kaldas and others (2003) demonstrated that resveratrol uptake remained linear, for an hour, and transportation was nondirectional. Metabolites identified in the Caco-2 cells were resveratrol sulfate and resveratrol glucuronide, with resveratrol sulfate being predominant. The ease of absorption and accumulation of resveratrol suggests gastrointestinal cells as a possible biological target in vivo. Henry and others (2005) also observed passive diffusion of resveratrol in Caco-2 cells; however, the transport of trans-piceid required the sodium-dependent glucose co-transporter SGLT1. Similar to Caco-2 cells uptake, normal and tumor human hepatic cell lines utilize both passive diffusion and active transport for resveratrol uptake (Lançon and others 2004). In the rat small intestine model, resveratrol was absorbed on the serosal side of the jejunum, with the majority being metabolized to resveratrol glucuronide (Kuhnle and others 2000).
Recently, several studies have examined resveratrol absorption in vivo. High absorption of resveratrol, at least 70%, has been observed in human subjects after oral doses; however, only trace amounts of the parent compound were found in subjects' plasma. Resveratrol sulfate was the major metabolite found in human plasma (Walle and others 2004). Sulfation may be the primary limiting factor in the bioavailability of resveratrol. Vitaglione and others (2005) found resveratrol was absorbed by human subjects after moderate wine consumption, though only trace amounts of the parent compound were identified in some of the blood serum samples. Resveratrol absorption and bioavailability varied between subjects, but was not affected by meal type or quantity of lipids in the meal. The food matrix in which resveratrol is consumed may have an effect on absorption and bioavailability. Following oral administration of pure resveratrol to human subjects, resveratrol glucuronide was the major metabolite detected in the plasma and urine. High oral doses of grape juice resulted in the detection of the glucuronide and sulfate conjugates in subjects' plasma and urine (Meng and others 2004). Grape juice consists of mostly resveratrol glucosides, cis- and trans-piceid, with low amounts of the free resveratrol, suggesting a lower bioavailability of the glucosides compared to the pure compound. The administration of polyphenols through different matrices (aqueous, ethanol, and vegetable suspension) has been shown to have no effect on human absorption of total resveratrol (free and conjugated forms) (Goldberg and others 2003).
Antioxidant Activity of Resveratrol
Many compounds with aromatic groups are able to function as antioxidants by forming stable radicals via resonance structures, thereby preventing continued oxidation. Resveratrol contains 2 aromatic groups and has been shown to have a higher 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and hydroxyl radical-scavenging capacity than propyl gallate, vitamin C, and vitamin E (Soares and others 2003). A positive correlation has been established between the antioxidant power of wine and resveratrol content (Alonso and others 2002).
As an antioxidant, resveratrol may delay and/or prevent oxidative stress-induced cellular damage and disease. Excessive damage induced by oxidative stress can induce cells to undergo apoptosis. Resveratrol has been shown to inhibit oxidative-induced apoptosis in a variety of cell lines including Swiss 3T3 mouse fibroblasts, rat pheochromocytoma (PC12), human peripheral blood mononuclear (PBM), and human retinal pigment epithelium (RPE) cells (Jang and Surh 2001; Losa 2003; Kutuk and others 2004; King and others 2005). Reduced oxidative stress in RPE cells by resveratrol may be associated with reduced incidence of age-related macular degeneration (AMD), a leading cause of blindness in the elderly.
The antioxidant activity of resveratrol may also be associated with protection against the progression of atherosclerosis. The oxidation of low-density lipoproteins (LDL) is an important event in the development of this disease. Resveratrol has been shown to inhibit copper-initiated and, to a lesser extent, 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH)-initiated oxidation of porcine LDL (Belguendouz and others 1997; Frémont and others 1999). In vivo, resveratrol inhibited copper-catalyzed LDL oxidation in 2 healthy human subjects by 81% and 70%, respectively (Frankel and others 1993). At the site of early atherosclerosis lesions, blood platelets can also be found. When activated, these platelets can generate reactive oxygen species (ROS). Resveratrol was found to inhibit ROS production and lower lipid peroxidation, though not to the extent achieved by vitamin C, in blood platelets (Olas and Wachowicz 2002). While there was no synergism between resveratrol and vitamin C, resveratrol was able to reduce the prooxidant effects of vitamin C.
Cardiovascular Health and Resveratrol
The incidence of coronary heart disease in the French population is low despite their high fat consumption and the heavy use of tobacco products. This observation is referred to as the “French paradox,” and may be associated with increased dietary consumption of red wine. Red wine is one of the few dietary sources of resveratrol and it is believed that this compound is responsible, in part, for the positive cardiovascular effects associated with moderate wine consumption (Constant 1997). The most accepted mechanism of cardioprotection by resveratrol is the inhibition of platelet aggregation (Bhat and others 2001a). Platelets are cells without nuclei that are made in the bone marrow and function to stop bleeding via aggregation at the wound site. Platelets can be activated by several different factors, including adenosine diphosphate (ADP), collagen, and thrombin. When activated platelets change morphology they aggregate and seal damaged blood vessels. Excessive aggregation can lead to the development of cardiovascular disease. Pretreatment of platelets with resveratrol has been shown to inhibit lipopolysaccharide (LPS) and LPS + thrombin-stimulated platelet adhesion to collagen and fibrinogen in a non-dose-dependent manner (Olas and others 2002). Using in vitro and in vivo models, Wang and others (2002) have demonstrated that resveratrol inhibits ADP, collagen, and thrombin-stimulated human platelet aggregation in vitro. Furthermore, rabbits receiving resveratrol supplementation had reduced rates of platelet aggregation when fed a high cholesterol diet. The prevention of calcium influx through the stored operated calcium channels has been suggested as a target for resveratrol in the inhibition of thrombin-induced platelet aggregation (Dobrydneva and others 1999).
The cardioprotective effects of resveratrol may also be due, in part, to its vasorelaxation properties. Chen and Pace-Asciak (1996) examined the vasorelaxation effect of resveratrol on rat aortic rings with and without intact endothelium. Pretreatment with resveratrol resulted in a dose-related decrease in noradrenaline (NA)- and phenylephrine (PE)-induced contraction in endothelium-intact rat aortic rings. Endothelium-independent rings required higher concentrations of resveratrol before relaxation was observed. The authors concluded that resveratrol mediates vasorelaxation in endothelium-intact and endothelium-independent aortic rings via nitric oxide-dependent and -independent mechanisms, respectively. In a similar study, resveratrol relaxed endothelium intact rat aortic rings constricted by PE and potassium chloride (KCl) via NO release; however, there was no vasorelaxation effect with endothelium-independent rings (Orallo and others 2002). The release of NO has also been suggested as a mechanism for the reduction of ischemia-reperfusion injury experienced by rat hearts after resveratrol treatments (Hung and others 2000; Bradamante and others 2003).
Other methods of resveratrol cardioprotection include the prevention of LDL oxidation (Frankel and others 1993; Frémont and others 1999). LDL oxidation can cause the formation of fatty streaks in arteries and lead to the development of atherosclerosis. Resveratrol also interacts with signal transduction pathways within vascular tissue affecting cellular gene expression, proliferation, and differentiation.
Inflammatory processes are mediated by prostaglandins (PGs). Inhibition of PG activity may be partially responsible for the chemopreventative and cardioprotective effects of resveratrol. Resveratrol has been shown to dose-dependently inhibit induced production of PGE2 in human peripheral blood leukocytes (Richard and others 2005), while in vivo resveratrol significantly decreased elevated levels of rat PGD2 (Martín and others 2004). In the in vitro and in vivo model, resveratrol decreased the expression of cyclooxygenase-2 (COX-2), an enzyme that catalyzes PG synthesis and is induced by inflammation (Martín and others 2004; Richard and others 2005). Resveratrol has been observed to decrease induced COX-2 activity by inhibiting the expression of the enzyme via signal transduction pathways (Subbaramaiah and others 1998; Kundu and others 2004). Resveratrol also inhibits the inflammatory actions of cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) (Pendurthi and others 1999; Culpitt and others 2003).
Nuclear factor-κB (NF-κB), a transcription factor, regulates genes involved in inflammation and tumorigenesis (Baeuerle and Baichwal 1997). Inhibition of NF-κB activity is a possible mechanism by which resveratrol exerts its anti-inflammatory activity. Inhibition of TNF-induced NF-κB activation by resveratrol has been observed in several cell lines (Holmes-McNary and Baldwin 2000; Manna and others 2000). A recent study found resveratrol decreased induced mammary tumor incidence, number of tumors, and extended cancer latency in female Sprague- Dawley rats by suppressing the expression of NF-κB and 2 enzymes regulated by NF-κB: COX-2 and matrix metalloprotease 9 (Banerjee and others 2002). The exact mechanism by which resveratrol inhibits NF-κB activation remains uncertain. Manna and others (2000) determined that resveratrol inhibited TNF-induced NF-κB activation by preventing phosphorylation and nuclear translocation of the NF-κB subunit p65. Resveratrol has also been shown to prevent NF-κB activity by blocking NF-κB DNA binding and inhibiting IκB kinase (IKK) activity (Holmes-McNary and Baldwin 2000). IκB is an inhibitory protein that is bound to inactive NF-κB. Upon activation, IKK phosphorylates IκB, degrading it and allowing NF-κB to translocate to the nucleus. IKK activity is not directly inhibited; resveratrol inhibits an upstream signaling component (Holmes-McNary and Baldwin 2000).
Phytoestrogenic Activity of Resveratrol
Phytoestrogens are nonsteroidal dietary compounds that, like estrogen, are able to bind to estrogen receptors resulting in the transcription of estrogen-responsive genes. The structural similarity of resveratrol to the synthetic estrogen diethylstilbestrol (DES) (Figure 5) suggests that it may have estrogenic activity. The estrogenic activity of resveratrol has been proposed as a mechanism for cardioprotection and prevention of estrogen-dependent cancers. In the vascular smooth muscle cells of stroke-prone spontaneously hypertensive rats, resveratrol inhibited advanced glycation end products (AGEs)–induced cellular events (Mitzutani and others 2000). AGEs are involved in a variety of vascular complications. ICI 182780, an estrogen receptor antagonist, partially reversed the inhibition of the AGEs-induced events, suggesting that the inhibitory effects of resveratrol were partially facilitated by estrogen receptors (Mitzutani and others 2000).
In the human breast carcinoma MCF-7 cell line, the estrogenic properties of resveratrol vary. Stimulation of estrogen-regulated progesterone receptor (PR) expression in MCF-7 cells by resveratrol has been observed in several studies (Gehm and others 1997; Lu and Serrero 1999; Bhat and others 2001b). Though not as potent as the natural estrogenic hormone, estradiol, Gehm and others (1997) found that resveratrol exhibited a superagonist property by inducing reporter gene activity 2- to 3-fold greater than estradiol. Cis- and trans-resveratrol were both observed to have superagonist activity in MVLN cells, estrogen-dependent MCF-7 cell line (Basly and others 2000). In contrast, resveratrol has been shown to have no superagonist activity (Bhat and others, 2001b) and demonstrated anti-estrogenic activity, via estradiol-induced gene expression inhibition, in the MCF-7 cell line (Lu and Serrero 1999; Bhat and others 2001b). The estrogenic properties of resveratrol also appear to vary among different cell lines. In a comparison of 4 mammary cancer cell lines (MCF-7, T47D, LY2, and S30), resveratrol acted as an agonist in the MCF-7 and the S30 cell lines, while antagonizing estrogen activity in T47D and LY2 cells. The anti-estrogenic activity of resveratrol was also observed in human endometrial adenocarcinoma (Ishikawa) via the inhibition of estradiol-induced alkaline phosphatase activity, PR expression, and estrogen receptor-mediated reporter gene (Bhat and Pezzuto 2001).
The estrogenic activity of resveratrol may also help prevent bone loss in post-menopausal women. Resveratrol was shown to increase the proliferation of osteoblastic MC3T3-E1 cells and induce alkaline phosphatase activity, an enzyme believed to be involved in bone mineralization (Mizutani and others 1998). The anti-estrogen tamoxifen blocked these effects, suggesting an estrogen-dependent mechanism. Resveratrol-treated ovariectomized rats have been found to have significantly greater femur bone length, epiphysis bone mineral density, and bone calcium content than ovariectomized rats without treatment (Liu and others 2005).
Resveratrol as Signal Transduction Modulator
Mitogen-activated protein kinase (MAPK) pathways are well-characterized mammalian signal transduction pathways and include p38, c-Jun N-terminal protein kinase (JNK), and extracellular signal-regulated kinase (ERK). MAPK pathways consist of several kinases, which activate one another via a phosphorylation cascade in the cell cytoplasm and end with the activation of various transcription factors. The JNK and p38 pathways are associated with cellular apoptosis, while ERK is linked to cell proliferation and differentiation. The interaction between resveratrol and MAPK pathways provides possible explanations for many of the beneficial effects of resveratrol.
Several studies have suggested the cardioprotection offered by resveratrol is due to the modulation of MAPK pathways. In porcine coronary arteries resveratrol was found to inhibit the induced activation of p38, JNK1, and ERK1/2 by endothelin-1 (ET-1), a cardiovascular disease mediator (El-Mowafy and White 1999). Resveratrol was also able to prevent ET-1-induced translocation of phosphorylated ERK1/2 to the nucleus. The modulation of ERK signaling by resveratrol has been shown to play an important role in angiotensin II (Ang II)–induced proliferation and ET-1 gene expression in rat aortic smooth muscle cells (Haider and others 2002; Chao and others 2005). Excessive secretion of extracellular matrix (ECM) proteins by cardiac fibroblasts and myofibroblast and differentiated cardiac fibroblasts can lead to the development of cardiac fibrosis. Resveratrol has been found to target MEK, an ERK pathway kinase, and ERK activation in the inhibition of cardiac fibroblast mitogenic signaling, proliferation, and differentiation into myofibroblasts (Olson and other 2005).
The anticancer properties of resveratrol are due, in part, to the activation of the p53 protein and the suppression NF-κB and AP-1 through the inhibition of signaling cascades. The p53 protein is a transcription factor and an important tumor suppressor. It has been postulated that over half of human cancers are related to the loss or mutation of this protein (Dong 2003). In JB6 mouse epidermal cells resveratrol inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF)-induced cell transformation while inducing apoptosis, activating p53 transcription activity, and increasing p53 levels (Huang and others 1999). Resveratrol was able to induce apoptosis in mouse fibroblast cells with wild-type p53, but could not induce apoptosis in p53-deficient fibroblast cells (Huang and others 1999). Studies have shown resveratrol-induced apoptosis and p53 activation (via phosphorylation) are mediated by the ERK and p38 pathways (She and others 2001; Lin and others 2002).
Activator protein-1 (AP-1) is a dimeric transcription factor and is responsible for the modulating expression of several tumor-promoting genes (Kundu and Surh 2004). For this reason, AP-1 has been offered as a potential target for resveratrol. Yu and others (2001) induced AP-1 activity in human cervical squamous carcinoma (HeLa) cells using 12-myristate 13-acetate (PMA), a tumor promoter, and ultraviolet light-C (UV-C), a tumor promoter/initiator. PMA was a strong activator of the ERK1/2 pathway, while UV-C activated all 3 of the MAPK pathways. Resveratrol inhibited both PMA-induced and UV-C-induced AP-1 activity. Suppression of PMA-induced activity was associated with the inhibition of the ERK1/2 pathway. Similarly, UV-C-induced AP-1 activity suppression was associated with the inhibition of all 3 MAPK pathways. In vivo, topical application of resveratrol to mouse skin inhibited TPA-induced expression COX-2 via the inhibition of AP-1 activity and ERK phosphorylation (Kundu and others 2004). In human breast epithelial cells, Subbaramaiah and others (1998) found that resveratrol suppressed induced COX-2 activity. However, in these cells, resveratrol targeted the protein kinase C (PKC) signal transduction pathway, preventing the translocation of PKC into the membrane. Resveratrol also suppressed the expression of the c-Jun, part of the AP-1 dimer.
Resveratrol is a polyphenol produced in plants as a response to fungal infection and exists in cis and trans form, with the trans form being more biologically active. Peanuts, peanut butter, grapes, and red wine are the primary dietary sources of this compound. Easily absorbed in both in vitro and in vivo models, resveratrol has exhibited antioxidant, cardioprotective, chemopreventative, anti-inflammatory, and estrogenic properties, as well as interaction with signal transduction pathways. Future research should focus on translating in vitro findings regarding potential health benefits into in vivo models. In addition, further understanding of the potential toxicity, health effects, bioavailability, and metabolism of resveratrol is necessary before dietary and supplement recommendations can be made.