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Current Medicinal Chemistry, 2013, 20, ????-???? 1

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From Resveratrol to Its Derivatives: New Sources of Natural Antioxidant

Shan He*,1 and Xiaojun Yan*,2

1School of Marine Sciences, Ningbo University, Ningbo 315211, China;

2Key Laboratory of Applied Marine Biotechnology (Ningbo

University), Ministry of Education, Ningbo 315211, China

Abstract: Resveratrol, a star natural product from red wine, has attracted increasing attention around the world. In recent years, resvera-trol derivatives (including its oligomers) have shown amazing chemical diversity and biological activities. They have been emerging to be promising new sources of natural antioxidant. This review summarizes recent finding on antioxidant activities of resveratrol deriva-tives and the structure-activity relationship for the first time. Scientific evidences have highlighted their potential as therapeutic agents for cerebral and cardiovascular diseases. In our opinion, more effort should be devoted to the synthesis of resveratrol oligomers. Based on the structure-activity relationship, screening for resveratrol derivatives with higher antioxidant activity than trans-resveratrol is war-ranted, and these molecules may have greater therapeutic potential in future investigations.

Keywords: Antioxidant, Biological Activity, Chemical Diversity, Derivative, Resveratrol, Structure-Activity Relationship

1. INTRODUCTION

Reactive oxygen species (ROS), including superoxide anion (O2

), hydroxyl radical (OH), peroxyl radicals (ROO), and sin-glet oxygen (1O2), are highly reactive molecules generated during cellular respiration and normal metabolism, which play a dual role as both deleterious and beneficial species [1]. Beneficial effects of ROS occur at low/moderate concentrations and involve physiologi-cal roles in cellular responses to noxia, in the function of a number of cellular signaling pathways, and the induction of a mitogenic response [2]. However, overproduction of ROS results in oxidative stress (OS), a state of imbalance between ROS production, and the ability of cells endogenous antioxidants to defend against them, leading to progressive oxidative damage to cell structures, including lipids and membranes, proteins, and nucleic acids [3]. Therefore, ROS have been implicated as being important causative agents of aging and various human diseases, such as stroke, cancer, heart diseases, multiple sclerosis, Parkinson's disease, and autoimmune disease [4]. For example, in the past 20 years, the study of ROS dependent damage to DNA has become a major thrust of carcino-genesis research. ROS are able to attack the bases or the deoxyribo-syl backbone of DNA, or attack other cellular components such as lipids to generate reactive intermediates that couple to DNA bases. The resulted endogenous DNA lesions are genotoxic and induce mutations that can contribute to the development of cancer [5].

In the normal physiological state, ROS are regulated by cellular endogenous antioxidants both enzymatically and non-enzymatically, which constitute a complex and efficient antioxida-tive defense system. Enzymatic antioxidants include superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), while non-enzymatic antioxidants are represented by glutathione (GSH), ascorbic acid (Vitamin C), -tocopherol (Vitamin E), caro-tenoids, flavonoids, and other antioxidants. Under normal condi-tions, there is a balance between ROS and the intracellular levels of these antioxidants, which is essential for the survival of living or-ganisms and their health [6]. However, under OS the impaired anti-oxidative defense system is unable to control the level of ROS, and demand exogenous supplement of antioxidant to scavenge exces-sive ROS to restore the original state of cellular redox homeosta-sis [7]. Therefore, antioxidants that effectively scavenge these ROS are potential preventive or therapeutic agents against ROS-mediated diseases.

Resveratrol (3,5,4-trihydroxystilbene; Fig. (1)) was first iso-lated from the roots of white hellebore (Veratrum grandiflorum

*Address correspondence to these authors at the 818 Fenghua Road, Ningbo Univer-sity, Caoguangbiao Sci&Tech Hall, Ningbo 315211, China; Tel: +86 574 87600458; Fax: +86 574 87600570; E-mail: [email protected] and Ningbo University, Post Box 71, Ningbo 315211, China; Tel: +86 574 87600738; Fax: +86 574 87600590; E-mail: [email protected]

Loes. fil.) in 1940 [8]. Now most of the commercial resveratrol products are isolated and purified from a traditional Chinese and Japanese medicine, the roots of Polygonum cuspidatum [9]. Initially characterized as a phytoalexin of grapevines (Vitis vinifera) [10], resveratrol attracted little interest until 1992, when it was linked to the low incidence of heart diseases in some regions of Francethe so-called French paradox, that is, despite a high fat intake, mor-tality from coronary heart disease is lower due to the regular con-sumption of red wine [11]. In 1997, a seminal paper reporting the cancer chemopreventive activity of resveratrol [12] has triggered considerable attention on this natural polyphenol. The past 15 years have witnessed intense research devoted to the biological activities, especially the antioxidant activity, of this star natural product [13], which has become a dietary supplement and a candidate for drug development, and its biological activities have been extensively reviewed [14]. Its potent antioxidant activity is empowered by its unique structure. It has recently become clear that the three phenol groups with remarkable H-transfer capacity [15] and the tran-sisomery of the double bond [16] are responsible for its antioxidant activity (Fig. (2)).

Since ROS play an important role in carcinogenesis, antioxi-dant activities of 700 plant extracts were assessed by J. M. Pez-zuto's Lab in 1990s, to discover and characterize natural antioxi-dants with cancer chemopreventive activity. Bio-assay guided isola-tion of the 28 plant extracts found active in the primary screening resulted in the characterization of many potent antioxidants. Among them, resveratrol and its derivative piceatannol showed the highest cancer chemopreventive activities determined by a 7,12-dimethylbenz[a]anthrancene (DMBA)-induced preneoplastic lesion formation in mammary gland organ culture model [17]. This report indicated that resveratrol derivatives may offer comparable or even stronger biological activities. Other investigation also demonstrated that some derivatives exhibited higher antioxidant activities than resveratrol [18]. In recent years, research interests are shifting from resveratrol to its derivatives (including its oligomers), which are emerging to be promising new sources of natural antioxidant. This review summarizes findings in the past 15 years, which docu-mented the discovery of resveratrol derivatives as new antioxidants and their therapeutic applications, with special attention to the structure-activity relationship (SAR).

2. CHEMICAL DIVERSITY

Resveratrol and its derivatives belong to a group of plant poly-phenolic compounds stilbene, which are distributed in particular families of plants including Vitaceae, Dipterocarpaceae, Gnetaceae, Cyperaceae, and Leguminosae [19]. The stilbene nucleus is based on a 14-carbon skeleton composed of two phenyl rings linked by an ethylene bridge. Stilbenes are derived from the general phenylpro-panoid pathway, starting from phenylalanine. The biosynthesis of

wasimFinal

2 Current Medicinal Chemistry, 2013, Vol. 20, No. 4 He and Yan

HO

R5

R1 R2

R3

R4

R1 R2 R3 R4 R5

1 resveratrol H H OH H OH

2 isorhapontigenin H OMe OH H OH

3 oxyresveratrol OH H OH H OH

4 piceatanol H OH OH H OH

5 rhapontigenin H OH OMe H OH

6 gnetol OH H H OH OH

7 piceid H H OH H OGlu

8 pinosylvin H H H H OH

9 astringin H OH OH H OGlu

10 rhapontin H OH OMe H OGlu

A

B

Fig. (1). Chemical structures of resveratrol and its natural monomeric derivatives. resveratrol in plant is catalyzed by stilbene synthase via a single reaction. Stilbene synthase uses three malonyl-CoA and one p-coumaroyl-CoA as substrates and synthesize a linear tetraketide intermediate, which is then cyclized via an aldol condensation, followed by an additional decarboxylation to afford resveratrol (Fig. (3)) [20]. Then further modifications, including glycosylation [21], methylation [22], oligomerization [23], isomerization [24], and isoprenylation [25], generate various resveratrol derivatives with intriguing chemical diversity (Fig. (4)).

OH

HO

HO

A

B

C

Fig. (2). The meta-hydroxyl groups (A), the para-hydroxyl group (C) and transisomery of the double bond (B) are essential for the potent antioxidant activities of resveratrol against different ROS.

There have been several reviews concerning the chemical di-versity of natural stilbenes [26]. From 1995 to 2008, about 400 new naturally occurring stilbenes have been isolated and characterized [26d]. To the best of our knowledge, there are at least 500 natural stilbenes reported, most of which are resveratrol oligomers. The chemical diversity of resveratrol oligomers mainly lies in the fol-lowing aspects:

(1) Degree of polymerization (DP). DP is the number of units of resveratrol monomers forming an oligomer, which could range

from 2 to 8. Representative structures of oligomers with differ-ent DPs are shown in Fig. (5). Dimers, trimers and tetramers constitute the major members of resveratrol oligomers. Highly condensed stilbene oligomer (HCSO), which was composed of more than five stilbene monomers, is rare. Hitherto, there have only been six HCSOs discovered from the plant kingdom, namely vaticanol D [27], vaticanols H-J [28], vateriaphenol A [29], and chunganenol [30]. All of them are resveratrol oli-gomers from Dipterocarpaceous plants, with exception of chun-ganenol (Fig. (5)), which was the first resveratrol hexamer from Vitaceae family [30]. Vateriaphenol A (Fig. (5)), a resveratrol octamer from Vateria indica has the highest DP ever reported.

(2) Variety of skeleton. Resveratrol oligomers are produced by oxidative coupling between resveratrol monomers via different patterns, thus generating distinct skeletons. Ten patterns have been reported when two monomeric units linked by only one CC or COC bond (with two linkage points). Another ten patterns have been found, when two monomeric units linked by two CC or COC bonds (with four linkage points), com-monly forming a ring. Seven patterns have been observed, when two monomeric units linked by three CC or COC bonds (with six linkage points), usually forming two rings. And there are only two patterns discovered, when two monomeric units linked by four CC or COC bonds (with eight linkage points) [26d]. In sum, there are at least 29 different patterns, leading to the formation of dozens of skeletons. Some examples with different linkage points are shown in Fig. (6). Readers are encouraged to consult the recent review by Dr. H. X. Lous lab for further details [26d]. It is worth noting that chunganenol,

H2N

OH

O

phenylalanine

PALOH

O

cinnamic acid

C4HOH

O

HO4CL

S-CoA

O

HO

p-coumaric acid p-coumaroyl-CoA

3 malonyl-CoASTS

OH

HO

HO

trans-resveratrol

CoA-S

O O O O

OH

tetraketide intermediate

STS

aldol condensation

Fig. (3). Biosynthesis of resveratrol. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, hydroxycinnamoyl CoA ligases; STS, stilbene synthase.

From Resveratrol to Its Derivatives Current Medicinal Chemistry, 2013, Vol. 20, No. 4 3

OH

HO

HO

trans-resveratrol

glycosylation

OH

HO

GluO

trans-piceid

methylation

pterostilbene

OH

MeO

MeO

oligomerization

trans--viniferinOH

OHO

HO

HO

OH

isomerization

isoprenylation

OH

HO

HO

arachidin-3

OH

HOHO

cis-resveratrol

Fig. (4). Common modifications of resveratrol.

which was discovered in our lab, features a unique skeleton interunit lingkage where two stilbene units are connected by a methylene bridge (Fig. (5)) [30].

(3) Complex stereochemistry. The planar structures of monomeric resveratrol derivatives do not possess any chiral carbon. When two monomeric units couple to form an oligomer, opening of the ethylene bridge could generate asymmetry. For example, catalyzed by horseradish peroxidase (HRP), dimerization of resveratrol yields the racemic mixture of resveratrol-trans-dehydrodimer (Fig. (7)) [31]. As the largest resveratrol oli-gomeric molecule, the octamer vateriaphenol A have 16 chiral carbons. In most cases, hydrogens from the same ethylene bridge located as trans configuration in oligomers. All the natu-ral resveratrol oligomers with chiral centers are optically active, indicating that biosyntheses of them in plants are enantioselec-tive (formation of dimer) or diastereoselective. Some of these selectivities are family specific. For instance, (+)--viniferin (Fig. (4)), a resveratrol dimer, and (+)-hopeaphenol, a resvera-trol tetramer, are isolated from Vitaceaeous plants, however their enantiomers are from other families such as Dipterocar-paceae and Gnetaceae. Based on the biomimetic transformation from (+)--viniferin to many other oligomers, Takaya et al. fur-ther proposed that the stereochemistry of resveratrol oligomers from Vitaceaeous plants may be originated from the stereo-chemistry of (+)--viniferin [32]. The amazing chemical diversity of resveratrol derivatives

shows again the great imagination of nature. Synthesis of mono-meric resveratrol derivatives has been a hot topic in both organic and medicinal chemistry. A number of catalytic methods have been

developed to generate chemical diversity synthetically (for review, see [33]). Even though considerable achievement has been made in recent years [34], the synthesis of resveratrol oligomers remains a great challenge due to their chemical complexity as discussed above. Advance in organic chemistry to provide a general and facile approach is still highly needed at the following points: (1) Enanti-oselective or diastereoselective oligomerization. (2) Regioselective coupling at specific location. (3) Regioselectivity in forming spe-cific coupling pattern.

3. ANTIOXIDANT ACTIVITY

3.1. DPPH (1,1-Diphenyl-2-Picrylhydrazyl) Radical

DPPH is a stable free radical, which has been widely used for the evaluation of antioxidant activities of natural products in the micromolar range [35]. The DPPH assay is simple and rapid, thus highly suitable for in vitro screening for antioxidants [36]. Resvera-trol and its derivatives with DPPH scavenging activities are summa-rized in (Table 1). Apparently, resveratrol is not the best DPPH scavenger. Many derivatives showed comparable or stronger DPPH scavenging activities than resveratrol. They are astringin, as-tringinin, piceatannol, scirpusin A, quadrangularin A, parthenocis-sin A, laetevirenol A, laetevirenol B, chunganenol, -viniferin, and vitisin A. Among them, laetevirenol A, laetevirenol B, and chun-ganenol with novel structures were discovered in our lab [37]. There seems to be a relationship between DP and DPPH scavenging activity, and the average activities of different DPs follow the order: monomer dimer > tetramer > trimer [38]. It is also observed that the antioxidant activity of the same compound could vary signifi-cantly under different assay conditions. Normally, lower DPPH

4 Current Medicinal Chemistry, 2013, Vol. 20, No. 4 He and Yan

Parthenocissin A(Dimer)

Parthenocissin B(Trimer)

OH

HO

OH

OH

OH

O

HO

OH

HO

Kobophenol A(Tetramer)

Amurensin E (Pentamer)

Vaticanol J(Heptamer)

Vateriaphenol A(Octamer)

OH

OH

OH

HO

HO

OH

H

HO

HO

OH

O

HO

OH

O

OH

HO

O

OH

OH

HO

O

HO

O

OH

OH

OH

OH

O

HO HO

OH

OH

H

OH OH

HOO

H

HO

HO HO

OH

OH

H

HO

HO

HO

O

O

HO

HO

OH

OH

HO

OH

HO

Chunganenol(Hexamer)

O

HO

HO

HO

HO

HO

HOO

H

H

OH

HO

HO

OH

H

HOOH OH

OHH

HO

OH

OH

HO

OH

O

HO

HO

HO

HO

H

H

OH

O

OH

OHHOHO

HH

HO

HO

OH

O

HO

HO

HOH

HO

H

OH

O

OH

HO

OH

Fig. (5). Chemical structures of typical resveratrol oligomers with different DPs.

From Resveratrol to Its Derivatives Current Medicinal Chemistry, 2013, Vol. 20, No. 4 5

HO OH

HO

OH

OH

OH

OH

Amurensin A(Two linkage points)

MeO H OH

OH

OH

OH

HO

OH

Parthenostilbenin A(Four linkage points)

H H

OHHO

OH

HOOH

HO

Pallidol(Six linkage points)

O

O

HO

OH

HO

OH

Betulifol A(Eight linkage points)

H

H

H

H

Fig. (6). Chemical structures of representative resveratrol oligomers with different linkage points.

OH

HO

HO

horseradish peroxidase

H2O2

O

HO

HO

OH

OH

OH

+

O

HO

HO

OH

OH

OH

Fig. (7). Dimerization of resveratrol to give resveratrol-trans-dehydrodimer.

concentration, longer reaction time and higher temperature would decrease IC50 value. We strongly recommend that future investiga-tion of resveratrol derivatives should include trans-resveratrol as positive control, so that data from different reports could be com-pared.

3. 2. Hydroxyl Radical

The hydroxyl radical has a high reactivity, making it a very dangerous radical with a very short half-life of approximately 109 s in vivo [43]. Resveratrol has been reported to scavenge hydroxyl radical as determined by electron paramagnetic resonance (EPR) spin-trapping technique [13d], which is the most accurate and reli-able method for measuring scavenging effect against ROS. Leonard et al. used the Fenton reaction (Fe2+H2O2Fe3+OHOH) as a source of hydroxyl radical, which was trapped by DMPO to generate DMPO-OH adducts, a much more stable free radical de-tectable by EPR. Resveratrol was found to scavenge hydroxyl radi-cal in a dose dependant manner with reaction rate constant calcu-lated as 9.45108 M-1s-1 [13d]. However, as we look into the data presented in Ref 13d, addition of 1.3 mM resveratrol only caused 40% inhibition of hydroxyl radical signals in EPR spectrum. This result has been confirmed in our recent investigation [44]. We have screened many resveratrol derivatives for hydroxyl radical scaven-ger by EPR spin-trapping technique, however, none of them showed potent scavenging effect (unpublished data).

3.3. Superoxide Anion Radical

Molecular oxygen has a unique electronic configuration and is itself a radical. The addition of one electron to molecular oxygen forms the superoxide anion radical (O2

) [1a]. Although it is less reactive than hydroxyl radical, O2

is considered the primary ROS, and can further interact with other molecules to generate much more toxic secondary ROS, such as H2O2,

OH, and ROO [45]. It has been implicated in the pathology of various diseases [46]. O2

is usually generated using a xanthine/xanthine oxidase system and measured spectrophotometrically or using ESR spin-trapping technique. Leonard et al. have reported the superoxide anion scavenging effect of resveratrol in a dose dependant manner [13d]. In 2000, vaticanol D, a novel resveratrol hexamer isolated from Vatica rassak, showed a scavenging activity of super oxide at IC50 = 7.4 M. It was also the first occurrence of resveratrol hex-

amer [27]. In 2002, trans--viniferin, the major dimer of resvera-trol, isolated from Vitis vinifera (grape), exhibited stronger superox-ide anion scavenging activity than resveratrol and some of its monomeric derivatives [47]. In 2003, resveratrol along with ten derivatives were isolated from Gnetum gnemon. Most of them showed potent scavenging activities toward O2

[48]. Two years later, Kim et al. reported superoxide anion scavenging activity of twelve stilbenes from Parthenocissus tricuspidata, where piceatan-nol exerted the best activity among the isolates [40]. The scaveng-ing activities of resveratrol and its derivatives against O2

are listed in (Table 2). It is worth noting that superoxide anion scaveng-ing effect can differ with the test used, and there are more determin-ing factors in the assay condition and measurement than the DPPH assay.

3.4. Peroxyl Radicals

Another ROS derived from oxygen is peroxyl radicals(ROO). They can initiate fatty acid peroxidation, which is detrimental to cell structure and function [49]. Therefore, it is very important to evaluate an antioxidants capacity to inhibit lipid peroxidation [50]. Antioxidant activities of resveratrol and its monomeric derivatives have been assessed by their capacity to prevent Fe2+-induced lipid peroxidation in microsomes or Cu2+-induced lipid peroxidation in low-density lipoproteins (LDL). Astringin and astringinin (also known as piceatannol) showed similar or even stronger effect than resveratrol in both assay system [18b]. This result has been confirmed in another investigation where twelve stilbenes were evaluated for their lipid peroxidation inhibitory activity in rat liver homogenates [40]. 4,4'-Dihydroxystilbene has been reported to exert 81.5% inhibition in a -carotene bleaching assay, which was comparable to that of -tocopherol (Vitamin E) tested in the same conditions [47]. Eight stilbenes derivatives from Gnetum gnemon showed better lipid peroxide inhibition than that of resveratrol [48]. In our previous report, the antioxidant activities of quadrangularin A and parthenocissin A, two isomeric resveratrol dimers, were markedly stronger than that of vitamin C, as determined by -carotene bleaching assay [51]. In addition, there are a handful syn-thetic resveratrol monomeric derivatives are effective antioxidants against lipid peroxidation. These results are discussed in the latter structure-activity relationship section. (Table 3) summarizes in-hibitory effects of naturally occurring resveratrol derivatives toward lipid peroxidantion.

6 Current Medicinal Chemistry, 2013, Vol. 20, No. 4 He and Yan

Table 1. DPPH Scavenging Activities of Resveratrol and Its Derivatives.

Compounds DPs Assay Condition IC50 Refs.

trans-Resveratrol 1 74.0 M cis-Resveratrol 1 97.0 M

trans-Piceid 1 200 M cis-Piceid 1 140 M Astringin 1 30.6 M

Astringinin 1

100 M DPPH Reaction time = 10 min Trolox IC50 = 10.1 M

29.0 M

[16b]

trans-Resveratrol 1 94.6 g/ml Piceatannol 1 68.4 g/ml Scirpusin A 2

300 M DPPH Reaction time = 10 min

Temperature = 37 oC 78.0 g/ml

[15]

,-Dihydrorhaponticin 1 8.98 g/ml

6-O-(7,8-Dihydrocaffeoyl)- ,-dihydrorhaponticin 1

40 g/ml DPPH Reaction time = 30 min

Temperature = 25 oC Ascorbic acid IC50 = 2.29 g/ml

9.04 g/ml [37]

Resveratrol 1 12.9 g/ml Piceatannol 1 7.42 g/ml

Pallidol 2 36.1 g/ml Parthenocissin A 2 43.9 g/ml

Piceid 1 44.2 g/ml Cyphostemmin B 2

100 M DPPH Reaction time = 30 min

Temperature = 37 oC Quercetin IC50 = 3.37 g/ml

34.9 g/ml

[38]

Resveratrol 1 71.9 M Quadrangularin A 2 66.9 M Parthenocissin A 2 57.9 M Laetevirenol A 2 38.4 M Laetevirenol B 3 37.3 M Laetevirenol C 3 110.8 M Laetevirenol D 3 128.0 M Laetevirenol E 3 158.2 M

Parthenocissin B 3

150 M DPPH Reaction time = 30 min

Temperature = 37 oC Vitamin E IC50 = 28.3 M

172.7 M

[35a]

Chunganenol 6 37.3 M Amurensin B 3 188 M

Gnetin H 3 251 M -Viniferin 2 62.2 M

[35b]

Amurensin G 3 138 M Vitisin A 4 42.4 M

Hopeaphenol 4 115 M Resveratrol 1

150 M DPPH Reaction time = 30 min

Temperature = 37 oC Vitamin E IC50 = 33.6 M

73.2 M

[39]

Wilsonol A 3 103.5 M Wilsonol B 3 195.4 M Wilsonol C 4 182.2 M

Diviniferin B 4 175.3 M Resveratrol 1 75.2 M

Pallidol 2 146.8 M -Viniferin 2 127.3 M

Ampelopsin B 2 194.7 M Ampelopsin D 2 96.9 M Miyabenol C 3 89.7 M

Dividol A 3 175.0 M Hopeaphenol 4 94.3 M

Gnetin H 3 184.5 M Heyneanol A 4

150 M DPPH Reaction time = 30 min

Temperature = 37 oC Vitamin C IC50 = 32.3 M

144.6 M

[40]

From Resveratrol to Its Derivatives Current Medicinal Chemistry, 2013, Vol. 20, No. 4 7

(Table 1) contd.

Compounds DPs Assay Condition IC50 Refs.

Ampelopsin G 3 149.3 M Amurensin G 3

277.2 M

trans-Resveratrol 1 24.5 M cis-Resveratrol 1 24.1 M

trans-3,5-Dihydroxy-4'-methoxystilbene 1 48.6 M trans-3,5-Dimethoxy-4'-hydroxystilbene 1 30.1 M

,-Dihydroresveratrol 1

60 M DPPH Reaction time = 60 min

106.8 M

[41]

Table 2. Superoxide Anion Radical Scavenging Activities of Resveratrol and Its Derivatives.

Compounds DPs IC50 Refs.

Vaticanol D 6 7.4 M [25] trans--Viniferin 2 140 M

Resveratrol 1 950 M 4-Hydroxystilbene 1 1100 M

4,4'-Dihydroxystilbene 1 820 M 3,5-Dihydroxystilbene 1 1680 M

[46]

Gnemonol K 3 69 M Gnemonol L 3 59 M -Viniferin 2 20 M

Gnetol 1 66 M Isorhapontigenin 1 29 M

Resveratrol 1 15 M Latifolol 3 68 M

Gnemonol B 3 79 M Gnemonol I 3 57 M

[47]

Resveratrol 1 37.9 g/ml Piceatannol 1 0.45 g/ml

Tricuspidatol 2 38.7 g/ml Pallidol 2 42.5 g/ml

Parthenocissin A 2 39.9 g/ml Betulifol A 2 22.1 g/ml -Viniferin 3 19.8 g/ml

Cyphostemmin B 2 24.7 g/ml

[38]

3.5. Singlet Oxygen

Singlet oxygen (1O2) is molecular oxygen in its first excited singlet state, generated by the transfer of energy to ground state (triplet) molecular oxygen [52]. It is formed readily on exposure of a range of endogenous and exogenous sensitizers, including por-phyrins and dye molecules such as Rose Bengal, to ultraviolet and visible light [53]. 1O2 reacts with a wide range of cellular targets including proteins, DNA, RNA, lipids, and sterols [54]. Among these important biological molecules, proteins, Cys, Met, Trp, Tyr and His residues are major targets for 1O2, since the rate constants for reaction of 1O2 with proteins side-chains are higher than that with the others, and proteins are present at higher concentrations within cells [55]. Dr. M. J. Davies has estimated that 68.5% of 1O2 generated within cells may be consumed by proteins [56]. It has been postulated to play a role in the development of a number of light-induced diseases including cataract, sunburn and some skin cancers [57]. Therefore, antioxidants with potent 1O2 quenching effects may have potential in the treatment and prevention of these diseases. For a long time, the 1O2 quenching effects of stilbenes

have been ignored, until our investigation demonstrated that palli-dol (Fig. (6)), a resveratrol dimer from red wine, is a 1O2 oxygen quencher in 2009 [58]. Pallidol showed strong quenching effects on 1O2 at very low concentrations in aqueous system with IC50 = 5.5 M, which was even stronger than EGCG (Epigallocatechin gallate, a famous antioxidant from green tea with IC50 = 14.5 M). Our kinetic study has revealed that the reaction of pallidol with 1O2 had an extremely high rate constant (ka = 1.71 1010). It is recom-mended to be used as a preventive agent in 1O2-mediated diseases, which may contribute to the health beneficial effects of red wine. In the same year, another two novel resveratrol tetramers, laetevire-nols F and G, from Parthenocissus laetevirens has been emerged as effective 1O2 quencher with IC50 = 5.4 M and 6.3M respectively, which were comparable with pallidol and stronger than EGCG [59]. In another investigation in our lab, three resveratrol oligomers have been isolated and purified from Vitis chunganensis by high-speed counter-current chromatography (HSCCC). Among them, vitisin A, a resveratrol tetramer, showed similar selective quenching effect against 1O2 with IC50 = 6.9 M [41]. Furthermore the novel resvera-

8 Current Medicinal Chemistry, 2013, Vol. 20, No. 4 He and Yan

Table 3. Lipid Peroxidation Inhibitory Activity of Resveratrol and Its Derivatives

Compounds DPs IC50 Refs.

trans-Resveratrol 1 3.0 Ma 2.6 Mb

cis-Resveratrol 1 18.1 Ma 19.0 Mb

trans-Piceid 1 21.3 Ma 19.3 Mb

cis-Piceid 1 16.0 Ma 16.6 Mb

Astringin 1 1.9 Ma 3.1 Mb

Astringinin 1 1.0 Ma 1.9 Mb

[16b]

Gnemonol K 3 19 M Gnemonol L 3 7 M -Viniferin 2 33 M

Gnetol 1 61 M Isorhapontigenin 1 45 M

Resveratrol 1 75 M Latifolol 3 32 M

Gnemonol B 3 50 M Gnemonol I 3 25 M

[47]

Resveratrol 1 9.03 g/ml Piceatannol 1 2.67 g/ml

Tricuspidatol 2 46.40 g/ml Pallidol 2 12.46 g/ml

Parthenocissin A 2 10.86 g/ml Betulifol A 2 19.8 g/ml -Viniferin 3 16.43 g/ml

Cyphostemmin B 2 11.04 g/ml Parthenostilbenin A 2 20.35 g/ml Parthenostilbenin B 2 18.68 g/ml

[38]

a Fe2+-induced lipid peroxidation in microsomes b Cu2+-induced lipid peroxidation in LDL

Table 4. Singlet Oxygen Quenching Effects of Resveratrol and Its Derivatives.

Compounds DPs IC50 Refs.

Pallidol 2 5.5 M [57] Laetevirenols F 4 5.4 M Laetevirenols G 4 6.3 M [58]

Vitisin A 4 6.9 M [39] Chunganenol 6 1.4 M [28] Amurensin G 3 5.2 M [43] Wilsonol A 3 12.3 M Wilsonol B 3 23.8 M Wilsonol C 4 7.6 M

Diviniferin B 4 6.2 M Resveratrol 1 18.5M

[40]

trol hexamer chunganenol has exhibited the hitherto highest 1O2 quenching activity (IC50 = 1.4 M) [30]. (Table 4) summarizes quenching effects of resveratrol and its derivatives against 1O2. It is

observed that most of resveratrol oligomers assayed are stronger than resveratrol, especially chunganenol with at least 10-fold higher than the monomer.

From Resveratrol to Its Derivatives Current Medicinal Chemistry, 2013, Vol. 20, No. 4 9

Our previous investigations have proved that resveratrol and its derivatives are potent 1O2 quenchers, therefore we conducted a mechanistic study in 2010 [44]. In order to understand the mecha-nism under the quenching effect, our priority was to find out the active functional group(s) that may react with 1O2. A mimetic HPLC/ESI-MS2 assay was designed to identify the product(s) of reaction between resveratrol and 1O2. Based on detailed analysis of the MS fragmentation data, we were able to characterize the major product as resveratrol quinone (Fig. (8)). Additional analyses of the reaction products between resveratrol oligomers (Pallidol and Amurensin G) and 1O2 have lead to the identification of corre-sponding quinone structures, revealing that resveratrol and its de-rivatives undergo a similar reaction with 1O2. The reaction take place at the resorcinol group, that is the 3,5-dihydroxylbenzene moiety. The first step of the reaction involves a 1,4-cycloaddition of 1O2 to the resorcinol ring, yielding the endoperoxide intermediate IM-1. Then the subsequent reaction step diverts into two pathways: Pathway A undergo an intramolecular H-abstraction of IM-1 to generate IM-2; Pathway B involves hydrolysis and then consecu-tive loss of a molecular water to give an unstable hydroperoxide IM-2' and then IM-2. The two pathways maintain a competitive dynamic equilibrium. Our further theoretical calculation, performed with PM3 semiempirical molecular orbital calculations, indicated that pathway B played a predominant role in the second step. Fi-nally, the intramolecular loss of H2O in IM-2 yield resveratrol qui-nones (Fig. (8)) [44]. The 1O2 quenching effect of resveratrol has been corroborated by Jung et al.s recent report. Resveratrol has shown a protective effect on the methylene blue sensitized pho-tooxidation of -terpinene, which explains the mechanism of how resveratrol protects tissues and cells from photosensitized oxidation in biological systems [60].

4. THERAPEUTIC POTENTIAL

In the past 15 years, numerous publications have proved the therapeutic potential of resveratrol in various diseases including cancer, heart disease, diabetes and stroke [14c]. However, its ex-tremely low bioavailability and rapid clearance from the circulation have laid down some limitation in its applications. As Dr. D. A. Sinclair demonstrated in the review, developing analogues with improved bioavailability, or finding new, more potent compounds that mimic its effects will become increasingly important [14c]. Although still scarce, a few investigations have already revealed the therapeutic potential of resveratrol derivatives.

In 2001, Hung et al. have reported the beneficial effects of as-tringinin (3,3',4',5-tetrahydroxystilbene, also known as piceata-nol) on the ischemia and reperfusion (I/R) damage in rat heart [61]. Astringinin with an additional hydroxyl group in its structure have shown stronger antioxidant activity than resveratrol in differ-ent investigations. In the study, astringinin has been introduced to examine its cardioprotective effects in ischemia or I/R, where the left main coronary artery was occluded by three different proce-dures: (i) 30 min occlusion, (ii) 5 min occlusion followed by 30 min reperfusion, and (iii) 4 h occlusion. Rats were infused with and without astringinin before coronary artery occlusion to evaluate its preventive effects. Pretreatment of astringinin has significantly reduced the incidence and duration of ventricular tachycardia and ventricular fibrillation. Astringinin could completely prevent the mortality of animals during ischemia or I/R at the dosages of 2.5 10-5 and 2.5 10-4 g/kg. During the same period, astringinin pre-treatment has also increased nitric oxide (NO) and decreased lactate dehydrogenase (LDH) levels in the carotid blood. Therefore, as-tringinin is a potent antiarrhythmic agent with cardioprotective activity. It is also observed that the beneficial effects of astringinin are related to its antioxidant activity and upregulation of NO pro-duction [61].

In 2004, another resveratrol derivative, oxyresveratrol (trans-2,3',4,5'-tetrahydroxystilbene), has been indicated as a neuroprotec-

tive agent, which inhibits the apoptotic cell death in transient cere-bral ischemia [62]. The neuroprotective effect of oxyresveratrol was assessed in the transient rat middle cerebral artery occlusion (MCAO) model to mimic the onset of ischemic stroke. Oxyresvera-trol was administered twice intraperitoneally: immediately after occlusion and at the time of reperfusion. Oxyresveratrol has dra-matically reduced the brain infarct volume of MCAO rats at the dosages of 10 or 20 mg/kg. The neurological deficits induced by I/R have also been improved by oxyresveratrol treatment. His-tological analysis of apoptotic markers in the ischemic brain area has indicated that oxyresveratrol treatment inhibited cytochrome c release and caspase-3 activation in MCAO rats, and the number of apoptotic nuclei in ischemic brain was also reduced by oxyresvera-trol treatment. The dose dependent neuroprotective effect of oxyresveratrol suggests that it is a potent neuroprotectant in vivo, and is potentially useful in the treatment of stroke [62].

In the next year, isorhapontigenin (trans-3-methoxy-3',4,5'-tetrahydroxystilbene) has been reported to attenuates cardiac hyper-trophy via blocking oxidative stress-mediated pathways [63]. Isorhapontigenin inhibited angiotensin II (Ang II)-induced cardiac hypertrophy, which was associated with a decrease in ROS levels and intracellular malonaldehyde content and an increase of activi-ties of endogenous antioxidants such as SOD and GPx. Ang II in-duced phosphorylation of PKC, Erk1/2, JNK, and p38 was inhibited by isorhapontigenin. In addition, PKC-dependent PI3KAktGSK3/p70S6K pathway was also blocked by this resveratrol ana-log. Pretreatment with isorhapontigenin dramatically inhibited Ang II-mediated NF-B through regulating the degradation and phos-phorylation of IB and the activity of IKK and AP-1 activation by affecting the expression of c-Fos and c-Jun proteins. These re-sults were supported by further in vivo evidence. In an aortic-banded rat model, isorhapontigenin treatment significantly attenu-ated heart weight/body weight ratio by approximately 25%, de-creased posterior wall thickness and left ventricle diastolic and systolic diameters, increased 10% fractional shortening, and re-duced cardiac myocyte size and systolic blood pressure. Therefore isorhapontigenin could prevent the development of cardiac hyper-trophy through an antioxidant mechanism involving inhibition of different intracellular signaling transduction pathways. It may be used as a supplemental pharmacological agent for the prevention and treatment of cardiac hypertrophy [63].

Recently, we have studied the neuroprotective effects of parthenocissin A, a novel antioxidant and free radical scavenger, using a transient MCAO model in rats, which was the first in vivo therapeutic evidence of resveratrol oligomer [64]. MCAO rats treated with parthenocissin A showed dose-dependent reductions in brain infarction size with improved neurological and motor out-come. Parthenocissin A treatment inhibited lipid peroxidation and restored SOD activity in brain tissue. Furthermore, I/R induced elevation of NO production and nitric oxide synthase (NOS) activ-ity in brain tissue was also inhibited by parthenocissin A treatment. These findings indicated that the beneficial effect of parthenocissin A on neuroprotection was associated with a reduction of oxidative stress and an inhibition of NO production. Our results have opened new vistas in the potential use of resveratrol oligomers in stroke prevention and therapy [64].

5. STRUCTURE-ACTIVITY RELATIONSHIP

Investigation of the SAR of resveratrol and its derivatives is important for the understanding of their mechanism of antioxidative action and provide basis for designing compounds with better anti-oxidant activities. It has been the subject attracting medicinal chem-ists around the world in the past 15 years. As early as 1997, the number and position of hydroxyl groups have been revealed to play an important role in the antioxidant activity of stilbenes [18b]. In 2001, there were two reports appeared almost at the same time con-cerning the structural determinants of the antioxidant activity of resveratrol. In the stationary -radiolytic experiments in liposomes

10 Current Medicinal Chemistry, 2013, Vol. 20, No. 4 He and Yan

OH

HO

HO

trans-resveratrol

1O2

OH

HO

HO

O2

IM-1

Pathway A

OH

O

HO OOH

IM-2

Pathway B

H2O

OH

HO OOH

HOOH

IM-2'

- H2O

OH

O

HO O

resveratrol quinone

- H2O

Fig. (8). Proposed mechanism of reaction between resveratrol and 1O2.

and pulse radiolytic experiments in aqueous solutions, trans-resveratrol and 4-hydroxy-trans-stilbene (4-HS) showed almost the same effect and were much stronger than 3,5-dihydroxy-trans-stilbene (3,5-DHS) (Fig. (9)). Thus, Stojanovi et al. demonstrated that the para-hydroxyl group (Fig. (10)) made greater contribution to the peroxyl radical scavenging activity of resveratrol than the meta-hydroxyl groups (resorcinol group, Fig. (10)) [65]. This con-clusion has been supported by Caruso et al.s study using ab initio calculations and crystal structure of resveratrol. Their results dem-onstrated that the para-hydroxyl group is more acidic than the other two meta-hydroxyl groups, and H-atom transfer is the dominant mechanism by which resveratrol and its derivatives scavenge free radicals [66]. In another investigation, the SAR study has been extended to the stereoisomery and saturation of the stilbenic double bond of resveratrol. First, the important role of the para-hydroxyl group has been confirmed in this study. Nevertheless, it is clearly not the sole determinant for antioxidant activity. The double bond in the stilbenic skeleton and its transisomery are also important, since the cis-form and the derivative, in which the double bond is reduced, are significantly less effective than trans-resveratrol. In addition, partial methylation decreases the antioxidant activity of resveratrol, while complete methylation could cause significant loss of antioxidant activity, indicating that phenolic hydroxyl group is required [16]. These observations have been supported by another study, where stilbenes with para-hydroxyl group showed better antioxidant activities in both -carotene bleaching assay and superoxide anions scavenging assay, while resveratrol analogs without phenolic hydroxyl group did not have any antioxidative effect [47].

R3

R4

R5

R5'

R4'

R3'

R3 R4 R5 R3' R4' R5'

Resveratrol OH H OH H OH H

4-HS H OH H H H H

3,5-DHS OH H OH H H H

4,4'-DHS H OH H H OH H

3,4-DHS OH OH H H H H

3,4,5-THS OH OH OH H H H

3,4,4'-THS OH OH H H OH H

3,3',4',5-TTHS OH H OH OH OH H

3,4,4',5-TTHS OH OH OH H OH H

3,3',5,5'-TTHS OH H OH OH H OH

3,3',4,5,5'-PHS OH OH OH OH H OH

3,3',4,4',5,5'-HHS OH OH OH OH OH OH

Fig. (9). Monomeric trans-stilbenes for SAR investigations.

In 2002, Dr. Z. L. Lius lab has conducted a SAR investigation using more resveratrol analogs. 4-HS, 3,5-DHS, 4,4-dihydroxy-trans-stilbene (4,4-DHS), 3,4-dihydroxy-trans-stilbene (3,4-DHS), 3,4,5-trihydroxy-trans-stilbene (3,4,5-THS) and 3,4,4-trihydroxy-trans-stilbene (3,4,4-THS) have been synthesized (Fig. (9)). Their antioxidant activities against the peroxidation of linoleic acid have been studied in sodiumdodecyl sulfate (SDS) and cetyltrimethyl ammonium bromide (CTAB) micelles to mimic the microenviron-ment of biomembranes. In both assay systems, 3,4-DHS, 3,4,5-THS and 3,4,4-THS with ortho-dihydroxyl functionality showed stronger inhibition on linoleic acid peroxidation than resveratrol and molecules bearing no such functionality, which unveiling the exceptional antioxidant power of ortho-dihydroxyl group (Also known as catechol group (Fig. (10)). We herein propose a name ortho-dihydroxyl rule for this phenomenon). This can be under-stood because the ortho-hydroxyl phenoxyl radical, the oxidation intermediate, is more stable due to the intramolecular hydrogen bonding interaction, supported by both experiments [67] and theo-retical calculations [68]. Furthermore, it is easier for the ortho-hydroxyl phenoxyl radical to form the stable ortho-quinone in the oxidation process. It is for a similar reason, that para-hydroxyl group increase antioxidant activity by stabilizing the semiquinone radical-anion intermediate via resonance through the trans double bond. That is the reason why the antioxidant activity of 3,4,4-THS, bearing both functionalities, was the best among the tested com-pounds. Interestingly, resveratrol and its derivatives can work alone or synergistically with -tocopherol in the antioxidative action by trapping the propagating lipid peroxyl radical and reducing the -tocopheroxyl radical to regenerate -tocopherol [69]. In another investigation from the same lab, antioxidant activities of 4-HS, 3,5-DHS, 4,4-DHS, 3,4-DHS, along with resveratrol, have been evalu-ated for the free radical-induced peroxidation of rat liver micro-somes in vitro. And the activity sequence follows the order: 3,4-DHS > 4,4-DHS > resveratrol > 4-HS > 3,5-DHS. Again, 3,4-DHS with catechol group showed superior activity, which confirmed their previous conclusion [70]. The ortho-dihydroxyl rule is not restricted in the antioxidant activity against lipid peroxidation, a SAR study by Murias et al. has proved that it can also be applied to the O2

and DPPH scavenging activities. Stilbenes with catechol group or 3,4,5-trihydroxylbenzene group (pyrogallol group, Fig. (10)), including 3,3',4',5-tetrahydroxy-trans-stilbene (3,3',4',5-TTHS, IC50=2.69 M), 3,4,4',5-tetrahydroxy-trans-stilbene (3,4,4',5-TTHS, IC50=41.5 M) and 3,3',4,4',5,5'-hexahydroxy-trans-stilbene (3,3',4,4',5,5'-HHS, IC50=5.02 uM), exhibit a more than 6600-fold higher antiradical activity than resveratrol and its two other analogues, including 3,3,5,5-tetrahydroxy-trans-stilbene (3,3,5,5-TTHS) and 3,3,4,5,5-pentahydroxy-trans-stilbene (3,3,4,5,5-PHS) (Fig. (9)). Interestingly hydroxystilbenes with catechol group or pyrogallol group exerted a more than three-fold

From Resveratrol to Its Derivatives Current Medicinal Chemistry, 2013, Vol. 20, No. 4 11

OH

OH HO

OH

OH

R R

HO OH

R

OH

R

para-hydroxyl group catechol groupresorcinol group pyrogallol group

Fig. (10). Major functionalities of stilbenes with different hydroxylation patterns.

higher cytotoxic activity than other analogs without the functionali-ties in HL-60 leukemic cells, because oxidation of ortho-dihydroxyl group yield ortho-semiquinones, which undergo redox-cycling thereby consuming additional oxygen and forming cytotoxic oxy-gen radicals. These findings suggest that the cytostatic activities of resveratrol and its derivatives are linked to their antioxidant activi-ties [71].

Furthermore, Mikulski et al. have recently reported the quanti-tative structureantioxidant activity relationship of resveratrol and its derivatives, including seven oligomers and five glucosides [15]. Their free radical scavenging activities have been calculated using the density functional theory. According to the results, all oligomers and glucosides studied exhibit stronger antioxidant activity than trans-resveratrol. A dimer of 4,4'-DHS is stronger than its mono-mer. The H-atom transfer mechanism is more preferable than the single-electron transfer mechanism, and all the antioxidants showed higher ability to donate electron in water medium than in the gas phase. Although these theoretical results still await future experi-mental verification, the report has again highlighted the remarkable antioxidant activity of resveratrol derivatives and their potential therapeutic applications [15].

CONCLUSION

In the past 15 years, we have witnessed the research outbreak of resveratrol, a natural antioxidant from red wine and grape, which is under multiple clinical trials. Resveratrol has provided an entrance to the medicinal investigations of an important group of plant poly-phenols stilbene, where resveratrol derivatives have shown in-triguing chemical diversity. Numerous reports have proved resvera-trol derivatives to be a promising source of potent antioxidants, while some have shown higher activity than resveratrol. In recent years, in vivo studies of resveratrol derivatives have highlighted their therapeutic potential in the treatment in cerebral and cardio-vascular diseases, which is linked to their remarkable antioxidant activities. They may also be used as functional food supplements owing to their powerful antioxidant capacities. Finally, we would like to propose the following points to be considered in future in-vestigations:

(1) Screening of antioxidant capacity of resveratrol derivatives should include trans-resveratrol as a screening criteria. More interest should be directed towards compounds with higher ac-tivity than trans-resveratrol.

(2) SAR studies have revealed the so-called ortho-dihydroxyl rule, that stilbenes with ortho-dihydroxyl group possess higher antioxidant activities. These compounds may have greater therapeutic potential in future investigations.

(3) Different mechanisms have been observed when stilbenes react with different ROS. For example, the resorcinol group plays a predominant role in the quenching effect of resveratrol, while the para-hydroxyl group is more effective in scavenging per-oxyl radicals. Results from these mechanistic studies will pro-vide a basis for future screening and drug design.

(4) Our previous reports have shown that resveratrol derivatives, especially oligomers, are potent 1O2 quenchers. Further investi-

gations of their applications in light-mediated diseases includ-ing cataract, sunburn and skin cancers, are highly recom-mended.

CONFLICT OF INTEREST

The author(s) confirm that this article content has no conflicts of interest.

ACKNOWLEDGEMENTS

We thank support from Qianjiang Talent Plan (2012R10068), Zhejiang Marine Biotechnology Innovation Team (2012R10029-2), Ningbo Marine Algae Biotechnology Team (2011B81007), Talent Plan of Ningbo University (RCL2011718), and K.C.Wong Magna Fund in Ningbo University.

ABBREVIATIONS

ROS = Reactive oxygen species

OS = Oxidative stress

SOD = Superoxide dismutase

GPx = Glutathione peroxidase

CAT = Catalase

GSH = Glutathione

DMBA = 7,12-Dimethylbenz[a]anthrancene

SAR = Structure-activity relationship

PAL = Phenylalanine ammonia lyase

C4H = Cinnamate 4-hydroxylase

4CL = Hydroxycinnamoyl CoA ligases

STS = Stilbene synthase

DP = Degree of polymerization

HCSO = Highly condensed stilbene oligomer

HRP = Horseradish peroxidase

DPPH = 1,1-Diphenyl-2-picrylhydrazyl

EPR = Electron paramagnetic resonance

LDL = Low-density lipoproteins

EGCG = Epigallocatechin gallate

HSCCC = High-speed counter-current chromatog-raphy

I/R = Ischemia and reperfusion

NO = Nitric oxide

LDH = Lactate dehydrogenase

MCAO = Middle cerebral artery occlusion

Ang II = Angiotensin II

NOS = Nitric oxide synthase

SDS = Sodiumdodecyl sulfate

12 Current Medicinal Chemistry, 2013, Vol. 20, No. 4 He and Yan

CTAB = Cetyltrimethyl ammonium bromide

4-HS = 4-Hydroxy-trans-stilbene

3,5-DHS = 3,5-Dihydroxy-trans-stilbene

4,4-DHS = 4,4-Dihydroxy-trans-stilbene

3,4-DHS = 3,4-Dihydroxy-trans-stilbene

3,4,5-THS = 3,4,5-Trihydroxy-trans-stilbene

3,4,4-THS = 3,4,4-Trihydroxy-trans-stilbene

3,3',4',5-TTHS = 3,3',4',5-Tetrahydroxy-trans-stilbene

3,4,4',5-TTHS = 3,4,4',5-Tetrahydroxy-trans-stilbene

3,3',4,4',5,5'-HHS = 3,3',4,4',5,5'-Hexahydroxy-trans-stilbene

3,3,5,5-TTHS = 3,3,5,5-Tetrahydroxy-trans-stilbene

3,3,4,5,5-PHS = 3,3,4,5,5-pentahydroxy-trans-stilbene

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