Current Medicinal Chemistry, 2012, 19, 1663-1681 1663 Resveratrol in Medicinal Chemistry: A Critical Review of its Pharmacokinetics, Drug-Delivery, and Membrane Interactions

A.R. Neves, M. Lúcio, J.L.C. Lima and S. Reis*

REQUIMTE, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha, 164, 4099-030 Porto, Portugal Abstract: Resveratrol is a that among other sources occurs in and for this reason, red also contain considerable amounts of this compound. Resveratrol is thought to be responsible for the ‘‘’’ which associates red consumption to the low incidence of cardiovascular diseases. The interest in resveratrol has increased due to its pharmacological effects that include cardio and and several other benefic actions (e.g. , anti-inflammatory, anti-carcinogenic and anti-aging). Despite the therapeutic effects of resveratrol, its pharmacokinetic properties are not favorable since this compound has poor being rapidly and extensively metabolized and excreted. To overcome this problem, drug delivery systems have been developed to protect and stabilize resveratrol and to enhance its bioavailability. Herein is presented an up-to-date revision covering the literature reported for nano and microformulations for resveratrol encapsulation that include liposomes, polymeric nanoparticles, solid lipid nanoparticles, lipospheres, cyclodextrins, polymeric microspheres, yeast cells carriers and or zinc pectinate beads. Regarding the interaction of resveratrol with cell membranes, only few studies have been published so far. However, it is believed that this interaction can be implied in the biological activities of resveratrol since transmembranar proteins are one of its cellular targets. Indeed, resveratrol presents the capacity to modulate the membrane organization which may consequently affect the protein functionality. Therefore, the intracellular effects of resveratrol and the effects of this compound at the membrane level were also revised since their knowledge is essential for understanding the pharmacological and therapeutic activities of this bioactive compound. Keywords: Drug-delivery systems, drug-membrane interactions, pharmacokinetics, resveratrol.

1. INTRODUCTION this theory indicating that resveratrol plays a crucial role in cardiovascular protection provided by grapes and wines [25]. 1.1. Chemical Structure and Properties of Resveratrol (A) (B) Resveratrol, chemically known as 3,5,4’-trihydroxystilbene OH HO (two phenol rings linked by a styrene double bond), is a naturally occurring polyphenolic antioxidant compound produced by a wide HO variety of plants [1-4]. Resveratrol has been classified as a natural for being synthesized de novo by plants in response to: injury [5, 6], UV irradiation [7, 8], ozone exposure [9] and fungal OH attack [10]. Resveratrol belongs to the large group of biologically HO active substances found in plants as it exhibits pleiotropic health OH beneficial effects including anti-oxidant, anti-inflammatory, Fig. (1). Chemical structures of resveratrol isomers. (A) trans-3,5,4’- cardioprotective and anti-tumor activities [11]. trihydroxystilbene, and (B) cis-3,5,4’-trihydroxystilbene. Resveratrol is found in nature as both cis and trans isomers (Fig. 1), although the trans-isomer is believed to be the most 1.3. History of Resveratrol abundant and biologically active form [12, 13]. Resveratrol is a highly photosensitive compound susceptible to UV-induced Resveratrol got into prominence in early nineties in the context isomerization, since more than 80% of the trans-resveratrol in of “French Paradox” [16]. Initially resveratrol has been suggested solution is converted to cis-resveratrol if exposed to light for 1 h to play a role in the prevention of heart disease, associated with red [14, 15]. wine consumption. The cancer chemopreventive properties of resveratrol were then recognized in 1997 when Jang and colleagues 1.2. “French Paradox” demonstrated that this compound possesses activity against all the three major stages of carcinogenesis i.e. initiation, promotion and Epidemiological studies have revealed a correlation between progression [26]. The interest of the scientific community in the consumption and the low incidence of cardiovascular phytoalexin resveratrol has substantially increased over the last diseases, a phenomenon commonly known as the ‘‘French years and its broad biological and pharmacological activities at the Paradox’’ [16-21]. In fact, certain populations of France, in spite of cellular level have been demonstrated, resulting in its classification regular consumption of high fat diet and low exercise practice, as an antioxidant [27-31], anti-inflammatory [32-34], anti- appear to have less predisposition to heart diseases [16]. For carcinogenic [26, 35-46], cell cycle inhibitor [47, 48], anti-aging example, the incidence of heart infarction in France is about 40% [34, 49-53], neuroprotector [54, 55], and cardioprotector [56-63]. lower than in the rest of Europe [19, 22, 23]. The realization that a Recently, it has been also discovered a great potency of this moderate consumption of red wine is beneficial to health led to the compound in the treatment of obesity and has also been suggestion that the phenolic compounds could be responsible for discovered [4, 50, 64, 65]. the beneficial properties of red wine [22]. Accordingly, resveratrol, as a phenolic component of red wine, was thought to be responsible 2. INTRACELLULAR ACTIVITY OF RESVERATROL for these pointed benefits [24], and the most recent data reinforced 2.1. Anticarcinogenic Activity

*Address correspondence to this author at the REQUIMTE, Departamento de Química, Resveratrol has been shown to modulate a huge variety of Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha, 164, 4099-030 different intracellular signaling molecules involved in multi-stage Porto, Portugal; Tel: +351-222078966; Fax: +351-222078961; E-mail: [email protected]

1875-533X/12 $58.00+.00 © 2012 Bentham Science Publishers 1664 Current Medicinal Chemistry, 2012 Vol. 19, No. 11 Neves et al. carcinogenesis, , cell cycle, and apoptosis [26, 39, 66- cyclin inhibitor p21 and G1/S arrest [3]. In this context, resveratrol 70]. There is also evidence supporting the association between has been shown to induce accumulation, phosphorylation and antioxidant, anti-inflammatory and anticarcinogenic activities [71- of p53[106, 111-120] as an apoptotic response in cancer 74]. cells [3]. The mechanisms by which resveratrol exerts its The cyclin D1 constitutes another target for resveratrol. anticarcinogenic effects are still unclear but may include: This enzyme is required for cell cycle G1/S transition and is also scavenging of free radicals [26, 75]; suppression of overexpressed in cancers. Accordingly, it has been demonstrated (COX) activity [68, 76, 77], inhibition of cell proliferation [47, 78- that resveratrol downregulates cyclin D1, possibly by inhibiting the 80], induction of apoptosis [47, 81, 82], and inhibition of , expression and activities of p38 mitogen activated protein kinase namely ribonucleotide reductase [2, 83], DNA polymerases [84, 85] (MAPK) and the serine/threonine-protein kinases (Akt and Pak1). and protein kinase C (PKC) [86]. The different cellular mechanisms Moreover, resveratrol sensitizes the cells to apoptosis by increasing that may be responsible for the anticarcinogenic activity of extracellular signal-regulated kinase (ERK) activity [121]. resveratrol are described in the following subsections. 2.1.4. Carcinogenesis and Apoptosis 2.1.1. Antioxidant Hypothesis of Chemoprevention Resveratrol has also been suggested to down-regulate Resveratrol is an excellent scavenger of hydroxyl, superoxide, [3, 122], which belongs to the inhibitor of apoptosis proteins family and other radicals [31], protecting cell membranes against lipid (IAPs), that directly inhibits caspases 3 and 7 and activates caspase peroxidation and avoiding DNA damage caused by the generation 9 in the majority of cancer cells [3, 123, 124]. of reactive oxygen species (ROS) [1, 31]. The protective effects In acute lymphoblastic leukemia cells, resveratrol has also been have been demonstrated resulting from the downregulation of shown to induce mitochondria-mediated apoptosis through the Keap1 protein promoting the dissociation and activation of the depolarization of mitochondrial membranes [125-127], by nuclear factor (erythroid-derived 2)-like 2 (Nrf2) that is responsible inhibiting the F1 complex of the F0/F1 ATPase proton pump which by the elimination of ROS [87, 88] through the activation of is responsible for the synthesis of ATP from ADP during oxidative antioxidant enzymes, such as superoxide dismutase (SOD), phosphorylation [128]. catalase, reductase, glutathione peroxidase, glutathione transferase and oxidoreductases [3, 89]. 2.2. Neuroprotective Effects 2.1.2. Anti-Inflammatory Hypothesis of Chemoprevention Besides the anticarcinogenic effects, resveratrol has been shown Mediators of inflammation, such as COX-2, the enzyme that to have neuroprotective effects [3] in a few neurological disorders, catalyzes the production of ; inducible nitric oxide such as Alzheimer’s disease (AD), Parkinson’s disease (PD), synthase (iNOS); interferon-γ (IFN-γ), a potent activator of Huntington’s disease (HD), brain ischemia, and epilepsy [129]. macrophages for inflammatory response and cellular immunity; pro-inflammatory cytokines, like IL-1, IL-6, IL-8 and tumor In AD, resveratrol seems to lower the levels of secreted and necrosis factor-α (TNF-α), have also been involved in intracellular Aβ produced by different cell lines. In fact carcinogenesis, especially in the promotion and progression stages resveratrol does not inhibit Aβ production, but acts by promoting [90, 91]. Resveratrol has shown to exhibit anticarcinogenic effects the intracellular degradation of Aβ via the proteasome pathway [3, by blocking the expression of these various components of pro- 129, 130]. inflammatory signaling [26, 76, 77, 92-94], through the suppression Resveratrol was also found to overexpress the yeast silent of nuclear factor-κB (NF-κB) and the activator protein-1 (AP-1) information regulator gene (SIR2) that codifies for . Sirtuins [95-99]. are NAD-dependent histone deacetylases that participate in The reduction of intracellular levels of Ca2+ promoted by numerous age-related disorders and that have been found to extend resveratrol has also been associated to the decrease of the the lifespan [54, 129, 131-133]. SIRT1 is the human homologue of inflammatory mediators TNF-α, IL-8 and IL-6 [100], as Ca2+ acts as SIR2, and thus, resveratrol-induced SIRT1 expression may play an a secondary messenger during cell inflammatory activation. important role in protecting neurons from ROS, H2O2, nitric oxide (NO) and Aβ in AD brains [129]. In HD, it was also shown that The transforming growth factor-β (TGF-β) is also known to resveratrol-induced SIRT1 in neurons rescued neuronal dysfunction drive tumor progression [101] and it has been demonstrated that caused by polyQ toxicity [3, 54]. resveratrol can suppress this factor by downregulating the Akt/CREB activation, a pathway that responds to various signals On the one hand SIRT1 suppresses apoptotic activities of that drive the cell proliferation, differentiation, and adaptive Forkhead transcription factors (FOXO) and promotes neuronal responses [102]. survival; on the other hand SIRT1 inhibits NF-κB and subsequently suppresses iNOS and cathespin B, the two toxic factors of the 2.1.3. Carcinogenesis and Cell Cycle Arrest neurodegeneration, and protects AD neurons against Aβ-induced Multiple lines of evidence suggest that resveratrol induces cell toxicity [129, 134]. The antioxidant capacity of resveratrol is also cycle arrest in a multitude of human cancer cell lines [3, 103-108]. possibly related to its neuroprotective effect, since it prevents ROS For example, resveratrol was identified as an effective inhibitor of to induce Aβ production. ribonucleotide reductase (RR) which catalyzes the rate-limiting step of de novo DNA synthesis during the early S-phase of the cell cycle 2.3. Cardioprotective Activity [109], namely the reduction of ribonucleotides into the corresponding deoxyribonucleotides [2, 3, 83]. Inhibition of RR Several mechanisms have been suggested to explain the results in arrest of the cell cycle in the G1 phase. cardioprotective effect of resveratrol. For example, the ability of resveratrol to inhibit eicosanoid synthesis from Resveratrol also inhibits the oncogenic and oxidative stress leads to a decrease in platelet aggregation, contributing to protect activated tyrosine kinase Src and thereby blocks the activation of against atherosclerosis [135, 136]. Moreover, resveratrol was the signal transducer and activator of transcription Stat3 in thought to modulate the production of NO from vascular malignant cells resulting in cycle arrest and loss of viability [1, which might control the inflammatory responses and 110]. prevent vascular damage [3, 61, 137]. iNOS expression in Tumor suppressor protein p53 also plays an essential role in endothelial cells is regulated by NF-kB which is inhibited by regulating the cell cycle in response to stress, by activating the resveratrol [138]. Additionally, resveratrol inhibits TNF-α induced Resveratrol in Medicinal Chemistry Current Medicinal Chemistry, 2012 Vol. 19, No. 11 1665

Table 1. Resveratrol Content in Different Sources

Sources Servinga Total resveratrol/serving (µg) Reference

Itadori roots 1 g ~2200 [150]

Itadori tea 200 ml ~2000 [150]

Grapes 100 g 150-780 [150, 154]

Red wines 150 mL 80-2700 [150, 155-158]

Grape juice 240 mL 0.12-0.26 [155]

Blueberry 100 g 86-170 [156]

Cranberry 100 g ~90 [156]

Bilberry 100 g ~77 [156]

Pistachio 28 g 2.5-47 [150]

Raw 28 g 0.6-50 [150, 152, 155, 157]

Roasted peanuts 28 g 0.5-2.2 [152, 155]

Peanut butter 32 g 4.7-24 [152, 154, 155]

Cocoa powder 15 g 19-34 [155]

Dark chocolate 15 g 3.8-6.5 [153, 155]

Milk chocolate 15 g 0.8-2.6 [155] aserving corresponds to a typical consumed portion of each food source. expression of coagulation factors in vascular endothelial cells, synthase activity and to reduce glycogen phosphorylase activity in which suggests that resveratrol can inhibit thrombogenesis [139]. the of diabetic rats, with a consequent increase in liver Some studies also suggest that the upregulation of tumor suppressor glycogen storage [147]. p53 in the presence of resveratrol may be other mechanism by which it protects the cardiovascular system against atherosclerosis. 3. SOURCES OF RESVERATROL AND RESVERATROL Recently, it was shown that the overexpression of BIOSYNTHESIS catecholamines contribute to the increased risk of heart failures, atherosclerosis, coronary heart disease and hypertension. Since Resveratrol was first isolated in 1940 from the roots of white resveratrol is able to inhibit the ionic channels involved in hellebore (Veratrum grandiflorum O. Loes.) [1, 2]. Later, in 1963, catecholamine secretion, this was also considered as a possible resveratrol was identified as the active constituent of the dried roots mechanism by which resveratrol presents such important of Japanese knotweed (Polygonum cuspidatum), also called Ko-jo- cardioprotection activity. kon in Japan, which has been used since ancient times in traditional Chinese and Japanese medicine to cure vessels inflammation, 2.4. Obesity and Diabetes Prevention suppurative dermatitis, gonorrhea, favus, athlete’s foot, allergy, heart diseases, and hyperlipidemia [62, 148-150]. Even today, Resveratrol has recently been shown to present beneficial Itadori tea made from Japanese knotweed plant is a very common effects in obesity and diabetes, contributing to the prevention and source of resveratrol in Japanese diet [149]. treatment of resistance, or dyslipidemia [4, Afterwards, resveratrol has been found in a wide variety of 140, 141]. about 70 plant species and fruits [6, 150], including purple grapes, One of the mechanisms by which resveratrol can reduce the , mulberries, cranberries, , peanuts, groundnuts pathological consequences of a high-calorie diet is the activation of and [62, 150-152]. Recently, it has been confirmed that SIRT1 [142], but it has been also demonstrated that some benefits coconut and cocoa are new sources of resveratrol [153]. Given the of resveratrol result from phosphorylation and consequently numerous food sources where it is possible to find resveratrol, a activation of 5’-AMP-activated protein kinase (AMPK) which summary is presented in Table 1. contributes to the oxidation of fatty acids and to the decrease of Grapes are probably the most important source of resveratrol their synthesis [143]. Moreover, resveratrol potentiates the lipolytic for humans, since the compound is also found in one of the end response to epinephrine and reduces the insulin ability to counteract products of grapes: wine [159]. Resveratrol was first detected in lipolysis in [144]. grapevines () in 1976 [5], and afterwards in wine in In diabetic rats, resveratrol was able to reduce hyperglycemia. 1992 [159]. In grapes, especially when infected with Botrytis Recent studies have demonstrated that pancreatic β -cells are cinerea, resveratrol is synthesized almost entirely in the skin and its significantly influenced by resveratrol which enhances insulin content is maximum just before the grapes reach maturity [160]. secretion [4, 145]. Different mechanisms are proposed to be Therefore, resveratrol highest concentration is in the skin and seeds responsible for resveratrol-induced intracellular glucose transport of grapes (50–100 µg per gram, corresponding to 5–10% of their [4]. For example, the administration of resveratrol enhances the biomass) [151]. translocation of glucose transporter (GLUT4) to the plasma The enzyme responsible for the biosynthesis of resveratrol is membrane potentiating the internalization of glucose in the cells stilbene synthase (STS), which is rapidly activated in response to [146]. At the same time, resveratrol has shown to increase glycogen 1666 Current Medicinal Chemistry, 2012 Vol. 19, No. 11 Neves et al. exogenous/environmental stress factors, such as injury, UV Stervbo et al. showed that wines of the variety irradiation and chemical signals from fungi attack [12, contain the highest average levels of resveratrol and wines made 161]. STS catalyzes three condensation reactions between from the St. Laurent variety (grown for instance in Slovakia, the coumaroyl-coenzyme A (CoA) and three molecules of malonyl- Czech Republic, and Austria) contained the second highest average CoA via cleavage of three carbon dioxide molecules (Fig. 2). In levels of resveratrol, and were not significantly different from Pinot addition, STS also catalyzes the loss of the terminal carboxyl group, Noir [12]. which leads to the production of the C14 molecule resveratrol [161, Furthermore, although the average levels of cis-resveratrol are 162]. lower than trans-resveratrol, this former isoform may also be CoAS important. The levels of cis-resveratrol in wine are a result of SCoA OH vinification [171, 172], and isomerisation from the cis to the trans O isoform is facilitated by low pH [14]. Hence, the total level of + 3 OO available resveratrol after consumption may be the sum of trans- OH and cis-resveratrol. Coumaroyl-CoA 3 Malonyl-CoA Resveratrol- () may also be an important conjugate present in red wines once that its average concentration is STS about 120 µM (three times that of resveratrol) [12, 173]. 3 CO2 + 3 CoASH 3.2. Chemical Synthesis of Resveratrol SCoA Resveratrol can be obtained in the diet from plant tissues, fruits O and wine. However, when we need to isolate resveratrol to perfom laboratory studies or to produce formulations of the compound, the O OH extraction from the food sources requires large amounts of plant O materials and solvents, and involves laborious purification O procedures, resulting in very low product yields [174]. Chemical synthesis is available; however this method also shows some disadvantages such as a low yield and low purity of the resveratrol STS CO + CoASH obtained [175]. Reliable and efficient synthetic routes to obtain 2 resveratrol are therefore highly desirable. To overcome this OH problem, microorganisms (e.g. Saccharomyces cerevisiae [176, 177]) have been used to produce resveratrol by introducing the HO genes responsible for resveratrol biosynthesis. These transgenic implementations have resulted in highest resveratrol concentrations [174].

trans-resveratrol 3.3. Resveratrol Analogs HO Resveratrol has a number of naturally occurring analogs (Table Fig. (2). Schematic representation of resveratrol biosynthesis by STS 2). Many resveratrol derivatives, such as the viniferins, (Stilbene Synthase). , and piceid are also present in tissue and have been involved in plant defense mechanisms against biotic and In order to provide resistance against a stress factor, the abiotic stress [178]. In addition, several studies indicate that some phytoalexin accumulation must occur rapidly on the exposed site resveratrol analogs also have pharmacological benefits, such as [163]. The levels of resveratrol reach their peak approximately 24 h antioxidant effects, chemoprevention actions, and anti-aging after stress exposure, and decline after 42–72 h as result of properties [179-181]. activation of stilbene oxidase [6, 151]. Accordingly, the resveratrol content in grapes depends not only on the type of grape but also on Regarding the biological activity of resveratrol and its analogs, the stress exposure [7, 12]. several structural properties can be determinant, such as the number and position of hydroxyl and methoxyl groups, intramolecular 3.1. Resveratrol Content in Red Wine hydrogen bonding, and stereoisomery. For example, it was shown that trans-stilbenes possessing a 4’-hydroxyl group and bearing Since the grape skins are not fermented in the production ortho-diphenoxyl or para-diphenoxyl functionalities exert process of white wines, only red wines contain considerable remarkably increased activity [182, 183]. Moreover, antioxidant amounts of resveratrol [164, 165]. Resveratrol concentration and apoptotic activities of the analogs containing 3,4-dihydroxyl measured in a sampling of red wine ranged from 1 to 14 mg/L [12, groups, namely trans-3,4-dihydroxystilbene, trans-3,4,4’- 164], although higher or lower values are frequently found. trihydroxystilbene, and trans-3,4,5-trihydroxystilbene have shown to be significantly higher than those of resveratrol [181]. Another Because resveratrol is produced in response to exogenous stress important finding was the higher rate of COX-inhibition shown by factors, the levels in red wine are expected to vary between regions hydroxylated resveratrol derivatives when compared to and vintages depending on the local climate [162, 166-169]. When methoxylated analogs, which provide useful information for future comparing the average resveratrol content in wines with the latitude cancer drug design [184]. In this case, the most potent resveratrol of the wine producing region, it appears that in the northern analogs were (trans-3,4,3’,5’-tetrahydroxystilbene, hemisphere, the further north the higher level of resveratrol. In the PCA) and trans-3,4,5,3’,4’,5’-hexahydroxystilbene (M8). southern hemisphere, the regions that are closer to equator present wines with higher average levels of resveratrol [12]. Numerous 4. PHARMACOKINETICS factors, such as increased temperature, higher levels of SO2 and decreased pH during the wine making process also increase the As the pharmacology of resveratrol was subjected to extensive levels of resveratrol in the final red wines [12, 14, 170]. studies during the past decade, its pharmacokinetics has also been Resveratrol in Medicinal Chemistry Current Medicinal Chemistry, 2012 Vol. 19, No. 11 1667

Table 2. Resveratrol Analogs and their Properties

Name of analogs Chemical structure Properties References OH

HO - The additional hydroxyl group enhances its OH [183, 185- Piceatannol 1 . 187] - Potent LDL-antioxidant and cardioprotective effects.

OH OH

OH

HO - Radical scavenging and strong pro-apoptotic activity. M8 2 [188, 189] OH - Inhibitory effects on NF-κB.

HO

OH OH

H3CO - 3, 5-dimethoxy motif confers pro-apoptotic activity. Pterostilbene 3 [190-192] - Good antioxidant properties.

OCH 3 OH OH

- Resveratrol dimer has increased lipophilicity compared to the parent molecule and this fact increases its cellular HO [179, 193- ε-viniferin 4 uptake. O 196] HO - Proliferative and apoptotic effects. - Inhibits cytochrome P450 enzymes.

OH OCOCH3

- Higher hydrophobicity than the parent molecule and H3COCO consequent increased ability to cross the cellular [193, 197- Acetylated analog 5 membrane. 199] - Greater antiproliferative effect on cancer cell growth.

OCOCH3 OH OH

O - Glucoside . HO O HO - Inhibits platelet aggregation, LDL oxidation, and [136, 200- Piceid 6 OH eicosanoid synthesis. 203] - Antimetastatic activity.

OH 1trans-3,4,3’,5’-tetrahydroxystilbene, 2trans-3,4,5,3’,4’,5’-hexahydroxystilbene, 3trans-3,5-dimethoxy-4'-hydroxystilbene, 45-[(2R,3R)-6-Hydroxy-2-(4-hydroxyphenyl)-4-[(E)-2-(4- hydroxyphenyl)ethenyl]-2,3-dihydro-1-benzofuran-3-yl]benzene-1,3-diol, 5trans-3,5,4’-Tri-O-acetylresveratrol), 6trans-resveratrol 3-O-β-ᴅ-glucoside. investigated in preclinical models as well as in humans [35, 65]. metabolism without adverse effects in both rodents [204-215] and Unfortunately, the pharmacokinetic properties of resveratrol are not humans [216-220], resulting in only trace amounts of unchanged as favorable when compared to its beneficial pharmacological resveratrol in the systemic circulation. In humans, about 70 % of activities in various disease models [35]. In the next subsections, orally administered resveratrol (25 mg) is rapidly (< 30 min) the pharmacokinetics of resveratrol will be presented in more detail. absorbed and metabolized with a peak plasma level of ≈ 2 µM of resveratrol metabolites and a half-life of 9-10 h [216, 217, 221]. 4.1. Absorption and Bioavailability Furthermore, there is a significant person-to-person variability Recent literature on bioavailability of resveratrol suggests that in drug absorption and metabolic processes [217, 218]. The extent this polyphenol has high oral absorption but rapid and extensive to which the human colon can absorb and metabolize resveratrol 1668 Current Medicinal Chemistry, 2012 Vol. 19, No. 11 Neves et al.

OH HO

trans-resveratrol

HO SULTs

OH

SO3H O3SH HO

HO S HO S2 3

SO3H

O3SH

HO S1 Fig. (3). Metabolic pathway of resveratrol in liver by SULT enzymes. S1 - trans-resveratrol-3-O-4’-O-disulfate, S2 - trans-resveratrol-4’-O-sulfate, and S3 - trans-resveratrol-3-O-sulfate. depends on the hepatic function and on the metabolic activity of the 4.3. DISTRIBUTION local intestinal microflora. 4.3.1. Blood Transport 4.2. Metabolism The efficiency of a therapeutic substance is often related to its Resveratrol undergoes extensive phase I (oxidation, reduction, affinity to bind to protein transporters [228]. Resveratrol has poor and hydrolysis) and phase II (glucuronic acid, sulfate, and methyl water solubility and thus has to be bound to plasma proteins to conjugations) biochemical changes immediately after ingestion assure its body distribution and bioavailability [229]. Indeed, in its [210, 221-223], being metabolized into both glucuronic acid and transport, resveratrol can bind to serum proteins [230] such as sulfate conjugations of the phenolic groups in liver and intestinal lipoproteins, hemoglobin, and albumin which facilitate its carrier- epithelial cells [218, 224, 225]. Hydrogenation of the aliphatic mediated cellular uptake and then it can passively diffuse through double bond is also present [222]. While presystemic and systemic the plasma membrane [198]. conversion to major metabolites (glucuronic and sulfate Lu et al. [231] investigated the binding properties of resveratrol conjugations) occur in the intestine and liver very fast and to plasma proteins, such as human serum albumin (HSA) and efficiently in the so called enterohepatic recirculation [210], other hemoglobin (Hb) and confirmed that both complexes formed are metabolites such as dihydro-resveratrol and piceatannol, are spontaneous and exothermic. The binding constant of resveratrol– probably mediated by microbial fermentation of trans-resveratrol in HSA complex is larger than that of resveratrol-Hb, which indicates the gastrointestinal tract. the higher affinity of HSA to resveratrol. Hydrophobic interactions The of resveratrol in human liver by sulfotransferases seem to play a major role in the binding of resveratrol to the (SULTs) has been examined [226] and three metabolites were hydrophobic cavity of HSA, and hydrogen bonding is the main identified (Fig. 3): trans-resveratrol-3-O-4’-O-disulfate (S1), trans- force involved in the binding of resveratrol to the central cavity of resveratrol-4’-O-sulfate (S2), and trans-resveratrol-3-O-sulfate Hb where some residues interact directly with the hydroxyl groups (S3). The by uridine 5’-diphospho-glucoronosyl- of the compound. Electrostatic interactions can also be involved in transferases (UGTs) on intestinal absorption of resveratrol was also the formation of both complexes since residues with positive charge investigated [223] and it was possible to identify two metabolites are in the proximity of the binding compound. (Fig. 4), namely, trans-resveratrol-4’-O-glucuronide (G1), and 4.3.2. Liver Uptake trans-resveratrol-3-O-glucuronide (G2). It is known that liver plays a key role in the bioavailability of Modifications such as glucuronidation and sulfation typically resveratrol. Some studies show the highest accumulation of reduce the cell permeability to drugs and aid in their excretion. resveratrol in the liver of rats and mice after oral administration However, the in vivo efficacy of the administered resveratrol, [205, 212]. Nevertheless, no toxicity or hepatocyte lysis was despite its low bioavailability, has led some researchers to observed after treatment with high doses of resveratrol, which is interrogate whether metabolites would be the bioactive forms of the relevant because certain antineoplasic agents cause hepatotoxicity, parent compound [217, 221, 227]. Nevertheless, some studies show limiting their efficacy in anticancer therapy [26]. Moreover, the less pharmaceutical impact of the metabolites and indicate a large uptake of resveratrol by liver cells, along with its weak decrease in the expression of SULT enzymes in certain types of toxicity suggests its important role in the prevention of liver cancer cells, which indicates an overall low sulfation activity in diseases. these cells comparing to the normal ones. Therefore, the reduction of SULT expression may be a favorable factor for achieving better Besides a passive diffusion influx, it was also shown that therapeutic effects while an effective dose of trans-resveratrol is resveratrol enters the liver cells by an active process involving maintained in cancer cells. transporters, accounting for more than half of the total hepatic uptake [198]. This active process involves members of the family of Resveratrol in Medicinal Chemistry Current Medicinal Chemistry, 2012 Vol. 19, No. 11 1669

OH HO

trans-resveratrol

HO UGTs

CO H 2 HO2C O O OH HO OH O HO OH O HO OH HO

G1 G2 OH

HO Fig. (4). Metabolic pathway of resveratrol in intestine by UGT enzymes. G1 - trans-resveratrol-4’-O-glucuronide and G2 - trans-resveratrol-3-O-glucuronide. organic anion-transporting polypeptides (OATPs) which are with other dietary factors which might modulate resveratrol multispecific transporters or albumin binding proteins that bind functions synergistically (e.g., certain can effectively resveratrol–albumin complexes and then deliver resveratrol in a inhibit sulfotransferases [235], facilitating resveratrol bioavai- similar way to the fatty acid uptake [232]. lability), and developing more bioavailable analogs of the compound. In this last field, studies have been conducted to modify 4.4. Excretion the resveratrol molecule in order to increase its bioavailability while preserving its biological activity, and the use of other resveratrol In the last process of pharmacokinetics, all the metabolites of related molecules has also appeared as a promising approach [200]. resveratrol are eliminated from the organism and excretion is almost equally distributed between urine and feces. An almost 4.6. Toxicity complete elimination of resveratrol and its metabolites from tissues is observed 72 h after a single dose. The two major metabolites In several toxicity studies, resveratrol was orally administrated identified in urine of rodents were the mono-glucuronides of trans- at its maximum tolerated doses to access its adverse effects. The resveratrol and of dihydro-resveratrol [233]. In humans, the major results show the lack of carcinogenicity of resveratrol [236]. In metabolites were the glucuronide- and sulfate-conjugates of addition, studies revealed the absence of acute skin and eye resveratrol and of dihydro-resveratrol. The total recovery of irritation or other allergenicity signs that could be caused by the glucuronic and sulfate conjugations in humans urine and feces was compound [233]. Despite being an -like compound, studies about 71–98 % after oral doses and 54–91 % after intravenous provide further evidence that trans-resveratrol has low estrogenic doses, whereas the aglycone form of resveratrol presented a near potency in vivo. In fact, oral administration of high doses of zero recovery [218]. These findings clearly evidence that the resveratrol had no effect on reproductive capacity and there were no circulating form of resveratrol is predominantly the modified significant changes in bone density. metabolite, and not the original aglycone. Commercial dietary supplements contain on average between 4.5. Future Bioavailability Studies 50 and 500 mg of trans-resveratrol and human clinical studies have also been performed up to single doses of 5 g of the same Taken together, pharmacokinetic data from several studies compound without observing adverse effects [219]. These data suggest that, in general, trans-resveratrol is rapidly absorbed, suggests that trans-resveratrol is well tolerated in humans and that metabolized and excreted in humans and animals, indicating a poor 450 mg/day of resveratrol can represent a safe dose for a 70 kg bioavailability of resveratrol which compromises its biological and individual. pharmacological benefits inside the organism. The only exception A recent epidemiology study suggested that resveratrol to this behavior occurs, if we consider the tissues where there is consumption is inversely related to breast cancer risk since 50% or high accumulation of resveratrol after the oral administration, such greater reductions in breast cancer were observed in women with as at the upper digestive tract and also at the intestinal epithelium. the highest levels of resveratrol consumption [237]. Given the weak Moreover, we cannot forget the evidence that the in vivo toxicity of resveratrol, it was suggested that this bioactive concentrations of the individual metabolites are much higher than compound should be a promising candidate for chemoprevention in those of the native compound. Therefore, further studies are needed humans [26]. Moreover, this compound is able to penetrate the to determine whether the metabolites represent inactivated forms of blood–brain barrier [238], suggesting its potential therapeutic value the drug that act as a pool from which free resveratrol can be in brain diseases. hydrolyzed once it reaches the target tissues, or are themselves active in promoting many of the health benefits attributed to resveratrol [234]. 5. CARRIERS USED FOR THE ENCAPSULATION AND DELIVERY OF RESVERATROL In addition, other studies are also needed to enhance the bioavailability of resveratrol by modulating its metabolism, In most experiments resveratrol has been used in a free form devising appropriate formulations, identifying possible interactions dissolved in different organic solvents (i.e., DMSO, acetone, and 1670 Current Medicinal Chemistry, 2012 Vol. 19, No. 11 Neves et al. ) that are not suitable for drug delivery [239-241]. However, preparation process and the biological applications of the particles it seems desirable to stabilize resveratrol to preserve its biological produced. Properties like: composition, morphology, particle size, and pharmacological activities and enhance its bioavailability. polydispersity index (PI, related to the size distribution), zeta Indeed, resveratrol has several disadvantageous properties that potential (related with the surface charge), and encapsulation justify its encapsulation in carriers, such as: poor water solubility efficiency are some of the parameters determined for the [242]; short biological half-life; chemical instability (tendency to characterization of resveratrol carrier systems (Table 3). suffer oxidation and extreme photosensitivity [243]); and its extensive and rapid metabolism and elimination [217]. 5.1.1. Liposomes

The development of site-specific drug delivery systems that Liposomes have been widely used to deliver drugs, based on protect resveratrol during its transit inside the organism is the assumption that liposome membranes fuse with cell membranes extremely important and requires a multidisciplinary approach. and then release their content into the cytoplasm. Liposomes are Microencapsulation has a growing application in pharmaceuticals, optimal carriers for the entrapment and cellular delivery of drugs cosmetic and food industries [244-247] due to the resultant because they can incorporate a lipophilic drug within the membrane extension of shelf-life, protection against oxidation and control bilayers [267-270]. Liposomes are also able to promote a controlled release of active component. Hence, the disadvantageous properties release of the drugs at the target site over a prolonged period of of resveratrol can be partially overcome by microencapsulation time [271]. strategies applied to protect resveratrol from light and oxidative stress and to increase the compound solubility in an aqueous Besides acting as drug carriers, liposomes may play a role in the environment [242]. On the other hand, nanotechnology has led to treatment of diseases because these aggregates interact with cells the development of nanocarrier delivery systems that are able to thereby being able to regulate cell differentiation. Since liposomal transport resveratrol to target tissues [248]. A promising strategy is incorporation of resveratrol enhances its effect, it may be a the development of colloidal carriers that can transport resveratrol promising and powerful tool to increase the efficacy of transport and release it, at specific desired locations. Nanoemulsions, and delivery of this compound. polymeric nanoparticles, liposomes, and solid lipid nanoparticles Aiming to achieve an optimal system for the entrapment and (SLN) composed of pharmacological acceptable excipients have all delivery of resveratrol, different liposomal formulations were been examined for this purpose [249-254]. developed and no substantial differences were observed between liposomes of different lipid compositions [254, 264, 272]. 5.1. Properties and Characterization of Resveratrol Carriers Liposomes were obtained by extrusion, which is reported to be the most suitable technique to get homogeneous distribution of vesicle In recent times, different resveratrol formulations were size [273]. Indeed, the extruded liposomes were uniform in size, developed, characterized and tested on different cell systems [251- with an average diameter close to the filter pore size of the extruder 265]. Furthermore, recent research involves studies of the (100 nm) and the P.I. was 0.1-0.2 which reveals an acceptable size morphological changes of the cells treated by free and/or distribution, hence a good quality for all formulations. The zeta resveratrol-loaded particles, as well as, the cellular uptake of the potential measurements presented no significant changes during particles. Characterization methods are necessary to control the storage time and liposome size analysis revealed that there were no

Table 3. Properties of Different Resveratrol-Loaded Carriers

Zeta Potential Polydispersity Encapsulation Particle Type Composition Morphology Size References (mV) Index efficiency (%)

Spherical P90G, DCP, CHOL; PC; Liposomes oligolamellar < 100 nm ≈ -40 0.1 to 0.2 50 to 80 [254, 264] DPPC, EPC, PEG, CHOL vesicles Polymeric Spherical shape, mPEG-PCL < 100 nm -5.5 to -6.5 ≈ 0.1 ≈ 90 [252, 263] nanoparticles smooth surface Solid lipid Compritol, phospholipon, Spherical ≈ 200 nm ≈ -38 ≈ 0.3 ≈ 60 [253] nanoparticles lutrol Soybean PC, CHOL, coconut, Spherical and Lipospheres 200 to 350 nm -50 to -80 n.i. 75 to 99 [265] perfluorocarbons homogeneous Regular spherical β-cyclodextrin, Cyclodextrins shape, highly 400 to 500 nm -16 to -20 ≈ 0.2 30 to 40 [260] carbonyldiimidazole porous structure Polymeric Polystyrene, DVB, AN, BPO, Porous particles, ≈ 6 µm n.i. n.i. ≈ 80 [262] microspheres CDD, PVA crystalline structure Eukaryotic Yeast cells S. cerevisiae unicellular micro- 3 to 4 µm n.i. n.i. ≈ 19 [266] organism Spherical shape; Calcium- [251, 255, Pectin, Ca2+ rough and rugged ≈ 1 mm n.i. n.i. 60 to 99 pectinate beads 256] surface Spherical shape; Zinc-pectinate Pectin, Zn2+ rough and rugged ≈ 1 mm n.i. n.i. 94 to 99 [257-259] beads surface n.i. – not indicated; P90G – phospholipon 90G; DCP – dicetyl phosphate; CHOL – cholesterol; DPPC – dipalmitoylphosphatidylcholine; EPC – egg-phosphatidylcholine; PEG – poly(ethyleneglycol); mPEG-PCL – methoxy poly(ethyleneglycol)-poly(caprolactone); PC – phosphatidylcholine; DVB – divinylbenzene; AN – acrylonitrile; BPO – benzoyl peroxide; CDD – 1-chlorododecane; PVA – polyvinyl alcohol. Resveratrol in Medicinal Chemistry Current Medicinal Chemistry, 2012 Vol. 19, No. 11 1671 changes (i.e., increase in size due to aggregation) in the long term significant changes in cell morphology, metabolic activity or cell stability study, thus providing evidence of the stability of the cycle [281]. Furthermore, SLN have been reported as excellent prepared formulations. Transmission electron microscopy (TEM) delivery systems for resveratrol being able to carry this bioactive analysis showed small, spherical and oligolamellar vesicles [254, compound until the nuclear target site. Indeed, on the one hand, 264, 272]. resveratrol has multiple actions in cell environment, especially Resveratrol was incorporated into liposomes at a percentage around the nuclear membrane and, on the other hand, SLN suffer around 70%, showing that the liposomal formulations developed intracellular trafficking [282]. Therefore, it is possible to conclude present good encapsulation capability. The amount and the that SLN can be used as carrier systems to enhance the intracellular chemical stability of resveratrol in liposomes were also investigated delivery of resveratrol. by high precision liquid chromatography (HPLC) over a 60-day 5.1.4. Cyclodextrins Complexes period at 4 ⁰C and no changes were detected, indicating absence of Cyclodextrins (CDs), contain a hydrophobic inner cavity and a trans/cis isomerization [254]. hydrophilic outer surface, being capable of interacting with a large 5.1.2. Polymeric Nanoparticles variety of molecules to form non-covalent inclusion complexes Nanoparticles are an ideal way to deliver drugs because of their [283]. Chemically, CDs are cyclic oligosaccharides with six, seven high loading efficiency and superior ability to penetrate cell or eight glucose residues linked by glycosidic bonds in a cylinder- membranes. Among the biodegradable nanoparticles used as drug shaped structure [284]. CDs and their derivatives have received carriers, amphiphilic block copolymers (e.g. methoxy poly(ethylene considerable attention in pharmaceutical applications and have been glycol)-poly(caprolactone) (mPEG–PCL)), composed of a used as complexing agents to increase the water solubility, stability hydrophilic segment and a hydrophobic segment, are capable to and bioavailability of various compounds, such as drugs, vitamins load lipophilic drugs by self-assembling into nanospherical and food additives [285-287]. structures with a hydrophilic outer shell and a hydrophobic inner The type of CD can influence the formation and the core [274, 275]. Accordingly, being resveratrol a lipophilic performance of drug:CD complexation [288, 289]. For instance, the compound it is possible to develop resveratrol-loaded nanocarriers cavity size of CD should be suitable to accommodate a drug based on polymeric nanoparticles. Additionally, these drug-loaded molecule of a particular size [290, 291] and the charges of the drug polymeric nanoparticles are not bigger than 100 nm, thus being and CD should be opposite to enhance the complexation affinity easily internalized by cells [248]. In recent works, nano- [292, 293]. The degree of substitution of CDs also plays an precipitation methods were used to obtain mPEG–PCL resveratrol- important role in balancing the CD water solubility and its loaded nanoparticles [252, 276]. The morphology of the complexing ability [294]. nanoparticles was characterized by TEM and atomic force Recent studies have demonstrated that CDs can be used as microscopy (AFM) and both images show the spherical shape of resveratrol complexing agents, to increase total resveratrol the polymeric nanoparticles with a smooth surface [252, 276]. concentration in aqueous solution, while maintaining its biological Regarding the loading efficiency, it depends mainly on the activities [260, 284]. For example, CD-based nanosponges have lipophilicity of resveratrol and its affinity to the hydrophobic core been obtained by cross-linking different types of CDs with (PCL). The highest drug loading content of resveratrol into carbonyldiimidazole [260]. The resultant solid particles present polymeric nanoparticles was about 20% and the encapsulation spherical morphology and have a very high solubilization effect for efficiency was 90% [252, 276]. poorly soluble molecules [295, 296]. Moreover, these particles have The cellular uptake of the polymeric nanoparticles occurs by shown to increase solubility and stability of resveratrol entrapped in endocytosis, which enhances the drug penetration in cells and the matrix together with a good resveratrol encapsulation [260]. demonstrates higher therapeutic efficiency than the obtained with The size of the particles was analyzed by dynamic light scattering free drug administration. (DLS) and is around 400 to 500 nm with a unimodal distribution. 5.1.3. Solid Lipid Nanoparticles TEM results were in agreement with DLS studies revealing an average diameter of 400 nm and a regular spherical shape for the Solid lipid nanoparticles (SLN) are composed of solid lipids complexes. Scanning electron microscopy (SEM) studies have and surfactants and can be used as carriers for resveratrol because shown highly porous structured complexes [260]. It was also seen a of their size, hydrophobic core with hydrophilic surface, delay in the resveratrol oxidation and photodegradation while this biocompatibility, and also because drug loaded SLN protect drugs bioactive compound was strongly entrapped in the internal cavity of from hydrolysis and thus increase drug stability [249, 277]. cyclodextrins [284]. Therefore, CDs can improve the stability and Resveratrol loaded-SLN were studied regarding the release the shelf life of resveratrol by protecting it against several profile of resveratrol from SLN and also regarding the cellular degradation processes [286]. uptake of the nanoparticles [253]. The controlled release profile of 5.1.5. Yeast Cell Microencapsulation resveratrol-loaded SLN presents two phases. The first phase corresponds to an initial burst release of 40 % of resveratrol that is The eukaryotic structure and the natural properties of yeast cell associated with the particle shell. This phase is followed by a wall have made it a potential encapsulating material with prolonged release of the remainder resveratrol that is located in the advantages over other microencapsulation technologies [246, 297]. lipid matrix [253]. Moreover, baker’s yeast (S. cerevisiae) has emerged as a convenient host for the development of a new kind of drug delivery The efficiency of the SLN cellular uptake depends on the system [298, 299]. Some studies have also suggested that the surface properties of these nanocarriers [278]. Several molecular membrane bilayer of yeast cell may act as a liposome during the interactions are expected when SLN encounters the microencapsulation of compounds [246, 297]. and these interactions may be dependent on several factors such as the biological membrane organization and the existence of lipid One of the purposes of microencapsulation is to improve the rafts [279], as well as the formation of actin cytoskeleton stability of active compounds. Taking into account the easily invaginations for the mediated entrance [280]. oxidizable and extremely photosensitive properties of resveratrol and the hygroscopic characteristic of yeast cells, it has been SLN may also be considered as a physically stable and well investigated the storage and protective capacity of this later system. tolerated nanocarrier system. Indeed, with sizes below 180 nm, It is possible that the increased water solubility of yeast SLN are able to pass rapidly through membranes causing no encapsulated resveratrol is due to the interactions established 1672 Current Medicinal Chemistry, 2012 Vol. 19, No. 11 Neves et al. between this bioactive compound and the yeast protein and/or 6. INTERACTION OF RESVERATROL WITH MEMBRANE polysaccharide. Thus, the bioavailability of resveratrol can be MODEL SYSTEMS enhanced by yeast cell encapsulation processing due to the increased water solubility obtained with this microencapsulation Transmembrane proteins, such as: ATPases [308], voltage- system [261]. gated potassium channels [309], PKC [310] and ABC transporters (e.g. P-glycoprotein (PgP), multidrug resistance-associated protein The structure of the yeast cell wall is permeable to both small 1 (MRP1) or breast cancer resistance protein (BCRP) [311-315]) polar and nonpolar molecules with a molecular radius smaller than were identified among the cellular targets of resveratrol. Despite of 0.81 nm or molecular weight lower than 620 Da [300, 301]. these membrane targets being implicated in resveratrol biological Accordingly, the encapsulation of resveratrol (molecular weight of effects; only few studies dealing with the effect exerted by 228 Da) into yeast cells is expected to be possible. resveratrol on model membranes were published [310, 316-319]. The loading process of resveratrol into the yeast cell particles The generally accepted model of the cell membrane is a liquid- occurs by passive diffusion [297] driven by the concentration ordered lipid phase, with dispersed, smaller liquid-disordered discrepancy found between the external medium and the internal domains where most proteins reside. Domain formation is a yeast cellular medium. This loading process is further enhanced by dynamic process, as some components transiently accumulate in a the hydrophobic interactions and hydrogen bonds established particular area. The weakness of lipid–lipid interactions leads to a between the -OH group of resveratrol and the -NH2, -OH and - mosaic of dynamic and reversible lipid domains in the bilayer of COOH groups from the polar headgroups of the membrane biological membranes [279]. Given the fact that lipids play at least phospholipids. Shi et al. [261] have shown an experimental two roles in cellular regulation – a structural role as the building encapsulation yield of ≈ 4.5 % of resveratrol, indicating that this blocks of membranes and a role in cellular signaling – it was bioactive compound was successfully incorporated into the yeast suggested that phospholipid turnover in cells can result in the cells. In addition, HPLC retention time and UV-Vis spectrum activation of multiple pathways and that phospholipid metabolism revealed that no chemical changes have taken place during plays a crucial role as signal transducers. In this context, resveratrol resveratrol encapsulation process. may also interfere in cellular regulation given that this compound In addition, the in vitro releasing profile of resveratrol from the has been described as potentially able to perturb membrane yeast cells has been investigated to show the potential of these phospholipids possibly impairing or altering their structural and systems as effective control drug delivery carriers. In this context, it signaling role. In fact, Sarpietro et al. [318] studied the partition was reported that the outer protein layer and the plasma membrane behavior of resveratrol between liposomes of dimyristoylphos- of the yeast cells are responsible for establishing a permeability phatidylcholine (DMPC) and water and observed that 90 % of barrier to the permeating molecule [297, 300]. This permeability resveratrol was present in the lipid phase. Fabris et al. [316] also barrier can be destroyed by acid [302], making possible the release showed that the percentage of membrane-bound resveratrol was 93 of the encapsulated compound (e.g. resveratrol). Therefore it was %, 97 % and 98 % in membranes of DMPC, dipalmitoylphosphati- described that 90 % of resveratrol is released within 90 min in dylcholine (DPPC) and distearoylphosphatidylcholine (DSPC), simulated gastric fluid [261] favoring its bioavailability. respectively. Garcia-Garcia et al. [310] have also carried out some 5.1.6. Pectinate Beads studies in membrane models made of mixtures of palmitoyl- oleoylphosphatidylcholine and palmitoyl-oleoylphosphatidylserine Among polysaccharide polymers, pectins exhibit numerous (POPC/POPS) at a 4:1 lipid:resveratrol molar ratio and the results advantages such as their low cost, biodegradability, wide variety of revealed that resveratrol is able to be incorporated into the types, and flexibility in use. Furthermore, pectins are resistant to phospholipid membrane in a location that is inaccessible to the gastric and intestinal enzymes, but can be almost completely hydrophilic fluorescence quencher, acrylamide. In this work, the degraded by the colonic bacterial enzymes, which makes them authors took advantage of the fluorescence properties of resveratrol suitable carriers for colon-targeted delivery [303]. The low and studied the effect of acrylamide, which is able to quench solubility of these polymers has led to the development of pectinate fluorescence of compounds that are located in the aqueous external beads that can be prepared according to the pectin type or the media of the membrane, while not being able to reduce their presence of additives. For example, the degree of methoxylation fluorescence if these compounds are embedded in the membrane can be altered to tune the physicochemical properties of pectins. [320]. Therefore, this study suggests that resveratrol incorporates Indeed, highly methoxylated pectins are poorly soluble, whereas into membranes and that the phenolic rings, which act as pectins with low degree of methoxylation are more soluble. Pectins fluorophores, are not accessible to acrylamide. These evidences are can also be cross-linked with divalent cations (e.g., calcium, or in agreement with other experiment using DPPC liposomes as zinc) to increase their solubility. Ionic interactions between the membrane models and the fluorescence probes 6-AS, 9-AS (buried negatively charged carboxylic groups of pectin molecules and the in the core of membrane) and 1,8-ANS (located at the surface of the positively charged divalent cations lead to intermolecular cross- membrane). This study indicates that resveratrol is deeply located linking (known as ‘‘egg-box’’ conformation [304]) and formation in the membrane core, with no significant effect at the membrane of gelled spheres. Hence, when pectin is cross-linked with calcium, surface [321]. In contrast, another study in membrane models of it forms a multiparticulate Ca-petinate bead system which can be a DMPC and DPPC labeled with fluorescence probes revealed that viable carrier for site-specific resveratrol delivery to the colon [305- resveratrol interacts preferably with the headgroup region of 307]. Zn-pectinate beads are also used, exhibiting a prolonged drug membrane where Prodan fluorophore is located, and to a lesser release profile, better than the obtained when Ca-pectinate beads extent with the regions at a certain depth in the bilayer where are used as carrier systems [257-259]. Laurdan is incorporated [319]. The observations that resveratrol Das et al. have developed spherical resveratrol loaded Ca- could affect the bilayer at different depths point to the relatively pectinate beads [251, 255, 256] and Zn-pectinate beads [257-259] high mobility of this bioactive compound inside the membrane; with a mean diameter of 1 mm for dried beads, and above 2 mm for however, more studies are needed to confirm this ubiquitous the undried beads. Resveratrol has shown to be effectively membrane location of resveratrol. entrapped and has also presented a prolonged release profile due to Once incorporated into the membrane, resveratrol considerably the increased stability conferred by the optimized beads. In fact, affects the gel to liquid-crystalline phase transition of multilamellar even after 6 months of storage, the stability was still around 99 % in vesicles made of POPC/POPS with a decrease of the main phase the case of storage at room temperature or 4 °C and > 90 % when transition temperature from 4 °C in the pure phospholipid to 1 °C in the formulations where stored at 40 °C. Resveratrol in Medicinal Chemistry Current Medicinal Chemistry, 2012 Vol. 19, No. 11 1673 the presence of 120 µM of resveratrol and to -8 °C when a sphingomyelin and cholesterol. However, membrane model concentration of 300 µM of resveratrol is reached [310]. The systems with a single phospholipid component represent an observed decrease of the main phase transition temperature important first step to understand the biophysical effects of suggests that the membrane fluidity is increased by the presence of resveratrol. Nevertheless, further studies with more complex resveratrol, which preferably partitions into the fluid domains [321, membrane model systems are needed to fully elucidate the details 322]. Sarpietro et al. [318] and Wesolowska et al. [319] have also of the interaction of resveratrol and biomembranes and to correlate shown that resveratrol interacts with DMPC membranes causing the the results obtained with the biological effects of resveratrol. disappearance of the lipid pretransition, the reduction of the main phase transition temperature and the decrease of the transition 7. CONCLUDING REMARKS cooperativity. Fabris et al. [316] have demonstrated that in DSPC model system resveratrol influences the mobility of the stearic acid Resveratrol has emerged as one of the most promising naturally molecules while Tsuchiya et al. [317] reported a rigidifying effect occurring compound with a great therapeutic potential, due to its of resveratrol on the inner membrane regions. evident value as a cancer preventive, cardioprotective, antioxidant, anti-inflammatory and neuroprotective dietary substance. Studies Furthermore, it has long been known that a molecule that is are being conducted to determine the preventive and therapeutic inserted in a membrane may affect the phospholipid phase efficacy of dietary or supplemented resveratrol on tumor polymorphism, e.g. by hindering or promoting the transitions from development and progression, as well as, in the prevention of lamellar to non lamellar phases. Since, nonbilayer lipid phases, coronary disease and neurodegenerative pathologies. In fact, such as the inverted hexagonal HII phase [323], have been resveratrol and its analogs present pharmacological safety and may associated to a great variety of biological processes [324], the effect be used in combination with other agents to enhance therapeutic of resveratrol in lipid polymorphism may also be considered as an efficacy and minimize toxicity. additional explanation for its biological activity. Unfortunately, the pharmacokinetic properties of resveratrol are Compounds may interact with membranes changing the not as favorable as its beneficial pharmacological activities. Several packing parameters of the phospholipids in a way that the studies suggest that, in general, trans-resveratrol is rapidly hydrophobic tails present a greater volume than the hydrophilic absorbed, metabolized and excreted in humans and animals, headgroups. Consequently, upon interaction with compounds, indicating a poor bioavailability of resveratrol that compromises its phospholipids may change their geometry from a cylinder shape to biological effects. The question of whether resveratrol can a conical shape that will tend to destabilize membrane lamellar accumulate up to bioactive levels in target organs remains to be phases, eventually leading to the formation of a nonlamellar addressed. Several studies have tried to answer this question with inverted hexagonal (HII) phase [325, 326]. Other compounds are opposing results. To overcome this problem, resveratrol carriers able to interact with the membrane occupying the free spaces and site-specific delivery systems have been developed to protect between the headgroups and promoting a cylinder-like shape that and stabilize resveratrol and to enhance its bioavailability, stabilizes the lamellar phase. Resveratrol seems to produce a similar preserving its biological and pharmacological activities. Despite the effect of stabilization of lamellar phases, since it occupies a large progress made in this field, the development of size-tuned carrier space at the polar part of the membrane near the headgroup region systems with optimized lipophilicity is still required for the delivery as proposed by Fabris et al. [316] and Wesolowska et al. [319]. In of resveratrol to more difficult targets, such as brain. fact, differential scanning calorimetry (DSC) and 31P-nuclear magnetic resonance (NMR) studies revealed that resveratrol Concerning the molecular mechanisms of resveratrol interacts with multilamellar vesicles of dielaidoyl phosphatidyl- bioactivity, only few studies were published about its interaction ethanolamine (DEPE) and increases the temperature (TH) at which with the lipid membranes. Additionally, several aspects of the the lamellar phase suffers transition to the non lamellar inverted interplay between resveratrol, membranes and enzymes remain hexagonal phase (HII) [310]. elusive. The reported studies indicate that resveratrol is able to be incorporated into lipid membrane models penetrating the It is known that the tendency to form nonlamellar lipid hydrophobic core of bilayers, but its exact location needs to be structures may be associated with increasing activity of membrane- determined by further investigations. Additionally, resveratrol has dependent enzymes such as phospholipase A [327] and PKC [328], 2 been shown to modulate the membrane biophysical properties by which is measured by assessing the decrease of T in phospholipids H decreasing enthalpy and by changing phase transition temperature such as DEPE and palmitoyl-oleoylphosphatidylethanolamine (POPE) [329-331]. Accordingly, the capacity of resveratrol to influencing membrane fluidity and affecting the lipid polymorphism. The effects of resveratrol in the membrane increase TH and stabilize the lamellar phase may be related to its effect as an inhibitor of the abovementioned enzymes, and biophysical properties are of great importance when trying to ultimately may constitute an additional explanation to its anti- explain the different biological activities described for this inflammatory effect. Indeed, it was confirmed that resveratrol compound. Indeed, the biophysical changes induced in membranes inhibited PKCα when activated by POPC/POPS vesicles with an indirectly affect the activity of membrane proteins or interfacial IC of 30 µM, whereas when the enzyme was activated by Triton enzymes. Hence, further biophysical interaction studies of 50 resveratrol with model membranes could provide a rational X-100 micelles the IC50 was 300 µM [310]. These results indicate that resveratrol is a better inhibitor of PKCα in the presence of a approach to a better understanding of the overall mechanism of lipid bilayer, probably because this bioactive compound is able to action that is behind the pharmacological and therapeutic activities stabilize the lipid lamellar phase thus reducing the interfacial of this compound. activation of the enzyme. Therefore, further studies would be needed to fully understand the enzymatic inhibition promoted by ACKNOWLEDGEMENTS resveratrol that might be due not only to a direct interaction The authors acknowledge the FCT (Fundação para a Ciência e a between the enzyme and the compound, but can also result from the Tecnologia) for financial support through the Ph.D. grant biophysical effects of resveratrol affecting the interfacial lipid (SFRH/BD/73379/2010) conceded to ARN. environment that is required for the enzyme activation. The in vitro membrane interaction studies are usually criticized ABBREVIATIONS for being performed in lipid systems with a simplified composition in comparison to the real biological membranes that contain AMPK = 5’-AMP-activated protein kinase different types of phospholipids, as well as, significant amounts of AN = acrylonitrile 1674 Current Medicinal Chemistry, 2012 Vol. 19, No. 11 Neves et al.

AP-1 = activator protein-1 PVA = polyvinyl alcohol AD = Alzheimer’s disease PKC = protein kinase C AFM = atomic force microscopy ROS = reactive oxygen species BPO = benzoyl peroxide RR = ribonucleotide reductase BCRP = breast cancer resistance protein SEM = scanning electron microscopy CDD = 1-chlorododecane SLN = solid lipid nanoparticles CHOL = cholesterol STS = stilbene synthase CoA = coenzyme A SULTs = sulfotransferases CDs = cyclodextrins SOD = superoxide dismutase

COX = cyclooxygenase TH = transition temperature from lamellar phase to the DCP = dicetyl phosphate non lamellar inverted hexagonal phase DEPE = dielaidoyl phosphatidylethanolamine TGF-β = transforming growth factor-β DSC = differential scanning calorimetry TEM = transmission electron microscopy DMPC = dimyristoylphosphatidylcholine TNF-α = -α DPPC = dipalmitoylphosphatidylcholine UGTs = uridine 5’-diphospho-glucoronosyltransferases DSPC = distearoylphosphatidylcholine SIR2 = yeast silent information regulator gene

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Received: September 05, 2011 Revised: January 03, 2012 Accepted: January 04, 2012