coumarins as antioxidants

Current Medicinal Chemistry, 2011, 18, 3929-3951 3929

0929-8673/11 $58.00+.00 © 2011 Bentham Science Publishers Ltd.

Coumarins as Antioxidants

I. Kostova*,1, S. Bhatia

2, P. Grigorov

1, S. Balkansky

1, V.S. Parmar

2, A.K. Prasad

2 and L. Saso

3

1Department of Chemistry, Faculty of Pharmacy, Medical University, 2 Dunav St., Sofia 1000, Bulgaria

2Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi – 110 007, India

3Department of Physiology and Pharmacology “Vittorio Erspamer”, Sapienza University of Rome, P. le Aldo Moro 5, 00185 Rome,

Italy

Abstract: Coumarins, a well-known class of naturally occurring compounds, display a remarkable array of biochemical and pharmacol-

ogical actions, some of which suggest that certain members of this group of compounds may significantly affect the function of various

mammalian cellular systems. The development of coumarins as antioxidant agents has attracted much attention in recent years. Coumar-

ins afford an opportunity for the discovery of new antioxidants with truly novel mechanisms of action. This review updates and expands

the 2006 review by the same author. The review considers and incorporates the most recently published literature on coumarins as related

to their antioxidant properties. A lot of coumarins have been identified from natural sources, especially green plants. These natural com-

pounds have served as valuable leads for further design and synthesis of more active analogues. Beyond doubt, a deep understanding of

the mechanisms of existing synthetic and natural coumarins will build the basis for the rational design.

Keywords: Synthetic and natural coumarins, antioxidants.

INTRODUCTION

Free radicals and other reactive oxygen species are derived ei-ther from normal essential metabolic processes in the human body or from external sources. Free radicals are atoms, molecules or ions with unpaired electrons, which are highly active to chemical reactions with other molecules. In the biological system, the free radicals are often derived from oxygen-, nitrogen- and sulphur-containing molecules. These free radicals are parts of groups of molecules called reactive oxygen species (ROS), reactive nitrogen species (RNS) and reactive sulphur species (RSS). ROS includes free radicals such as superoxide anion, perhydroxyl radical, hydroxyl radical, nitric oxide and other species such as hydrogen peroxide, singlet oxygen, hypochlorous acid and peroxynitrite. RNS are derived from nitric oxide. RSS are easily formed from thiols by reaction with ROS. ROS are produced during cellular metabolism and functional activities, and have important roles in cell signalling, apoptosis, gene expression and ion transportation [1]. However, excessive amounts of ROS can have deleterious effects on many molecules including proteins, lipids, RNA, DNA and carbohydrates since they are very small and highly reactive. Damaged cells func-tion wrongly, which may result in further escalation of the oxidative stress. Simultaneous ionic disbalance, mitochondrial dysfunction and activation of the caspase/calpine cascades, result in cell death [1-3].

Cells are normally able to defend themselves against ROS damage through the use of intracellular enzymes to keep the homeostasis of ROS at a low level. However, during times of environmental stress and cell dysfunction, ROS levels can increase dramatically, and cause significant cellular damage in the body. Thus, oxidative stress significantly contributes to the pathogenesis of inflammatory disease, cardiovascular disease, cancer, diabetes, Alzheimer’s disease, cataracts, autism and aging [4-8]. In order to prevent or reduce the ROS induced oxidative damage, the human body and other organisms have developed an antioxidant defence system that includes enzymatic, metal chelating and free radical scavenging activities to neutralize these radicals after they have formed. In addition, intake of dietary antioxidants may help to maintain an adequate antioxidant status in the body. Antioxidants may be molecules that can neutralize free radicals by accepting or donating electron(s) to eliminate the unpaired condition of the radical. The antioxidant molecules may directly react with the

*Address correspondence to this author at the Department of Chemistry, Faculty of

Pharmacy, Medical University, 2 Dunav St., Sofia 1000, Bulgaria; Tel: +359 2 92 36 569; Fax: +359 2 987 987 4; E-mail: [email protected]

reactive radicals and destroy them, while they may become new free radicals which are less active, longer-lived and less dangerous than those radicals they have neutralized [1]. Small molecules, such as vitamin C, vitamin E, uric acid and glutathione play important roles as cellular antioxidants. Many synthetic antioxidants have been widely used in the food industry to retard lipid oxidation. However, lots of them are not preferred for pharmacological use due to toxicological concerns. Thus, more and more interest has focused on identifying plant products and their synthetic analogs to use as dietary antioxidant supplements.

In the recent years, antioxidant research has expanded dramatically due to its potential benefit in disease prevention and health promotion. Many research models have been established for the studies of mechanisms of action of antioxidants as well as identification of new antioxidants especially from natural sources. Most of the natural antioxidants come from fruits, vegetables, spices, grains and herbs which contain a wide variety of antioxidant compounds, that may help to protect cellular damages from oxidative stress and also lower the risk of chronic diseases.

Coumarins (known as 1,2-benzopyrones), consisting of fused benzene and pyrone rings, are an important group of low-molecular weight phenolics and have been widely used for the prevention and treatment of many diseases. Coumarins comprise a group of natural compounds found in a variety of plant sources. The coumarins pos-sess anti-inflammatory, antioxidant [2,3], anticancer [9,10], antivi-ral [11,12], anticoagulant, etc. activities. Thus, they have excellent pharmaceutical potential. Several recent reviews summarize ad-vances in the application of coumatins, especially concerning their antioxidant properties [9-17]. The coumarins are variable in struc-ture, due to the different types of substitutions in their basic struc-ture, which can influence their biological activity. A careful struc-ture -activity-relationship study of coumarins with special respect to their antioxidant and anticancer activities should be conducted.

The naturally occurring coumarins as well as their synthetic

analogs will be the primary focus of this review. The pharmacol-ogical and biochemical properties and therapeutic applications of simple coumarins depend upon the pattern of substitution. Cou-marin (1,2-benzopyrone, (1)) is the simplest compound of a large class of naturally occurring phenolic substances made of fused ben-zene and -pyrone rings. The hydroxycoumarins, as typical pheno-lic

compounds, act as potent metal chelators and free

radical scav-

engers. Hydroxycoumarins have attracted intense interest in recent years because of their diverse pharmacological properties. Among these properties, their antioxidant effects were extensively exam-ined.

3930 Current Medicinal Chemistry, 2011 Vol. 18, No. 25 Kostova et al.

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In this review, plant derived coumarins and their synthetic ana-logues will be systematically evaluated based on their plant origin, structure-activity relationship and antioxidant efficacy. Owing to their diverse effects and inconclusive results from different in vitro studies, the mechanism of their action has not yet been fully under-stood and the correlation of effects with chemical structure is not conclusive at the moment. It is the objective of this review to sum-marize experimental data for different coumarins used as antioxi-dant agents, because promising data have been reported for a series of these agents. In addition, their ability to bind metal ions repre-sents an additional means of modulating their pharmacological responses. Thus, the aim of this review is to update the current approaches to study properties and mechanisms of action of cou-marins-based antioxidants with emphasis on the chemical and biological systems. This review may greatly benefit researchers and scientists who are interested in the study of detailed chemical and molecular mechanisms of the antioxidant activity of coumarins. It is also helpful for physicians who are interested in alternative antioxidant therapy or prevention for certain oxidative stress related diseases or conditions.

STRUCTURE–ACTIVITY RELATIONSHIP OF COUMARIN

DERIVATIVES

Natural polyphenols can be divided into several different classes depending on their basic chemical structure which ranges from simple molecules to highly polymerized compounds. Coumarin (known as 1,2-benzopyrone), consisting of fused benzene and a-pyrone rings, is an important group of low-molecular weight phenolics. Antioxidant activity of phenolic compounds was correlated to their chemical structures. Structure-activity relation-ship of many phenolic compounds (e.g. flavonoids, phenolic acids, coumarins, tannins, etc.) has been studied. In general, free radical scavenging and antioxidant activity of these classes of compounds mainly depends on the number and position of hydrogen-donating hydroxyl groups on the aromatic ring of the phenolic molecules, and is also affected by other factors, such as glycosylation of aglycones, other H-donating groups (-NH, -SH), etc.

Very few systematic studies have been reported on structure-antioxidant activity correlations in coumarins. To select or design better antioxidants, researchers have to study their structure–activity relationships (SAR). In fact, there has been plenty of work devoted to this topic. The SAR and even quantitative structure–activity relationships (QSAR) for most of the natural antioxidants have been elucidated by means of experimental determinations and theoretical calculations. However, there still lacks the elucidation of SAR for coumarins. Through comparing the peroxyl radical-scavenging activity of a series of coumarins in sodium dodecyl sulfate micellar system, Foti and co-workers indicated that coumarins containing a catechol moiety were stronger than the others to scavenge peroxyl radical [18], which was supported by the observations from other groups [19]. It is not surprising to see catechol moiety playing a key role in enhancing the antioxidant activity of coumarins and a large number of experimental and theo-retical studies confirmed the same, revealing that the catechol group was beneficial to enhance the radical-scavenging activity of natural antioxidants. On the other hand, the ring B of coumarin, a 1,2-pyrone, is unique in natural antioxidants and has also been investigated in previous studies [20]. Considering the success of density functional theory (DFT) calculations used in elucidating the SAR for a large number of antioxidants, Zhang et al. evaluated the

1,2-pyrone effect on the radical-scavenging activity. By means of density functional theory calculation on B3LYP/6-31G (d,p) level, two kinds of theoretical parameters, i.e. O–H bond dissociation enthalpy (BDE) and adiabatic ionization potential, were obtained to characterize the distinct peroxyl radical-scavenging mechanisms of coumarins, i.e. H-atom transfer and proton concerted electron trans-fer, respectively. It was found that the former mechanism was pre-ferred in the radical-scavenging process of coumarins, thus O–H BDE was a suitable index to measure the radical-scavenging activ-ity. As coumarins without catechol moiety have little structural factors beneficial to reduce O–H BDE, they are weaker antioxidants than the counterparts containing a catechol group. The unique 1,2-pyrone in ring B of coumarins is roughly an electron-withdrawing group, however, its electronic effect is rather weak. Consequently, 1,2-pyrone, being a weaker electron-withdrawing group than 1,4-pyrone (a group ubiquitous in flavonoids and isoflavonoids), has little influence on the antioxidant activity of coumarins. However, this group may play an important role in determining the pharmacological effect of coumarins [20].

Xanthine oxidase (XO) is an important source of free radicals and has been reported in various physiological and pathological models. XO causes gout and is responsible for oxidative damage to living tissues. This enzyme reduces molecular oxygen. Regulation of XO activity is important during inflammation.. XO catalyses the oxidation of hypoxanthine and xanthine to uric acid yielding superoxide radicals and raises the oxidative level in an organism. Coumarins acting to inhibit XO inhibition have been reported and the structure–activity relationships (SARs) of coumarins interacting with this enzyme have also been discussed [21-23]. Lin et al. [23] employed 1,1-diphenyl-2-picrylhydrazyl hydrate (DPPH)– and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)–electron spin resonance (ESR) to study the effects of suppression of reactive oxygen species (ROS) by eight selected coumarin derivatives under oxidative con-ditions and the most potent radical scavengers among the tested compounds were identified. The results suggested that the number of hydroxyl groups on the ring structure of coumarins is correlated with the effects of ROS suppression. The authors also investigated the effect of the derivatives on the inhibition of xanthine oxidase (XO) activity, and the structure–activity relationships (SARs) of these derivatives against XO activity were further examined using computer-aided molecular modeling. All determined derivatives competitively inhibited XO. The results of the structure-based mo-lecular modeling exhibited interactions between coumarins and the molybdopterin region of XO. The carbonyl pointed toward the Arg880, and the ester O atom formed hydrogen bonds with Thr1010. Coumarins, which bear two hydroxyl moieties on its ben-zene rings, had the highest affinity toward the binding site of XO, and this was mainly due to the interaction of hydroxyl with the E802 residue of XO. The hypoxanthine/XO reaction in the DMPO-ESR technique was used to assess the combined effect on enzyme inhibition and ROS suppression by these coumarins. The authors combined the ROS-scavenging and XO inhibition roles of coumarins in order to identify which compounds are more vital for therapeutic applications. They eventually applied the results to living cells to confirm the conclusions drawn from the in vitro experiments.

Up to now, a lot of mechanistic studies have been carried out on phenolic antioxidants, which have indicated that the chain reaction is controlled mainly through free radical scavenging by phenolic hydroxyl of the antioxidants. So, if a proper theoretical parameter to characterize the ability of antioxidants to scavenge free radicals can be found, it will be possible to predict their antioxidant activity, which will undoubtedly improve the selection of new antioxidants.

7-HYDROXYCOUMARINS AND THEIR DERIVATIVES

Plant derived phenolic coumarins might play a role as dietary antioxidants because of their consumption in the human diet

Coumarins as Antioxidants Current Medicinal Chemistry, 2011 Vol. 18, No. 25 3931

especially in fruits and vegetables. Umbelliferone (7-hydroxy-coumarin, (2), Table 1), a natural antioxidant is the main coumarin (1,2-benzopyrone) metabolite of coumarin and is a therapeutically active molecule. It exhibits antioxidant properties in vitro and may share with other coumarin derivatives vasodilator effects. It is present in the edible fruits such as golden apple (Aegle marmelos Correa) and bitter orange (Citrus aurantium) [24]. Recently the effect of umbelliferone on glycemic control and lipids [25,26] and antioxidant properties [27-29], have been reported. Ramesh et al. [24] have also analyzed the effect of umbelliferone on fatty acid composition and histopathological studies of liver and kidney. The results showed that umbelliferone has a protective effect on mem-brane fatty acid composition of liver and kidney as supported by antioxidant and antihyperlipidemic effects of umbelliferone re-ported earlier as evidenced by improved histopathological changes, hepatic and nephritic markers, indicating recovery from the risk of diabetic complications. Furthermore, umbelliferone and its derivatives have shown to have lipid lowering potential [30,31].

The genus Heptaptera (umbelliferae) possesses ten species in the world mainly distributed from Europe to the Middle East including Italy, Balkans, Turkey, Syria, and Palestine [32]. Although limited phytochemical study has so far been carried on the Heptaptera species, the genus has been reported to be rich in coumarin derivatives; i.e. umbelliprenin derivatives. Orhan et al. screened several coumarin derivatives including umbelliferone and found some of them having moderate acetylcholinesterase inhibitory activity [33]. Although no traditional use of this genus has been reported up to date, the genus is known to contain coumarins in major amounts, which were tested for their antioxidant potential [32]. For this purpose, the ethyl acetate and methanol extracts prepared from the fruits, aerial parts, and roots of these species were tested in vitro for their acetylcholinesterase (AChE) inhibitory and antioxidant activities. Antioxidant activity of the extracts was determined by 2,2-diphenyl-1-picrylhydrazyl radical scavenging and ferrous ion-chelating power tests. The methanol extracts of the fruits and aerial parts of H. triquetra had the best scavenging effect against DPPH. All of the extracts screened displayed significant ferrous ion-chelating effect in a dose-dependent manner. It is most likely that coumarin-type of compounds in Heptaptera species could be responsible for antioxidant and mild anticholinesterase activity. The results showed that H. Triquetra showed the best radical scavenging and iron chelating effects among the extracts and could be a potential source for antioxidant compounds and this is the first report on anticholinesterase and antioxidant activities of Heptaptera species.

Several linear and angular coumarins have been synthesised as possible anti-inflammatory and antioxidant agents. They were found to interact with 1,1-diphenyl-2-picryl-hydrazyl stable free radical (DPPH) [34]. The anti-inflammatory activity seemed to be connected with their reducing activity. Khan et al. [35] reported the modulatory effect of coumarin (1,2-benzopyrone) on potassium bromate (KBrO3) mediated nephrotoxicity in Wistar rats and the results showed that coumarin may be used as an effective chemo-preventive agent against KBrO3-mediated renal oxidative stress, toxicity and tumor promotion response in Wistar rats. Structurally diverse coumarins were examined for their ability to inhibit lipid peroxidation induced either by Fe(II) and Fe(III) metal ions or by azo-derived peroxyl radicals in a liposomal membrane system [36]. The antioxidant effects of these compounds were evaluated on the basis of their ability to inhibit the fluorescence intensity decay of an extrinsic probe, 3-[p-(6-phenyl)-1,3,5-hexatrienyl]phenylpropionic acid (DPH-PA), caused by the free radicals generated during lipid peroxidation. All the coumarins tested exhibited higher antioxidant efficacies against metal-ion-induced peroxidations than peroxyl-radical-induced peroxidation, suggesting that metal chelation may play a larger role in determining the antioxidant activities of these compounds. The presence of hydroxyl substituents enhanced anti-

oxidant activity, whereas substitution by methoxy groups at the same positions diminished their activity [36].

The antioxidant effects of 7-hydroxycoumarin have been inves-tigated in a number of studies [37]. 7-Hydroxycoumarin and es-culetin (6,7-dihydroxycoumarin, (3), Table 1) were examined to determine their effect on the cellular metabolism of A431 cells over a 24-h exposure period [38]. 7-Hydroxycoumarin caused suppres-sion of the succinate dehydrogenase activity at concentrations larger than 10 μg/ml. Esculetin exerted a more serious effect with decrease in activity observed at greater than 1 μg/ml. The observed effects were dose-dependent. Using the cytosensor microphysiome-ter to assess metabolic activities, it was observed that esculetin was more detrimental to cellular metabolism than 7-hydroxycoumarin. 7-Hydroxycoumarin (100 μg/ml) caused the cellular metabolic rate to drop to 44.21 +/- 5.34% (n = 4) of the control metabolic rate, while 100 μg/ml esculetin caused the metabolic rate to fall to 21.5 +/- 4.54% (n = 4) of the control rate.

Connection between free radical-generating agents and inflam-matory processes suggests that accumulation of reactive oxygen species can cause hepatotoxicity [39]. t-Butyl hydroperoxide (t-BHP), can be metabolized to free radical intermediates by cyto-chrome P-450 in hepatocytes, which, in turn, can initiate lipid per-oxidation, affect cell integrity and result in cell injury. Lin W. L. et al. [40] used t-BHP to induce hepatotoxicity in vitro and in vivo and determined the antioxidative bioactivity of esculetin. Pretreatment with esculetin (5-20 μg/ml) significantly decreased the leakage of lactate dehydrogenase (LDH) and alanine transaminase (ALT), and also decreased the formation of malondialdehyde (MDA) in pri-mary cultured rat hepatocytes induced by t-BHP. An in vivo study in rats showed that pretreatment with esculetin significantly low-ered the serum levels of the hepatic enzyme markers (ALT and AST) and reduced oxidative stress in the liver. Histopathological evaluation of the rat livers revealed that esculetin reduced the inci-dence of liver lesions induced by t-BHP, including hepatocyte swel-ling, leukocyte infiltration, and necrosis. Thus, esculetin may play a chemopreventive role via reducing oxidative stress in living sys-tems.

Esculetin (6,7-dihydroxycoumarin) is an antioxidant coumarin derivative that inhibits the lipoxygenase and cyclooxygenase pathways of arachidonate metabolism [41,42]. Among other important pharmacological activities, esculetin promotes analgesic, immunomodulatory [43] and anti-tumoural effects [44,45]; decreases neutrophil infiltration [40], subepithelial fibrosis and TGF levels in the lung [46]; protects DNA against oxidative stress [41]; inhibits synthesis of leukotriene B4, thromboxane B2 [47], platelet aggregation [48], matrix metalloproteinases production [49,50], growth of human leukaemia cells [51] and the production of IL-6 and IL-8 [52]. The antioxidant [41], anti-inflammatory properties [47], inhibitory effect on vascular smooth muscle cells [53] and antitumour properties [54] of its derivatives have also been investigated. Based on the antioxidant and anti-inflammatory properties of esculetin and its derivatives, Witaicenis et al. [55] investigated the effects of esculetin and its substituted derivatives on the experimental model of trinitrobenzenesulphonic (TNBS) acid-induced rat colitis. For this purpose, macroscopic (diarrhoea, extension of lesion, colonic weight/length ratio and damage score) and biochemical parameters (myeloperoxidase, alkaline phospha-tase and glutathione) were evaluated. They assayed the effects of the compounds in preventing the inflammatory response induced by TNBS in two different experimental settings, i.e. when the colonic mucosa is intact and when the mucosa is healing after an initial insult. In the acute colitis model, esculetin promoted a reduction in the extension of the lesion accompanied by a reduction in the inci-dence of diarrhoea and restoration of the glutathione content. The effect of esculetin and its derivatives on the inflammatory process may be related to their antioxidant and anti-inflammatory proper-ties, as observed in this study. The antioxidant activity of esculetin


and its derivatives observed in [40,55], as well as their inhibitory activity on the cyclooxygenase and lipoxygenase pathways [42], suggest that these coumarins may be beneficial in the pathological inflammatory processes of the intestine. Additionally, the anti-inflammatory activities of esculetin and its derivatives could be associated with their inhibition of the cyclooxygenase and lipoxygenase pathways [56,57]. These coumarins have been reported to reduce eicosanoid generation by acting on 5-lipoxygenase and cyclooxygenase rather than by affecting phospholipase A2 [41]. Coumarins with two hydroxyl groups (and in some cases other substituents) present an additional interest in biological activities [41]. Conspicuously, all of the potentially active natural compounds possess at least two phenolic groups at either the 6, 7- or 6, 8-positions [9,10]. It has been found that ortho-dihydroxy coumarins are moderately powerful inhibitors of arachidonate metabolism via pro-inflammatory 5-lipoxygenase pathways and that 4-methylcoumarin derivatives are known to be less toxic when compared to other coumarins. Sharma et al. [58] demonstrated that the presence of adjacent phenolic hydroxyl groups is an important factor in the antioxidant and apoptosis-inducing activity of dihydroxy 4-methylcoumarins. Luchini et al. [59] suggested that the presence of the hydroxyl radical at the C-4 position in the coumarin molecule improves its anti-inflammatory effect in the TNBS rat colitis model. Sekiya K. et al. have studied the effects of coumarin and its derivatives on rat platelet lipoxy-genase and cyclooxygenase activities [60]. Esculetin was found to inhibit the lipoxygenase more strongly than the cyclooxygenase. The 6-glucoside of esculetin, esculin (4), Table 1 as well as umbel-liferone also selectively inhibited the lipoxygenase. The mechanism of the lipoxygenase inhibition by esculetin was non-competitive. It is interesting to mention that other antioxidants (hydroquinone, gallic acid and ascorbic acid) were less inhibitory to both enzymes and showed little selectivity [60]. Zhao J. et al. [61] determined DNA damage and oxidative stress in healthy term neonates at birth. The protective activities of five natural polyphenols against H2O2-induced DNA damage in mononuclear cells of umbilical blood were studied. The natural polyphenols, 7,8-Dihydroxycoumarin [(5), refer. Table 1], 7,8-dihydroxy-4-methylcoumarin [(6), refer. Table 1], quercetin (7) and resveratrol (8), were able to protect mononuclear cells of umbilical blood from oxidative attack, whereas other two polyphenols, rutin (9) and 7-hydroxy-4-methylcoumarin [(10), refer. Table 1], did not. The protective prop-erties of seven polyphenols against hydrogen peroxide induced DNA damage in human peripheral blood lymphocytes (PBL) were studied using single cell micro-gel electrophoresis [62]. Hydrogen

peroxide causes an increase in single cell DNA strand breakage in human PBL. Quercetin and 7,8-dihydroxy-4-methylcoumarin ex-hibited the strongest protection. Curcumin (11), resveratrol and vanillin protected against DNA damage induced by 50 μM H2O2, but rutin and 7-hydroxy-4-methylcoumarin failed to provide any protection even at concentrations up to 50 μM. Quercetin, 7,8-dihydroxy-4-methylcoumarin, curcumin, resveratrol and vanillin were found effective in protection of human single cell DNA from oxidative attack.

Kaneko et al. have examined the protective effects of coumar-ins against cytotoxicity induced by linoleic acid hydroperoxide [63]. Coumarins, esculetin (6,7-dihydroxycoumarin) and 4-methylesculetin [(12), refer. Table 1] protected cells from injury by linoleic acid hydroperoxide. Fraxetin [(13), refer. Table 1] and caf-feic acid showed weak, but significant, protection. Esculin, es-culetin and 4-methylesculetin were effective for protecting cells against linoleic acid hydroperoxide-induced cytotoxicity in the case of pretreatment for 24 h, however fraxetin and caffeic acid showed no protection. The sugar moiety in the esculin molecule appears to be hydrolyzed during pretreatment. Coumarins containing only one hydroxyl group (umbelliferone) showed no protective effect in pretreatment or concurrent treatment. Esculetin and 4-methylesculetin provided synergistic protection against cytotoxicity induced by linoleic acid hydroperoxide with alpha-tocopherol.

Fraxinus species have been used as anti-inflammatory drugs, possibly due to several phenolic acids and coumarins that possess free radical-scavenging activities [64]. Cortex fraxini (CF) is a widely and traditionally used herb in Taiwan. This herb also has anti-inflammatory properties and its ingredients include coumarins, such as esculin, esculetin, fraxin (14), and fraxetin [65]. Wu et al. [66] presented the antioxidant properties of crude extract, fractions and ingredients of CF and compared the antioxidant capacities of coumarin ingredients of CF and known antioxidants, including catechin (15), quercetin, ascorbic acid, trolox and BHT. The IC50 values for CF in the DPPH and trolox equivalent antioxidant capacity (TEAC) methods were 406 and 39.3 μg/ml, respectively. Among all fractions the chloroform fraction is the most active frac-tion in scavenging DPPH, ABTS and hydroxyl radicals, and there is a significant relationship between the antioxidant activities and the contents of the antioxidant phenolic ingredients. The amounts of esculetin and fraxetin in the chloroform fraction were 8.44 % and 11.1 %, respectively. Esculetin and fraxetin also had good radical-scavenging capacities, and esculetin was the best, among all test compounds, against the DPPH radical. Moreover, esculetin and

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fraxetin had selective scavenging activity upon hydroxyl radicals and hydrogen peroxide, and this potency was better than known antioxidants and equal to quercetin in scavenging hydrogen perox-ide. These results show that CF, partitioned with chloroform, gave a phenolic coumarin-enriched fraction, and that this fraction had the best free radical-scavenging activity and inhibition of Fe

2+/ascorbate-induced lipid peroxidation, mainly due to its reduc-

ing power. Some researchers [67] have indicated that glycosidic substitution decreases TEAC values of coumarins and flavonoids. Wu et al. [66] found similar results, in that there is lower free scavenging capacity in the two glycosidic coumarins, esculin and fraxin, than in their aglycones. Furthermore, esculetin and fraxetin also possess the better active oxygen-scavenging activity than do ascorbic acid and BHT, and the different potencies in scavenging various oxygen radicals, between esculetin and fraxetin, are similar to those reported earlier [14]. The active oxygen scavenging activities of two glycosidic coumarins, esculin and fraxin, were also lower than their aglycones. From these results [14,66] it may be suggested that ortho-dihydroxy substitution and the 3,4-double bond play an important role in the antioxidant property of simple coumarins that lack the B-ring of flavonoids. A hydroxyl group in the 7-position of simple coumarins is necessary for its antioxidant activity and any C6–C8 substitution, including glycosidic or methoxy groups, might decrease their free radical-scavenging capacity [66].

The activity of 4-methylcoumanrin [(16), refer. Table 1], 7-hydroxy-4-methylcoumarin and 7,8-dihydroxy-4-methylcoumarin in the peroxidation of human low-density lipoprotein (LDL) has been examined [68]. The peroxidation was initiated either thermally by the water-soluble initiator 2,2'-azobis(2-amidinopropane hydro-chloride) (AAPH), or photochemically by a triplet sensitizer benzo-phenone (BP) or its water-soluble analogue disodium 3,3'-disulfobenzophenonate (DSBP). Kinetic analysis of the peroxida-tion process demonstrated that 7,8-dihydroxy-4-methylcoumarin was a good antioxidant for both the AAPH-initiated and BP- and DSBP-photosensitized peroxidation; 7-hydroxy-4-methylcoumarin was a prooxidant for the AAPH-initiated and DSBP-photosensitized peroxidation, but an antioxidant for the BP-sensitized peroxidation; 4-methylcoumanrin was a prooxidant in all of these initiation condi-tions. It was suggested that he antioxidative action of the coumarin derivatives may include trapping the initiating radicals, trapping the propagating lipid peroxyl radicals, recycling alpha-tocopherol and/or deactivating the excited photosensitizer.

The antioxidant activity of 4-methylcoumarin, 6-hydroxy-4-methylcoumarin and 7,8-dihydroxy-4-methylcoumarin, respecti-vely, in the inhibition of peroxidation of linoleic acid in micellar systems has been studied by Yu et al. [19]. The peroxidation was initiated either thermally by water-soluble initiator 2,2’-azobis(2-methylpropionamidine)dihydrochloride (AMPAD) or photoche-mically by a triplet sensitizer benzophenone (BP) in the anionic micelle sodium dodecyl sulfate (SDS) or in the cationic micelle cetyl trimethylammonium bromide (CTAB). It was found that 7,8-dihydroxy-4-methylcoumarin is a good antioxidant for both AMPAD-initiated and BP-sensitized peroxidation in both SDS and CTAB micelles, while 6-hydroxy-4-methylcoumarin is only active for BP-sensitized peroxidation in SDS micelles. 4-Methylcoumarin was ineffective in any case. Thus, the antioxidative action of the

coumarin derivatives may include trapping the initiating radicals, trapping the propagating lipid peroxyl radicals, recycling a-tocopherol and/or deactivating the excited photosensitizer.

Free iron participates in the production of reactive oxygen spe-cies (ROS) and plays an important role in the pathogenesis of car-diovascular diseases. Therefore, chelation of iron may attenuate some ROS consequences, but on the other hand, reduction of ferric ions to ferrous ones is unfavourable and leads to intensification of ROS production. Mlad nka et al. [69] have examined the interac-tion of iron with coumarins which has been rarely analyzed. A se-ries of naturally occurring and chemically synthesized 4-methylcoumarins were analyzed for their ferrous and total iron-chelating properties and compared with standard iron chelator de-feroxamine. The authors have critically analyzed the structure-activity relationship of coumarins interaction with iron at different pH conditions. Emphasis is given not only to the potentially positive effects resulting from iron chelation but also on possible undesired involvement of coumarins in iron reduction. The study examines especially the effects of 4-methylcoumarin derivatives which are generally considered as safe substances and cannot be metabolized in human into mutagenic epoxides, in contrast to many other coumarins [70,71]. The iron chelation activity was assessed by a simple spectrophotometric approach based on the specific indicator for ferrous ions – ferrozine. The methodology was also extended for the measurement of total iron. Among the tested cou-marins, ortho-dihydroxy derivatives were the most potent iron chelators and 7,8-dihydroxy-4-methylcoumarin even reached the efficiency of deferoxamine in neutral pH. However, these ortho-dihydroxycoumarins did not bind iron firmly in acidic conditions (e.g., in acute myocardial infarction) and, moreover, they reduced ferric ions. Thus, the use of iron-chelating coumarins in acidic con-ditions may be disadvantageous in contrast to neutral conditions.

Three dihydroxy-4-methylcoumarin derivatives, namely 7,8-dihydroxy-4-methylcoumarin, 6,7- dihydroxy-4-methylcoumarin and 5,7-dihydroxy-4-methylcoumarin alone and complexed with Fe(III) and ADP have been tested for their antioxidative potential [58]. Chemical speciation studies and formation constants reveal the formation of strong dihydroxy-4-methylcoumarin–Fe–ADP (1:1:1) ternary complex. In vitro studies were done for their anti-oxidative property by scavenging the superoxide radicals (O 2

– )

generated by xanthine + xanthine oxidase (XO) reaction. The re-sults indicate that O 2

– scavenging potential of all the three dihy-

droxy-4-methylcoumarins increased significantly after forming the ternary complex with Fe (III) and ADP. The structure-activity rela-tionship studies suggest that the introduction of hydroxyl group at C-7 and C-8 positions in the coumarins, irrespective of Fe(III)–ADP complexation, increases the antioxidative efficacy. No change in uric acid production in the reactions done for all studies further reveals that the coumarin derivatives and their complexes were the only causative factors for O 2

– scavenging and not the suppression

of the enzyme, xanthine oxidase. Fe(II) (ferrous sulphate, freshly prepared aqueous solution) in place of Fe(III) (ferric chloride) was also used in order to verify if Fe(II) has a role in antioxidative mechanism. The experiments revealed no effect in antioxidative efficacy when dihydroxy-4-methylcoumarins were complexed with Fe(II)–ADP [58]. The pharmacological and biochemical properties and therapeutic usefulness of coumarins have been worked out to

O O

O-B-D-Glucose

14

OHO

OH

OH

OH

15

HO

O

OH


be a sequel of the different substitutions in the derivatives. It is well established that the antioxidant activity of a compound is more exactly assessed by providing a microenvironment of the reaction medium and nature of the involved ROS rather than by its structure alone. Hence it is very important to assess the structure-activity relationship by trying different reactions. Coumarin derivatives have been reported as potent inhibitors of 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) formation from arachidonic acid and 12-hydroxy-5,8,10-heptadecatrienoic acid in polymorpho-nuclear leukocytes (PMN). The presence of two adjacent phenolic hydroxyl groups at the C-6 and C-7 or C-7 and C-8 positions in coumarin structure is must for its potency for the inhibition of the 5-HETE. Monohydroxycoumarins, e,g, umbelliferone and scopo-letin [(17), refer. Table 1] also inhibited the formation of 5-HETE, but not very strongly, suggestive of the essentiality of the presence of a phenolic hydroxyl group at the C-7 position for this effect [72].

Raj et al. [73] have shown that formation of ADP–Fe-antioxidant (ligand) could assume a crucial role in the prevention of ROS. The sequestration of transition metal ions by antioxidant is one of the most powerful preventive mechanisms in vivo [74] and chelation of transition metal by antioxidant ligands can inhibit membrane lipid peroxidation. The polyphenolic structure of dihy-droxy-4-methylcoumarin facilitates the inhibition of lipid peroxidation by scavenging free radicals through chelation. Raj et al. [73] reported that the chelator 7,8- dihydroxy-4-methylcoumarin is 40 times more potent than vitamin E while performing the lipid peroxidation inhibition experiments. But 6,7-dihydroxy-4-methylcoumarin was found to be a weak antioxidant compared to 7,8- dihydroxy-4-methylcoumarin. Studies made with five dihydroxy derivatives of coumarin for antioxidative activity, as inhibitors of 5-lipoxygenase pathway of arachidonic metabolism revealed the high efficacy of these compounds owing to their two fold ability to chelate the iron ions and donating the electron for redox cycling of iron thus rendering the 5-lipoxygenase in catalytically inactive ferrous form [47]. Studies on chelating behavior of dihydroxycoumarin in Fe(III)-ascorbate–H2O2 system revealed ortho-dihydroxycoumarin to be a strong metal chelator as compared to metal substituted compounds [75]. Different deriva-tives of 4-methylcoumarins have been examined by Raj et al. for their effect on the NADPH-catalysed liver-microsomal lipid per-oxidation [76,77]. Dihydroxy- and diacetoxy-4-methylcoumarins produced remarkable inhibition of lipid peroxidation. 7,8-Diacetoxy-4-methylcoumarin [(18), refer. Table 1] and 7,8-dihydroxy-4-methylcoumarin were found to possess excellent anti-oxidant and radical scavenging activities.

Recently, a series of selected 4-methylcoumarins were synthe-sised and tested for radical-scavenging ability using the stable 1,1-diphenyl-2-picrylhydrazyl radical, and for reducing power ability with a test based on the reduction of ferric to ferrous cation [78]. Special attention was paid to the number and position of the functional groups attached to the benzenoid part of the coumarin molecule. The authors have examined 4-methylcoumarin possessing methyl-, methoxy- and hydroxy-, acetoxy-, and benzoxy- groups in the benzenoid ring. The majority of them are known as natural compounds. All studied compounds showed activ-ity comparable to Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), an already known antioxidant, which is used as standard in most of the testing methods. The examined compounds showed weaker activity in reducing Fe3+ ions compared to the scavenging radicals. This might be explained by the presence of aqueous media, where coumarins were less soluble and resonance structures of resulting phenoxyl radicals are less stable. However, coumarins still showed antioxidant activity, comparable with DPPH radical-scavenging method. 7,8-Dihydroxy-4-methylcoumarin posses the strongest antioxidant activity, both in radical-scavenging and reducing power tests [78]. Since a large number of experimental and theoretical studies revealed that the catechol

group was beneficial in enhancing the radical-scavenging activity of natural antioxidants, it is not surprising to see the catechol group playing a key role in enhancing the antioxidant activity of coumarins. The results showed that the position and type of the substituent attached to the aromatic moiety of coumarin molecule have an influence on the radical-scavenging ability [78]. In general, substitution at the C-6 position gives stronger feature to coumarin for scavenging free radical than substitution at the C-7 position. The results are consistent with a previous report which proved that resonance structures of the radicals derived from coumarins with substitution at the C-6 position are especially stable because of the ortho-quinone form [23]. In addition, the type of substituent has an intense influence on the action of coumarins as antioxidants. The results indicate that the hydroxy group has the strongest influence, while the methyl group has the smallest. Previously published data indicate that the pyrone ring of the coumarin moiety has little influence on the antioxidant activity of coumarins, due to its rather weak electronic effect [20]. Observations of avar et al. [78] were made with regard to structural features that regulated the behaviour of the compounds. In general, the antioxidative effectiveness of phenolic compounds depends on the reaction activity of the phenol towards the chain-carrying peroxyl radicals and on the stability of the phenoxyl radical formed in the reaction. The reaction mechanism is based on an electron transfer (ET) reaction. Whilst the hydrogen atom abstraction is a marginal reaction pathway, because it occurs slowly in strong hydrogen-bond accepting solvents, such as methanol and ethanol, it occurs in other ET-based assays, the scavenging capacity against DPPH is strongly influenced by the solvent and the pH of reaction [69]. The steric accessibility of the DPPH is a major determinant of the reaction, since small molecules that have better access to the radical site have relatively higher antioxidant capacity. On the other hand, many large antioxidant compounds that react quickly with radicals may react slowly or may even be inert, thus introducing a benzoxy group into coumarin molecule displays relatively weak antioxidant prop-erty against the DPPH [78].

4-Methylcoumarins having two hydroxyl groups placed ortho to each other in the benzenoid ring have shown to possess excellent antioxidant and radical scavenging properties in different experimental models [79]. In fact hydroxycoumarins can behave like classic phenol or quinol-based antioxidants, in which a hy-droxyl group on an aromatic ring structure can carry out the single-electron reduction of a free radical. The resulting phenoxyl radical or semiquinone can either be stabilized through the presence of bulky or electron-withdrawing groups on the ring system or be oxidized further through a consecutive single-electron reduction by a second hydroxyl group to produce a quinone-type end product. Furthermore, a positive property of 4-methylcoumarins is that they are not substrates for the liver P450 monoxygenases that epoxidize coumarins lacking the C4 methyl group and thus, during metabolic degradation, cannot form 3,4-coumarin epoxides, which are believed to be mutagenic and probably also have other toxic effects [80]. Morabito et al. [81] reported a study on the structure activity relationship of eight synthetic 4-methylcoumarins, carried out by employing a series of different chemical cell-free tests. These coumarins have been previously described as a promising radical scavengers in cell-based systems [58,71]. The compounds were tested by means of three assays involving one redox reaction with the oxidant (DPPH assay, ABTS

+ assay and FRAP) [81]. Other

assays were employed to evaluate the antioxidant properties of the coumarins under investigation against NO, O2 and HClO, which are some of the major reactive oxygen and nitrogen species causing damage in the human body. Morabito et al. [81] have measured the protective capacity of these coumarins against the oxidative damage in a simple biomimetic model of phospholipid membranes. The results confirmed the good antioxidant activity of the 7,8-hydroxy-4-methylcoumarin. In general, their activity was not significantly affected by the introduction of an ethoxycarbonylmethyl or an


ethoxycarbonylethyl moiety at the C-3 position. A discrete antioxi-dant activity is retained also by the 7,8-diacetoxy-4-methyl-coumarin, although they were less efficient than the corresponding 7,8-dihydroxy compounds. Two of the 4-methylcoumarins (7,8-dihydroxy-4-methylcoumarin and 7,8-dihydroxy-3-ethoxycarbo-nylethyl-4-methylcoumarin) (19), very interestingly, showed strong scavenging activities against the superoxide anion and were also very effective in protecting the lipid bilayer against peroxidation. On the basis of these findings, these 4-methylcoumarins may be considered as potential therapeutic candidates for pathological con-ditions characterized by free radical overproduction [81].

Another attempt for evaluating the structure–activity relation-ship of different dihydroxy 4-methylcoumarins has been recently done [82]. The chain-breaking antioxidant activities of eight coumarins [7-hydroxy-4-methylcoumarin, 5,7-dihydroxy-4-methylco-umarin, 6,7-dihydroxy-4-methylcoumarin, 6,7-dihydroxycoumarin, 7,8-dihydroxy-4-methylcoumarin, ethyl-2-(7,8-dihydroxy-4-methy-lcoumar-3-yl)-acetate (20), 7,8-diacetoxy-4-methylcoumarin and ethyl 2-(7,8-diacetoxy-4-methylcoumar-3-yl)-acetate] (21) during bulk lipid autoxidation at 37 °C and 80 °C in concentrations of 0.01-1.0 mM and their radical scavenging activities at 25 °C using TLC-DPPH test have been studied and compared. It was found that the o-dihydroxycoumarins demonstrated excellent activity as anti-oxidants and radical scavengers, much better than the m-dihydroxy analogue and the monohydroxycoumarin. The substitution at the C-3 position did not show any effect either on the chain-breaking antioxidant activity or on the radical scavenging activity of the 7,8-dihydroxy- and 7,8-diacetoxy-4-methylcoumarins. The comparison with DL- -tocopherol (TOH), caffeic acid (CA) and p-coumaric acid (p-CumA) revealed some structure-activity correlations. Theo-retical calculations and the “Lipinski’s Rule of Five” were used for explaining the structure–activity relationships and pharmacokinetic behavior. The importance of phenolic compounds and of coumarins, in particular as potential bio-antioxidants, i.e. compounds with antioxidant and biological activity, has been theo-retically demonstrated recently [82, 83].

Electrochemical and chemical oxidation of 7,8-hydroxy-4-methylcoumarin and 7,8-diacetoxy-4-methylcoumarin were studied to investigate the mechanisms occurring in their antioxidant activi-ties in acetonitrile, under electron transfer and H-atom transfer con-ditions [84]. Electrolysis and chemical reactions were followed on-line by monitoring the UV spectral changes with time. The anodic oxidation of 7,8-hydroxy-4-methylcoumarin, studied by cyclic volt-ammetry and controlled potential electrolysis, occurs via a reversi-ble one-step two-electrons process, yielding the corresponding sta-ble phenoxonium cation. Moreover, the chemical oxidation with an H-atom acceptor also follows a similar path, yielding the stable neutral quinonic product. Intermediates were never evidenced in both cases. Only in the presence of a strong base, an anodic oxida-tion product mono-electronic was evidenced, likely the 7,8-hydroxy-4-methylcoumarin radical anion. However, the anodic oxidation of the acetoxy derivative 7,8-diacetoxy-4-methylco-umarin occurs at very high potential values, ruling out the possibil-ity that the antioxidant activity observed in vivo might occur via an electron transfer mechanism and no reactions were evidenced with an H-atom acceptor.

As it was mentioned earlier, the unsubstituted coumarins can form 3,4-coumarin epoxides during their metabolism and these intermediates could be toxic [85]. Differently, synthetic 4-methylcoumarins do not induce the formation of the epoxide, as they are not substrates for liver P450 monoxygenase [85]. For this reason, they could be better candidates for pharmacological use, particularly for their antioxidant activity. However, the great part of the structure-antioxidant activity relationship studies analyze the antioxidant activity of 4-methylcoumarins toward ‘not physiological’ radicals (such as DPPH) [78,86] or in ‘not physiological’ system (such as linoleic acid lipid peroxidation) [18]. One study reports on structure/antioxidant activity relation of three 4-methylcoumarins in LDL oxidation model (oxidation was initiated thermally by AAPH or photochemically by a tripler sensitizer benzophenone) [68]. Very recently, the antioxidant activ-ity of eight synthetic 4-methylcoumarins was systematically studied [87] both in their peroxyl radicals scavenging activity and in modulating the in vitro resistance of human low-density lipoprotein (LDL) to oxidative modification initiated by 2,2’-azobis (2-amidinopropane) dihydrochloride (AAPH) or catalyzed by copper. The antioxidant capacity was measured using: (i) a competition kinetic test to measure the relative capacity to quench peroxyl radi-cal; (ii) the in vitro oxidative modification of human low-density lipoprotein, initiated by AAPH or catalyzed by copper. The authors studied the effect of acetoxy and hydroxy substituents (at the position C-5,C-7; C-6,C-7 and C-7,C-8) in 4-methylcoumarins that differed also for the presence of ethoxycarbonylethyl or ethoxycarbonylmethyl chain at the C-3 position. In both models, the ortho-OH substituents were found to be better antioxidants than the meta ones [87]. The most efficient antioxidant was the 7,8-dihydroxy-4-methylcoumarin and the corresponding diacetoxy derivative was unexpectedly a good antioxidant. Finally, the pres-ence of an ethoxycarbonylethyl substituent at the C-3 position in-creased the antioxidant capacity of both 7,8-dihydroxy-4-methylcoumarin and 7,8-diacetoxy-4-methylcoumarin.

Beillerot et al. [88] investigated the antioxidant effects of some known and new coumarin derivatives on oxidative stress induced by doxorubicin (DOX) in MCF7 cells. Doxorubicin, an anthracycline antibiotic, is a potent chemotherapeutic agent, often used in the treatment of solid tumors, especially breast and ovarian cancer. However, its therapeutic use is limited by its cardiotoxicity. The DOX-induced cardiomyopathy mechanisms are not completely understood but they include formation of oxygen free radicals [89]. Different antioxidants such as vitamins A, E, and C, probucol or N-acetylcystein, and S-allylcystein do not interfere with anthracycline activity in tumor cells but their cardioprotective efficacy was limited, thus it is well established that there is, at the moment, no universal ideal antioxidant.

The coumarin derivatives investigated displayed a protective antioxidant activity without affecting DOX antitumoral efficiency. A set of eighteen coumarin derivatives were synthesized and ana-lyzed [88]. Their antioxidant power was evaluated in vitro with the FRAP (ferric reducing ability of plasma) method and in human breast adenocarcinoma MCF7 cells using H2DCFDA (2 ,7 -dichlorodihydrofluorescein diacetate) in a cytometric analysis. 4-Methyl-7,8-dihydroxycoumarin was found to exhibit an important

O OOH

OH

O

O

19

O O

OH

O

O

20

O OAcO

OAc

O

O

21

HO


antioxidant strength, a low cytotoxicity, and could decrease ROS production generated by DOX treatment without affecting DOX cytotoxicity in MCF7 cells.

The study of Goel et al. [90] examined 7,8-diacetoxy-4-methylcoumarin and its thiocoumarin derivative 7,8-diacetoxy-4-methylthiocoumarin (22) for their effect on human non-small cell lung cancer A549 cells. They showed that both coumarins not only inhibited cell proliferation, but also induced apoptosis with an IC50 of 160 μg/ml as confirmed by morphological examination, annexin-V assay and flow cytometric analysis. Interestingly, it was observed that these two coumarin compounds exhibited little cytotoxicity towards peripheral blood mononuclear cells but induced apoptosis in malignant cells. Treatment with 7,8-diacetoxy-4-methylcoumarin and 7,8-diacetoxy-4-methylthiocoumarin also resulted in pro-nounced release of apoptogenic cytochrome c from mitochondria to cytosol, alteration of mitochondrial membrane potential ( m), and activation of caspase-9 and caspase-3. Although an increase in the levels of ROS was observed, pre-treatment with antioxidant showed no protective effect against 7,8-diacetoxy-4-methylcoumarin- and 7,8-diacetoxy-4-methylthiocoumarin-induced apoptosis [90]. Re-sults suggested that downregulation of Bcl-xl, Cox-2 and mitogen activated protein kinase pathway and upregulation of p53, Akt and NF- B pathway are involved in the underlying molecular mecha-nism of apoptosis induction by 7,8-diacetoxy-4-methylcoumarin and 7,8-diacetoxy-4-methylthiocoumarin in A549 cells. Goel et al. [91] have previously reported that 7,8-dihydroxy-4-methylcoumarin induces apoptosis of human lung adenocarcinoma cells by ROS-independent mitochondrial pathway through partial inhibition of ERK/MAPK signaling. As one of coumarin derivatives, daphnetin (7,8-dihydroxycoumarin) has exhibited broad biological activities, such as anti-inflammatory, antioxidant [14] as well as atitumor effects [92]. Moreover, recent studies demonstrated that daphnetin can act as a potent antiproliferative and differentiation-inducing agent in human renal cell carcinoma [93]. Esculetin (6,7-dihydroxycoumarin) showed lipoxygenase inhibitory effect on the proliferation response of cultured rabbit vascular smooth muscle cells by modulating P signal transduction pathway [94]. The structure-activity relationship of esculetin and eight other coumarin derivatives indicated that two adjacent phenolic hydroxyl groups at the C-6 and C-7 positions in the coumarin skeleton were necessary for the potent antiproliferative and antioxidant effects. Therefore, glycosylation and damage of the catechol structure (ortho-dihydroxy groups) had significant negative influence on the activity of the coumarins, for example, it considerably reduced the radical scavenging level [95]. The study of Cai et al. [96] characterized antioxidant activity and phenolic compounds of traditional Chinese medicinal plants associated with anticancer, comprising 112 species from 50 plant families. A posi-tive, significant linear relationship between antioxidant activity and total phenolic content showed that phenolic compounds were the dominant antioxidant components in the tested medicinal herbs. Major types of phenolic compounds from most of the tested herbs were preliminarily identified and analyzed, and mainly included phenolic acids, flavonoids, tannins, coumarins and curcuminoids. These medicinal herbs exhibited far stronger antioxidant activity and contained significantly higher levels of phenolics than common vegetables and fruits. Traditional Chinese medicinal plants associ-ated with anticancer might be potential sources of potent natural

antioxidants and beneficial chemopreventive agents. The influence of other coumarin derivatives in induced oxidative stress has been widely studied also in vivo and ex vivo [97].

3-HYDROXYCOUMARINS AND OTHER C-3 SUBSTI-

TUTED COUMARINS

The C-3 substituted coumarins have been extensively investi-gated for their antioxidant properties, for instance, 3-hydroxycoumarin [(23), refer. Table 1] [98,99], as well as 3-aminocoumarin [(24), refer. Table 1], 3-acetylaminocoumarin [(25), refer. Table 1], and coumarin-3-carboxilic acid [(26), refer. Table 1] [99]. The antioxidant capacity has been investigated with chemilu-minescence measurement and by the accumulation of TBA-active products [99]. All coumarins were found to be antioxidants, with 3-hydroxy-, 3-amino- and 3-acetylamino coumarins being capable of amplifying chemiluminescence. 5-Lipoxygenase and alpha-D-glucosidase inhibitory activities of coumarins greatly increased through 3-hydroxylation by 3-hydroxyscopoletin [(27), refer. Table 1] and 3-hydroxyisoscopoletin [(28), refer. Table 1] [100]. In these tests 3-hydroxyscopoletin and 3-hydroxyumbelliferone [(29), refer. Table 1] had a high inhibitory potency for 5-lipoxygenase and for alpha-D-glucosidase respectively. Some benzo[l]khellactone de-rivatives and analogues were prepared by Nicolaides et al. and their strong inhibition effect on the soybean lipoxygenase was investi-gate [101]. All the tested compounds were found to interact with DPPH in a concentration and time dependent manner. Some fused 1,3-dioxolocoumarins were synthesized and evaluated as antioxi-dants and antinflammatories [102]. The compounds were tested for their ability to interact with 1,1-diphenyl-2-picrylhydrazyl stable free radical (DPPH), to scavenge superoxide anion radicals, to compete with DMSO for hydroxyl radicals, and to inhibit proteoly-sis, -glucuronidase and soybean lipoxygenase activity in vitro. These compounds were also tested for their effect on the ferrous ion-stimulated peroxidation of linoleic acid. They showed a potent inhibitory effect (55-57 %) against inflammation induced by car-rageenan in the rat paw edema model. On the contrary, their reduc-ing ability was found to be low and no inhibition on soybean lipoxygenase was recorded. A sensitive fluorimetric assay has been used to examine whether free hydroxyl radicals (HO•) were gener-ated in the immediate vicinity of DNA by Fe(II)-bleomycin [103]. When aqueous solutions of SECCA (the succinimidyl ester of cou-marin-3-carboxylic acid) were irradiated with gamma rays or incu-bated with Fe(II)-bleomycin or Fe (II)-EDTA in the presence of ascorbate and H2O2, 7-hydroxy-SECCA, a fluorescent product of the interaction of HO• with SECCA was generated. On the con-trary, Cu(II)-bleomycin complexes under similar conditions fail to induce 7-hydroxy-SECCA fluorescence.

A series of coumarin analogues bearing a substituted phenyl ring on the C-3 position were synthesized via a novel methodology and the in vitro antioxidant activity of the synthesized compounds was evaluated [104]. The presence of a catechol system is well known to improve the antioxidant activity of a compound. Especially in the case of flavonoid analogues as well as in coumarin derivatives, it has been shown that the ortho-dihydroxy system forms a resonance stabilized radical which enhances the radical scavenging ability of the compound [105] and it was taken into consideration upon the synthesis in order to examine the influence of this structural feature on the biological activity of the new coumarin analogues. The in vitro antioxidant activity of the new compounds was evaluated using two antioxidant assays: the radical scavenging ability of the compounds was tested against the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) stable free radical and their ability to inhibit lipid peroxidation induced by the thermal free radical producer 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH) was evaluated. Moreover, the ability of the synthesized coumarins to inhibit soybean lipoxygenase was determined. The interaction of the synthesized compounds with the stable free

S OAcO

22

OAc


radical DPPH indicates their radical scavenging ability in an iron-free system. The compounds, which possessed the catechol structural feature are potent DPPH radical scavengers, showing high activity comparable to the reference compound. The corresponding methoxylated analogues had very low activity which is remarkably enhanced when the methyl groups were removed. In addition, the presence of only one free phenolic hydroxyl group does not favour activity. The catechol moiety has been identified as the structural requirement for efficient DPPH radical scavenging and lipid peroxidation activity, however it does not favour LO inhibitory ability.

A facile, convenient and high yielding synthesis of a combina-torial library of 3-alkanoyl/aroyl/heteroaroyl-2H-chromene-2-thiones has been developed [106]. The assessment of radical scav-enging capacity of the compounds towards the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) was measured and these compounds were found to scavenge DPPH free radical efficiently. Some of the compounds were able to protect curcumin from the attack of sulfur free radical generated by radiolysis of glutathione (GSH). The newly synthesized compounds exhibited profound antioxidant activities. Reverse-phase preparative high-performance liquid chromatography (RP-HPLC) of the methanol extract of the aerial parts of Euphorbia petiolata, an endemic Iranian medicinal plant, yielded ten free radical scavengers including coumarins [107]. The free radical scavenging properties of these compounds were assessed by the 2,2-diphenyl-1-picrylhydrazyl assay.

The experiments reported by Kennedy S. H. et al. [108] demon-strated that benzoyl peroxide (BP) can promote radiation-induced transformation in vitro. Benzoyl peroxide (BP) was shown to be capable of generating free radicals, determined by the kinetics of hydroxylation as measured by the fluorescence of coumarin-3-carboxylic acid. Although the mechanisms involved in the BP en-hancement of radiation transformation are unknown, the authors hypothesize that lipid peroxidation produced by benzoyl radicals in the vicinity of membrane associated unsaturated lipids could con-tribute to the promotion of transformation in vitro. Quantitative analysis for determination of hydroxyl radical generation has been performed with use of a highly specific fluorescent probe coumarin-3-carboxylic acid whose hydroxylation product, 7-hydroxyco-umarin-3-carboxylic acid, fluoresces intensely [109,110]. The car-dioprotective effects of red wine consumption has been reported and these properties of wine have been attributed to certain poly-phenolic constituents of grapes [111].

Some novel coumarin derivatives with a 7-azomethine linkage were synthesized by 7-formylcoumarin (30) [112-114] and investi-gated. The compounds were tested in vivo for their anti-inflammatory activity and in vitro for their antioxidant ability and possessed significant protection against carrageenin induced rat paw edema. Some of the compounds were found in vitro to inhibit lipid peroxidation and to strongly scavenge superoxide radicals [113]. An attempt was made to delineate the possible mechanism of action of the studied compounds. Hydrophilicity, the presence of the free C-7OH group, and steric requirements for the substituent at the C-8 position are the most important factors in terms of SAR [114].The authors reported the in vivo antiinflammatory activity (inhibition of carrageenin rat edema) and the in vitro antioxidant effects (DPPH interaction, OH radical scavenging activity) of some new coumarin derivatives [112]. It has been established that active oxygen species are implicated in various stages of the process of inflammation, e.g. stimulation of phagocytosis, leucotriene biosynthesis.

4-HYDROXYCOUMARINS

The 4-hydroxycoumarin [(31), refer. Table 1] moiety, widely spread among coumarin natural products, has been the molecular template for the synthesis of a variety of analogues exhibiting

important biological activity. The potential of 4-hydroxycoumarin derivatives as antiinflammatory and antioxidant agents, prompted researchers to design and synthesize a series of novel coumarin analogues. Some novel quinolinone-3-aminoamides bearing a methyl or phenyl group on the nitrogen heteroatom incorporating the alpha-lipoic acid (LA) moiety have been recently synthesized by Detsi et al [115] and led to compounds with combined antioxidant and anti-inflammatory activities. Incorporating alpha-lipoic acid to the coumarin scaffold revealed that some of the novel hybrid compounds are potent leads and may be useful in the prevention of human diseases attributed to free radical damage [116].

O OOHC

30

Coumarin derivatives including 4-hydroxycoumarin with antibacterial and antioxidant activities [117-119] have been recently synthesized. Different substituted 3,3 -arylidenebis-4-hydroxyco-umarins and tetrakis-4-hydroxycoumarin derivative were the final products when 4-hydroxycoumarin and aromatic aldehydes contain-ing different groups in ortho, meta or para positions condense in boiling ethanol or acetic acid [117]. The antioxidant activity of these compounds was studied. A new family of coumarin deriva-tives containing a chalcone moiety was synthesized by condensa-tion of 3-acetyl-4-difluoroboryloxycoumarin (32) with aryl and heteroaryl aldehydes with piperidine in chloroform [119] and their antioxidant activity against the stable free radical DPPH was evalu-ated.

The antioxidant properties of five new 4-hydroxy-bis-coumarins were studied and compared with 4-hydroxycoumarin [120]. It was found that compound 3,3 -[(3,4-dihydroxyphenyl) methylene]bis(4-hydroxy-2H-chromen-2-one) (33) with a cate-cholic structure in the aromatic nucleus showed the strongest anti-oxidant activity. The compound demonstrated radical scavenging activity towards DPPH radical by using TLC DPPH rapid test. The other compounds did not show significant capacity as radical scav-engers. The structure–activity relationship was discussed on the basis of comparable kinetic analysis of studied 4-hydroxy-bis-coumarins with the known and standard antioxidants as -tocopherol (TOH), caffeic acid (CA), sinapic acid (SA), ferulic acid (FA) and p-coumaric acid (p-CumA). It has been found for the first time that the substitution in the aromatic nucleus of the studied 4-hydroxy-biscoumarins is responsible for their antioxidant activity. The remaining structure of 4-hydroxy-bis-coumarin moiety is not of significance for the antioxidant and antiradical activity of compounds under study. Four new 4-hydroxycoumarin derivatives, substituted with electron-donating and electron-withdrawing substituents on the third place in the coumarin ring, were synthe-sized by the same group [121] and were tested in vitro for antioxi-dant activity in hypochlorous system. The assay was based on the luminol-dependent chemiluminescence of free radicals, which de-creased in the presence of 4-hydroxycoumarin derivative. Com-pound ethyl 2-[(4-hydroxy-2-oxo-2H-chromen-3-yl)(4-hydroxy-phenyl)methyl]-3-oxobutanoate (34) expressed the best scavenger activity at the highest concentration. The results show that 4-hydroxycoumarins with electron-donating groups on the third position and on the seventh position in the coumarin ring have the best scavenger activity. Earlier, differently substituted 4-hydroxycoumarins were tested for free radical scavenger activity in the system of 1,1-diphenyl-2-picrylhydrazyl (DPPH) stable radical [122]. Structure–activity relationship of some coumarin antioxi-dants as protective agents against linoleic acid hydroperoxide


(LOOH) toxicity in cultivated human umbilical vein endothelial cells was recently explored [123].

A series of imino and amino derivatives of 4-hydroxycoumarins were synthesised and evaluated for antioxidant potential, through different in vitro models such as (DPPH) free radical-scavenging activity, linoleic acid emulsion model system, reducing power assay and phosphomolybdenum method [124,125]. All prepared compounds possessed good antioxidant activity and among them a p-nitrophenol derivative with IC50 at 25.9 μM possessed radical-scavenging activity which was comparable to BHT, while the best reducing power was observed in the case of benzyl amino compound. Also, observed data indicated that compounds may serve as inhibitors of lipid peroxidation process [125]. A series of novel spiro-substituted 4-hydroxypyranocoumarins and their corre-sponding dihydropyrano cis-diols has been synthesized and investigated for their radical scavenging properties, in an effort to establish structure–activity relationships [126]. Among them the spiroadamantylpyranocoumarin (35) and the diols can interact with the stable free radical 1,1-diphenyl-2-picrylhydrazyl and scavenge superoxide anions generated in the xanthine–xanthine oxidase sys-tem. The synthesized compounds scavenged DPPH radical in a concentration and a time-dependent manner. Their activity is comparable with that of 4-hydroxycoumarin and significantly higher than that of 7-hydroxycoumarin.

OTHER SYNTHETIC COUMARINS

A series of aminoalcohol derivatives of benzocoumarins from nucleophilic opening of oxirane with different amines, furnishing a variety of 2-hydroxy amino derivatives as well as simple alkyl substituted benzocoumarins were synthesized and evaluated for their in vivo anti-dyslipidemic and in vitro antioxidant activities [127, 128]. In terms of SAR, the simple alkyl substituted benzocoumarins exhibited interesting activity. Preliminary screen-ing indicated that the compound, which incorporates an unbranched benzyl group, demonstrated significant lipid lowering and antioxi-dant activities [127]. The study revealed that such attempts on ben-zocoumarin-based pharmacophores which is a biologically impor-tant scaffold might result in identification of new lead for anti-dyslipidemia. Six novel 4-methylcoumarins bearing different func-tionalities such as amino, hydroxy, N-acetyl, acetoxy and nitro have been synthesized and examined for the first time for their effect on NADPH dependent liver microsomal lipid peroxidation in vitro, and the results were compared with other model 4-methylcoumarin derivatives to establish the structure–activity relationship [129]. The results demonstrated that amino group is an effective substitute for the hydroxyl group for antioxidant property and produced a dramatic inhibition of lipid peroxidation. Ortho dihydroxy and or-tho hydroxy-amino coumarins were found to possess highest anti-oxidant and radical scavenging activities, possibly by forming a stable mixed ligand complex with ADP and Fe2+ thereby preventing the production of ADP-perferryl radical responsible for ROS formation, as postulated earlier [77, 130].

The structure of Schiff base derivatives of coumarins in addition to the biologically active coumarin nucleus, possesses amine moiety and extended conjugation that is responsible for its highly fluorescent and antioxidant nature.

A series of Schiff bases have been synthesized from dicarbal-dehyde of benzocoumarin, in which the reactions were regioselec-tive and the products existed in the keto-enamine form, in which the aromaticity of the relevant ring was disrupted. The compounds were evaluated in vitro for their antioxidant and in vivo for their antidyslipidemic activity for the first time and possessed significant lipid lowering and antioxidant activity [131]. Sashidhara et al. [132,133] have reported the synthesis and potential antidyslipi-demic and antioxidant activity of novel Schiff bases from 7-hydroxy-4-methyl-2-oxo-2H-benzo[h]chromene-8,10 dicarbalde-hyde in which the reactions were regioselective and products were in the keto-enamine form and the aromaticity of the relevant ring was disrupted [132]. Further, the efforts to bring the free aldehyde at the C-4 position into reaction failed both with increasing molar ratio of the alkyl amine and also by increasing the reaction temperature to reflux [134]. Coumarin Schiff-bases (CSB) possess-ing different substituents on the 4-methyl-2-substituted phenyl imino-2H-chromene-7-ol molecule were evaluated for their in vitro antioxidant and plausible anti-inflammatory potential [135]. The antioxidant studies of selected CSB were carried out by determining their reducing power, OH radical scavenging activity, scavenging of stable 2,2-diphenyl-1-picrylhydrazine (DPPH ) radical and inhi-bition of the polyphenol oxidase (PPO) enzyme. The assessment of possible anti-inflammatory potential was performed by trypsin in-hibition assay and inhibition of -glucuronidase. All the CSBs un-der study showed significant reducing effects. The majority of the tested CSB was found to be effective scavengers of DPPH radical with moderate to low OH scavenging ability and significantly inhibited the activity of PPO. With few exceptions, results from the inhibition assay of trypsin and -glucuronidase were not encourag-ing, however they may be helpful in defining structure-activity relationships in further optimization of the lead molecules. Presence of electrondonating groups and/or electronegative environment may be the possible reasons for the inhibition of trypsin by some compounds, however the same cannot be said for all the CSBs showing trypsin inhibitory activity [135]. Introduction of an oxy-imino type linkage at the allylic position with respect to the biogenetic C-3, C-4 double bond in the form of oximes, amidoximes, oxadiazoles, isoxazolines, etc. has resulted in compounds with promising antiproteolytic, antioxidant, and antiinflammatory properties [136].

The coumarin nucleus incorporates the styryl carbonyl moiety into a rigid framework, the presence of which in coumarin is expected to affect the scavenging of reactive substances derived from oxygen and influence the process involving free radical mediated injury [136]. Literature review reveals the importance of pyrazoles as antioxidants by inhibiting the oxidation of LDL [137]. Keeping in view the biological importance of arylhydrazonogroup, arylazogroup, pyrazolones, pyrazoles and coumarin nucleus, design

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of arylazopyrazoles and arylhydrazonopyrazolones containing coumarin moiety was carried out [138]. This was followed by the synthesis of target compounds 1-(4-methylcoumarinyl-7-oxy-acetyl)-3,5-dimethyl-4(arylazo)pyrazoles (36) and 1-(4-methylco-umarinyl-7-oxyacetyl)-3-methyl-4-(substituted phenyl) hydrazono-2-pyrazolin-5-ones (37) and their in vitro antioxidant screening. Compound with 4-bromophenylsubstituent in the phenyl ring exhibited highest antioxidant activity from pyrazole series and pyrazolin-5-one bearing 3-nitrosubstituent in the phenyl ring exhibited enhanced antioxidant activity from the respective series [138].

Among the fused coumarins, furocoumarins are important as photochemotherapeutic agents and exhibit antioxidant and anti-inflammatory activities [14]. Pyranocoumarins are used also as photoactive drugs and possess anti-inflammatory [14,101] and antioxidant [14,101] activities. Symeonidis et al. [139] reported the synthesis of angular [7,8]- and linear [6,7]-coumarins fused to dihydrofuro-, 1,3-dioxolo-, 5-membered- or 6-membered alicyclic ring. These compounds were tested in vitro for antioxidant activity and they were found to present significant scavenging activity. Many non-steroidal anti-inflammatory drugs have been reported to act either as inhibitors of free radical production or as radical scavengers. The design and synthesis of hybrid molecules encompassing two pharmacophores in one molecular scaffold is a well established approach to the synthesis of more potent drugs. Using this approach, several research groups have recently designed and synthesized hybrid molecules by coupling coumarins with a number of bioactive molecules including resveratrol [61,62,140] and these efforts resulted in new molecules endowed with antioxidant and other properties.

Nowadays, a lot of studies report complexes of coumarin de-rivatives with metals, which possess biological activity. Metal ions are very promising agents, capable to induce ROS by many differ-ent ways. Cerium(III) and neodymium(III) complexes with 3,3 -benzylidenebis[4-hydroxycoumarin] (38) were synthesized [141]. Their cytotoxicity toward cancerous cell cultures correlated with the weakness of the coordinative bond between the cation and or-ganic ligand and with the capability to scavenge superoxide radicals as well. The antioxidant properties of the complexes were estimated by monitoring their effects on the reduction of MTT in presence of the Xanthine/Xanthine Oxidase (X/XO) – in vitro derived superox-ide radical •O2

-. It was concluded that cerium (III) complex with

3,3 -benzylidenebis[4-hydroxycoumarin] might induce intracellular acidification along with control over the extracellular oxidative stress. The high antioxidant activity of the Ce (III) complex might explain the elimination of cancerous cells via apoptotic pathway. By eliminating superoxide radicals, the Ce (III) complex would locally diminish the extra-cellular H2O2 and would increase the opportunity for apoptotic death of the cancerous cells [141]. Some

copper chelates with coumarins have been studied for their antioxi-dant capacity by iron-induced chemiluminescence [142]. The cop-per complexes were potent antioxidants, comparable to butylated hydroxytoluene. The mechanism of the antioxidant action of these copper-coumarin chelates was found similar to that of Cu-Mn-superoxide dismutase, with a coumarin portion of the complex be-ing involved as a free radicals trap. Considerable effort has now been given to the functionalisation of coumarins so that metal-coumarin complexes may be synthesized towards the development of artificial photosynthetic systems, chemical sensors and molecular level devices. Group-6 tetracarbonyl complexes, [M(CO)4(L)] (M= Cr, Mo, W; L= N-[(2-Pyridyl)methyliden]-6-coumarin) were synthesized and characterized [143]. The ligand and the complexes are fluorescent. The compounds showed radical scavenging activity and were investigated spectrophotometrically using 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl (OH·), superoxide (O2

-) and

nitroxyl (NO·) radicals. The complexes exhibited potential antioxi-dant property both in cell-free and in-vitro studies and highest ac-tivity is observed with [W(CO)4(L)].

NATURAL COUMARINS AS ANTIOXIDANTS

Plants have formed the basis for the treatment of diseases in traditional medicine systems for thousands of years, and continue to play a major role in the primary health care of about 80 % of the World's inhabitants. While the natural products isolated as the ac-tive compounds might not be suitable for development as effective drugs, these can provide suitable leads for conversion into clinically useful agents.

Coumarins are present in plants in the free form and as glycosides. In general, coumarins are characterized by great chemical diversity, mainly differing in the degree of oxygenation of their benzopyrane moiety [95]. More than 300 coumarins have been identified from natural sources, especially from green plants [144]. Most of the compounds based on the coumarin nucleus include the anticoagulants like dicoumarol (39), warfarin (40), aflatoxins and the psoralens (photosensitizing agents). In nature, most coumarins are C-7 hydroxylated [145,146]. Major coumarin constituents included simple hydroxycoumarins (e.g., aesculin, esculetin, scopoletin, and escopoletin), furocoumarins and isofurocoumarin (e.g., psoralen (41) and isopsoralen (42) from Psoralea corylifolia), pyranocoumarins (e.g., xanthyletin (43), xanthoxyletin (44), seselin (45), khellactone (46), praeuptorin A), biscoumarins, dihydroiso-coumarins (e.g., bergenin (47)), and others (e.g., wedelolactone from Eclipta prostrata) [147,148]. Plants, fruits, vegetables, olive oil, and beverages (coffee, wine, and tea) are all dietary sources of coumarins; for example, seselin from fruit of Seseli indicum, khellactone from fruit of Ammi visnaga, and praeuptorin A from Peucedanum praeruptorum [149]. Cai et al. have found coumarins occurring in the medicinal herbs Umbelliferae, Asteraceae,

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Convolvulaceae, Leguminosae, Magnoliaceae, Oleaceae, Rutaceae, and Ranunculaceae, such as simple coumarins from A. annua, furocoumarins (5-methoxyfuranocoumarin, (48)) from Angelica sinensis, pyranocoumarins from Citrus aurantium, and isocoumarins from Agrimonia pilosa [96]. Some Indian medicinal plants (e.g., Toddalia aculeata, Murraya exotica, Foeniculum vulgare, and Carum copticum) and dietary spices (e.g., cumin and caraway) also possess coumarins [150]. In addition, coumestans, derivatives of coumarin, including coumestrol, (49) a phyto-estrogen, are found in a variety of medicinal and dietary plants such as soybeans and Pueraria mirifica. Natural phenolic compounds, including coumarins, play an important role in cancer prevention and treatment. Various bioactivities of phenolic compounds are responsible for their chemopreventive properties (e.g., antioxidant, anticarcinogenic, or antimutagenic and anti-inflammatory effects) and also contribute to their inducing apoptosis by arresting cell cycle, regulating carcinogen metabolism and oncogenesis expres-sion, inhibiting DNA binding and cell adhesion, migration, prolif-eration or differentiation, and blocking signaling pathways. The review of Huang et al. [95] covers the most recent literature to summarize structural categories and molecular anticancer mecha-nisms of phenolic compounds from medicinal herbs and dietary plants.

Recently, there has been a great interest in natural antioxidants as bioactive components of food, nutraceuticals or potential drugs against several diseases. Chemical investigation of the EtOH extract of Morus alba L. (Moraceae), as guided by free radical scavenging activity, furnished 5-hydroxy-7-methoxycoumarin and oxyresvera-trol (50) [151]. Compounds showed superoxide scavenging effects with the IC50 values of 19.1 ± 3.6 and 3.81 ± 0.5 μM, respectively. Oxyresveratrol exhibited a DPPH free radical scavenging effect (IC50 = 23.4 ± 1.5 μM) and showed hepatoprotective effect with EC50 value of 32.3 ± 2.62 μM on tacrine-induced cytotoxicity in human liver-derived Hep G2 cells [151]. Two new 5-methylcoumarin glycosides named diosfeboside A (51) and diosfe-

boside B (52) were isolated from the leaves of Diospyros crassi-flora (Hiern). Their structures were established and in vitro cyto-toxic activity of the new compounds against human carcinoma cell lines (HL-60, Bel-7402, BGC-823, and KB) was evaluated [152]. Eighty eight extracts from various parts of plants from European Asteraceae and Cichoriaceae were assayed for radical scavenging activity by means of DPPH (1,1-diphenyl-2-picryl hydrazyl radical) test using the SIA (sequential injection analysis) method developed for this purpose [153]. DPPH radical scavenging activity of all tested plant extracts was evaluated according to the IC50 parameter. Twenty nine extracts exhibited IC50 value lower than 0.1 mg/mL. The leaves of Leuzea carthamoides (IC50 = 0.046 mg/mL) were chosen as the most promising sample for a subsequent phytochemi-cal study, which resulted in the isolation of seven natural com-pounds. Antioxidant activity of the isolated compounds was evalu-ated by DPPH test and ferric reducing antioxidant power (FRAP) test and compared with trolox and quercetin [153]. Extracts of 32 plants from the Brazilian northeastern semi-arid region called Caat-inga were evaluated through DPPH radical scavenging assay, -carotene bleaching, and brine shrimp lethality tests (BST) [154]. Among the extracts studied, Byrsonima cf. gardneriana, Mascagnia coriacea, Cordia globosa, Diodia apiculata and Hypenia salzman-nii showed the highest activities in DPPH radical scavenging test. In the -carotene bleaching test the highest activities were observed for Passiflora cincinnata, Chamaecrista repens, B. cf. gardneriana, Rollinia leptopetala, Serjania glabrata, Diospyros gaultheriifolia, C. globosa, Mimosa ophtalmocentra, M. coriacea and Lippia cf. microphylla. In contrast, R. leptopetala, Zornia cf. brasiliensis and Leonotis nepetifolia were the most active species in the BST.

The bioassay-guided fractionation of the CHCl3 extract from pigeon pea leaves led to the isolation of a new natural coumarin, named as cajanuslactone (53), together with two known phytoalex-ins [155]. The structure of cajanuslactone was elucidated. It was determined as 7-hydroxy-5-O-methyl-8-(3-methyl-2-butylene)-4-phenyl-9,10-dihydro-benzopyran-2-one. The authors also reported

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the extraction, quantitative analysis and antioxidant activity test of extracts of pigeon pea leaves [156-158].

The aerial parts of several Angelica species of the Apiaceae as natural medicine, are rich sources of various coumarins with bio-logical, to a lesser extent, toxicological activities. From the chloro-form extract of the aerial parts of Angelica urumiensis MOZAFF, two new coumarins, together with six known coumarins and two known flavonoids were isolated [159]. On the basis of comprehen-sive spectroscopic analyses, the structures of the new compounds were established as pyranocoumarin dimer and (+)-8,9-dihydro-8-(2-hydroxypropan-2-yl)-2-oxo-2H-furo[2,3-h]chromen-9-yl-3-met-hylbut-2-enoate. The eight known compounds were isosamidin (54), laserpitin (55), pteryxin (56), isolaserpitin(57), cis-khellactone, angelicin, genkwanin (58) and salvigenin (59), respec-tively. These known structures are isolated from the aerial parts of A. urumiensis for the first time. Antioxidant activities of the two new coumarins were evaluated by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and exhibited a moderate antioxidant activity [159]. The dried root of Angelica gigas Nakai (Umbelliferae), Cham-Dang-Gui (the Korean name), has been traditionally used in oriental herbal medicines for thousands of years. The curative potential of A. gigas has led to efforts to isolate bioactive secondary metabolites from this plant, and several coumarin derivatives [160]. Among these coumarins, decursin (60) and decursinol (61) angelate are the most abundant components in the root of A. gigas [161] and have been shown to exhibit several pharmacological chemotherapeutic properties [161-169].

The antioxidant activity of eight coumarins and two flavonols isolated from Haplopappus multifolius was studied with the DPPH radical method [170]. Results show that a high concentration of phenolic coumarins and the presence of quercetin and rhamnetin in the exudates could account for the protection of the plant against oxidative stress. The structures of the coumarins 6-hydroxy-7-[(E,E)-3 ,7 -dimethyl-2 ,4 ,7 -octatrienyloxy] coumarin (62) and 7-[(E)-3 -methyl-4 -hydroxy-2 -butenyloxy] coumarin (63) are pro-posed on the basis of spectroscopic evidence. Antioxidant-guided fractionation of Mammea americana L. seeds resulted in the identi-fication of three new isoprenylated coumarins, mammea B/BA hydroxycyclo F (64), mammea E/BC (65), and mammea E/BD (66) [171]. In addition, twelve known isoprenylated coumarins as well as two known flavanols, (+)-catechin and (-)-epicatechin were iden-tified. The fifteen isoprenylated coumarins were screened for their

cytotoxicity in the SW-480, HT-29, and HCT-116 human colon cancer cell lines and antioxidant capacities in the DPPH (1,1-diphenyl-2-picrylhydrazyl) free-radical assay. Compounds exhib-ited significant cytotoxic activities and displayed high antioxidant activity in the DPPH assay (IC50 range 86 - 135 μM). The results of these assays were used to study the structure-activity relationships for this class of compounds. In the SW-480 cell line, the three new coumarins also exhibited dose-dependent increases in sub-diploid cells by flow cytometry, indicating that they induce apoptosis [171].

The fruits of wampee [Clausena lansium (Lour.) Skeels] con-tain a significant amount of coumarins with many health benefits [172]. The activity-guided separation of an ethyl acetate-soluble fraction on a polyamide column, followed by silica gel column and high performance liquid chromatography (HPLC) preparation af-forded a pure compound, which was identified to be 8-hydroxypsoralen (67) [172]. This isolate exhibited good scavenging activities against DPPH radical and superoxide anion as well as significant reducing power. It also showed potent proliferation in-hibitory activity against human hepatocellular liver carcinoma cell line (HepG2), human lung adenocarcinoma epithelial cell line (A549) and human cervical carcinoma cell line (HELA). This is the first report on the antioxidant and cytotoxic properties of C. lansium fruit extract [172]. Alpinia officinarum is a rhizome belonging to the family zingeberaeceae, cultivated in South-east Asia [173]. Qualitative phytochemical analysis of the extract of Alpinia offici-narum rhizome showed a majority of the compounds. The extracts showed a concentration dependent radical scavenging activity by inhibiting diphenylpicrylhydrazyl free radical. The aqueous alco-holic extract prepared by hot maceration process showed better reducing and total antioxidant activity.

Four new compounds together with 16 other known compounds including coumarins were isolated from Ajania przewalskii [174]. The novel sesquiterpenolide possesses a rare carbon skeleton. Cyto-toxicity of the compounds was assessed by their effects on selected cancer cell lines, K562, K562/ADM, BGC-823, and Hep-G2 cells. Radical-scavenging activities of the compounds were determined by ABTS and DPPH radical-scavenging assays. Phenols and cou-marins displayed significant antioxidant properties (IC50 < 20 μg/ml) [174]. The potent antioxidant activity of A. przewalskii might be attributed to the presence of the important polyphenolics, flavonoids, and coumarin compounds, which are attractive additives for the food and drug industry and are of great interest for use in complementary medicine supplements. Ryu et al. [175] isolated 18

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polyphenols with neuraminidase inhibitory activity from methanol extracts of the roots of Glycyrrhiza uralensis. These polyphenols consisted of four chalcones, nine flavonoids, four coumarins, and one phenylbenzofuran. Structure–activity analysis showed that the furan rings of the polyphenols were essential for neuraminidase inhibitory activity, and that this activity was enhanced by the apioside group on the chalcone and flavanone backbone. In addi-tion, the presence of a five-membered ring between C-4 and C-2 in coumestan (68) was critical for neuraminidase inhibition. All neuraminidase inhibitors screened were found to be reversible, non-competitive inhibitors. The antioxidant properties of methanol extracts of Lady’s Bedstraw (Galium verum L., Rubiaceae) herb from two different localities in Serbia were evaluated [176].

Antioxidant activity was assessed in four different model systems. Free radical scavenging capacity (RSC) was examined by measuring the scavenging activity of extracts on 2,2-diphenyl-1-pycrylhydrazil (DPPH) and hydroxyl radical (OH), as well as on hydrogen peroxide. In addition, the protective effects of lipid peroxidation (LP) in corn oil were evaluated by the TBA-assay using the Fe

2+/ascorbate system of induction. The amount of dried

extract, the content of total phenolics, flavonoids and chlorophylls was also determined. Extracts from both locations expressed very strong scavenger activity, reducing the DPPH (IC50=3.10 g/ml and 8.04 g/ml) and OH radical formation (IC50= 0.05 g/ml and 0.54

g/ml) and neutralising H2O2 (IC50= 4.98 g/ml and 3.80 g/ml), in a dose dependant manner. Also, examined extracts showed notable


inhibition of LP (IC50= 11.69 g/ml and 19.47 g/ml) [176]. The ethanolic extract of Symphyopappus casarettoi syn., Eupatorium casarettoi containing coumarins, was partitioned in hexane, chloro-form and methanolic fractions [177]. Extract and fractions were tested for their antioxidant activity in vitro and ex vivo assays. The methanolic fraction showed a higher antioxidant potential compared to the others fractions, which was correlated with its total phenol content. In addition, the ethanolic extract and the methanolic frac-tion attenuated ex vivo iron-induced cell death, quantified by lactate dehydrogenase leakage, and effectively protected against lipid damage induced by iron. These findings suggest that the ethanolic extract of S. casarettoi inflorescence and its methanolic fraction have in vitro and ex vivo antioxidant properties. Moussonia deppeana (Schldl. & Cham) Hanst is a species of Mexican Medicinal Flora used in Veracruz state. EtOAc extract was the most active in free radical scavenging test DPPH (CI50 18.3±3.4 μg/mL). The phytochemical screening revealed the presence of phenolic compounds (flavonoids and coumarins) in EtOAc and EtOH extracts [178]. Anti-inflammatory activity was evaluated by topical application of the extracts (doses 2 mg/ear) giving a greater inhibition in hexane and EtOAc extracts. The model of paw edema was evaluated in EtOAc extract, observing a similar inhibition to indomethacin. These results support the biological effect attributed in their traditional use. Antioxidant activity assay-guided chemical analysis, using a rat pancreas homogenate model, of aerial parts from Eysenhardtia subcoriacea, led to the isolation of the new compound subcoriacin [3-(2 -hydroxy-4 ,5 -methylendioxyphenyl)-6-(3 -hydroxymethyl-4 -hydroxybut-2 -enyl)-7-hydroxycoumarin] (69) together with the known substances: (+)-catechin, ( )-epicatechin, (+)-afzelechin (70), eriodictyol (71), (+)-catechin--O-

-d-galactopyranoside and quercetin 3-O- -d-galactopyranoside as bioactive constituents [179]. The bioactive compounds showed moderate to strong radical scavenging properties against diphen-ylpicrylhydrazyl radical (DPPH). In addition, subcoriacin, (+)-catechin, ( )-epicatechin and (+)-afzelechin improved the reduced glutathione levels in rat pancreatic homogenate. Grapefruit juice has been shown to increase the oral bioavailability of several clini-cally important drugs by inhibiting first pass metabolism [180]. Several compounds in grapefruit juice have shown different bio-logical activities. Unique among them are furocoumarins with po-tent inhibitory activity against cytochrome P450 enzymes. Two bioactive compounds were isolated from grapefruit juice and grape-fruit peel oil [180]. Structures of the compounds were elucidated and identified as bergaptol (72) and geranylcoumarin (73). The isolated compounds were tested for their radical scavenging activity

using 2,2 -azobis (3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazil (DPPH) methods at different concentrations. Bergaptol showed very good radical scavenging activity at all the tested concentrations. Furthermore, these com-pounds were evaluated for their inhibitory activity against CYP3A4 enzyme. Bergaptol and geranylcoumarin were found to be potent inhibitors of debenzylation activity of CYP3A4 enzyme with an IC50 value of 24.92 and 42.93 μM, respectively.

The isocoumarins, paepalantine (9,10-dihydroxy-5,7-dime-thoxy-1H-naptho(2,3c)pyran-1-one) (74), 8,8 -paepalantine dimer, and vioxanthin (75) isolated from Paepalanthus bromelioides, were assessed for antioxidant activity using isolated rat liver mitochon-dria and non-mitochondrial systems, and compared with the flavon-oid quercetin [181]. The paepalantine and paepalantine dimers, but not vioxanthin, were effective at scavenging both 1,1-diphenyl-2-picrylhydrazyl (DPPH•) and superoxide radicals in non-mitochondrial systems, and protected mitochondria from tert-butylhydroperoxide-induced H2O2 accumulation and Fe2+-citrate-mediated mitochondrial membrane lipid peroxidation, with almost the same potency as quercetin. These results point towards pa-epalantine, followed by paepalantine dimer, as being a powerful agent affording protection, apparently via superoxide radicals scav-enging, from oxidative stress conditions imposed on mitochondria, the main intracellular source and target of those reactive oxygen species. This strong antioxidant action of paepalantine was repro-duced in HepG2 cells exposed to oxidative stress condition induced by H2O2 [181]. Acacia confusa Merr. (Leguminosae) is traditionally used as a medicinal plant in Taiwan [182]. The XOD-inhibitory activity of ethanolic extracts from A. confusa was investigated for the first time. Results demonstrated that the ethanolic extract of A. confusa heartwood had a strong XOD-inhibitory activity. Okanin and melanoxetin showed excellent inhibition on XOD in non-competitive and competitive mode, respectively, and their inhibi-tory activity is better than that of allopurinol. This is the first study that demonstrates the XOD-inhibitory performance of okanin and melanoxetin [182]. Cucurbita maxima Duch. ex Lam.( Cucurbitaceae) known as Dadhiphala in Sanskrit, contains triterpenoids, flavonoids, coumarins, saponins, cucurbitacins, carotenoids, vitamins, minerals and amino acids. Attarde et al. [183] evaluated in vitro antioxidant activities of petroleum ether, chloroform and methanolic extract of pericarp of C.maxima. All extracts were tested for DPPH radical scavenging, nitric oxide radical scavenging and hydrogen peroxide scavenging activities. Cortex fraxini was extracted with 95 % ethanol to obtain a crude antioxidant extract [184]. The antioxidant activity was evaluated

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O

O


using the linoleic acid peroxidation method and the free radical scavenging assays, namely 2,2 -diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radicals. Cortex fraxini extract (CFE) showed high inhibition of peroxidation of linoleic acid when compared with butylated hydroxytoluene (BHT). CFE also exhibited excellent scavenging activity on DPPH and hydroxyl radicals. Total antioxi-dant activity was measured by the reduction of Mo(VI) to Mo(V) by the extract, and subsequent formation of a green phos-phate/Mo(V) complex at acidic pH. CFE had significant total anti-oxidant activity and the effects were increased with increasing reac-tion time. The suitability of CFE as an antioxidant was determined in peanut oil, and the decrease of lipid oxidation was monitored using thiobarbituric acid-reactive substances (TBARS) assay. CFE treatment significantly (P < 0.05) reduced lipid oxidation in peanut oil compared to the control. No significant differences (P = 0.05) in lipid oxidation were detected between CFE antioxidant and BHT antioxidant samples [184]. Stingless bee honey samples from west Amazonian Ecuador were studied for their physiochemical, chemi-cal and functional properties [185]. Coumarins and flavonoids were determined by densitometric HPTLC: fraxin and bergamotin (76) among coumarins & luteolin, quercitrin and isoramnetin among flavonoids. Among the vitamin E isomers, evaluated by HPLC, the occurrence of only -tocopherol was noted. All these results were compared with those acquired for two multifloral Apis mellifera honeys. DPPH and -carotene bleaching tests were performed, showing interesting values for Ecuadorian honey samples, higher than those shown by multifloral A. mellifera honeys. Ecuadorian Meliponinae honey samples also showed anti-mutagenic activity assayed with Saccharomyces cerevisiae D7 strain, inhibiting back mutation over the entire range of concentrations. The acetone ex-tract of the aerial parts of the plant Salvia cedronella Boiss. was screened for its total phenolic content and flavonoid content [186]. The antioxidant potential was evaluated, in vitro by using three different assays; -carotene–linoleic acid test system for total anti-oxidant activity, DPPH for free radical scavenging activity, Fe

2+–

ferrozine test system for metal chelation. A high content of phe-nolics, potent radical scavenging ability and significant iron chelat-ing effect were observed. However, the inhibition of lipid peroxida-tion was not significant in -carotene–linoleic acid test system. A phytochemical analysis yielded a new coumarin, 3-methoxy-4-hydroxymethylcoumarin, together with p-hydroxyphenylethyl docosanoate, and two triterpenoids, namely oleanolic acid and betu-linic acid. Mansonones (mansorin A (77) and mansorin B (78)) isolated from the heartwood of Mansonia gagei were tested for their antifungal activities against Cladosporium cucumerinum and Candida albicans, as well as for their larvicidal activities against Aedes aegypti and radical scavenging properties in a DPPH assay [187].

A variety of bisbenzyl coumarins isolated from the Chinese herbs were tested for antioxidant activity as reflected in the ability to inhibit lipid peroxidation in rat brain and kidney homogenates and rat erythrocyte hemolysis [188]. The pro-oxidant activities of the compounds were assessed by their effects on bleomycin-induced DNA damage. The coumarin xanthotoxol (79) exhibited potent antioxidative activity in both lipid peroxidation and hemoly-sis assays. The coumarins bergapten (80) and angelicin (81) slightly inhibited lipid peroxidation in brain and kidney homogenates. It is worth stressing that the compounds with antioxidant effects in this assay have at least one free aromatic hydroxyl group in their struc-ture. Obviously, the aromatic hydroxyl group is very important for antioxidative effects of the compounds.

Many new compounds were isolated from different plants and tested for their antioxidative activity [189-191]. The structure-activity relationships among the identified coumarin analogs sug-gest that hydroxyl groups at the C-6 and C-7 positions of the cou-marin skeleton played an important role in the expression of ty-rosinase inhibitory activity. Phytochemical studies on the aerial parts of Prangos uloptera, an endemic Iranian species of the genus Prangos, yielded five coumarins – xanthotoxin (82), prangenin (83), scopoletin, deltoin (84) and prangolarin (85) [192]. The structures of these coumarins were elucidated by spectroscopic means, and their antioxidant potential was evaluated by the DPPH assay. Their chemotaxonomic significance was also discussed. In the quantitative DPPH assay, scopoletin was the most active among all compounds, and displayed a significant antioxidant activity. Scopoletin and cleomiscosins A (86), C (87), and D (88), have been isolated from the root bark of Hibiscus syriacus [193]. Scopoletin inhibited monoamine oxidase with moderate IC50 values. Cleomis-cosin C showed lipid peroxidation inhibitory activity comparable to vitamin E. The quantification of coumarin derivatives such as sco-poletin, 7-hydroxycoumarin and 4-hydroxycoumarin in Noni (Morinda citrifolia) was described by Ikeda et al. [194]. Further-more, the quenching effects of Noni products and coumarin deriva-tives on reactive oxygen species were evaluated by a luminol chemiluminescent assay. Both Noni samples and coumarin deriva-tives dose-dependently quenched ROS such as superoxide (O2

-),

singlet oxygen (1O2), hydroxyl radical ( OH) and peroxynitrite

(ONOO ). The EC50 of scopoletin for O2-,

1O2, OH and ONOO

were 1.27 ± 0.22 mg/ml, 0.68 ± 0.04 mg/ml, >4.00 mg/ml, and 0.042±0.002 mg/ml, respectively. The contribution ratio of sco-poletin for ROS in Noni juices was also evaluated. Nahata et al. [195] investigated the effects of Evolvulus alsinoides (EA) on learn-ing and memory in rodents. The ethanol extract of EA and its ethyl acetate and aqueous fractions were evaluated for their memory enhancing properties. The chromatographic analysis of the ethanol extract of Evolvulus alsinoides led to the isolation of a marker

O

OOHOH

MeO

OMe

74

O

OHOHO

OMeMeO

O

OOHOH

MeO CH3

75

O

OO O

76

OO

OMe

77

OO

OH

78


compound, i.e. scopoletin. The presence of scopoletin is reported for the first time in this plant [195]. The presence of this furanocoumarin in EA further strongly support the view of involvement of antioxidant mode of action and the memory potentiating and cognition enhancing activity as reflected by the extracts. The occurrence of scopoletin has been reported in Convulvulus microphyllus and to have memory enhancing properties [196]. Hornick et al. [196] evaluated the effects of scopoletin on learning and memory, on the release of acetylcholine from brain synaptosomes and on long term potentiation in the hippocampus. Scopoletin has also been reported to possess sufficient anticholinergic potential [197]. Compounds, such as scopoletin and rutin are considered as key compounds of noni fruits [198,199]. In in vitro and in vivo (animal) experiments, noni fruit extracts or juice scavenges free radicals, inhibits oxidation of low-density lipoproteins, and shows anticancer and anti-inflammatory activity, and stimulates the immune system [194, 200]. Total phe-nolics, ascorbic acid, and antioxidant capacity of noni (Morinda citrifolia L.) juice and powder and the effects of light on its antioxidant capacity during storage under various conditions were recently determined [201]. The structure-activity relationships of the antioxidant activity of natural coumarins isolated from four Korean medicinal plants (F. rhynchophylla, A. dahuria, E. daniellii and P. japonicum) and four other coumarins has been recently stud-ied [202]. The free radical scavenging and lipid peroxidation assays revealed that five phenolic coumarins, scopoletin, esculetin, fraxetin, umbelliferone and daphnetin, possessed considerable anti-oxidant activities. The coumarins having a catechol group showed significant free radical scavenging activity and inhibitory effects on

lipid peroxidation, indicating that the catechol group significantly contributed to the antioxidant activities of coumarins. In contrast, the sugar moiety markedly reduced the activities of coumarin gly-cosides. The results also demonstrate that the -pyrone ring of coumarins significantly enhanced the capacity of inhibiting oxida-tive reactions of coumarins [202]. Many other reports on naturally occurring coumarins and their biological role appear in the litera-ture [203,204].

Tumor-modulating effects of coumarin antioxidants also have been studied using carcinogens [205,206]. Nishiyama et al. [205] compared the antioxidative activities of several hydrocoumarins with those of alpha-tocopherol for the oxidation of tetralin and lino-leic acid in a homogeneous solution. Hydrocoumarins exhibited a higher induction period than that of alpha-Toc in both systems. 6,7-Ddihydroxy-4,4-dimethylhydrocoumarin [(89), refer. Table 1] showed less cytotoxicity toward human fibroblasts than did 2,6-di-t-butyl-4-methylphenol. An antioxidant auraptene (7-geranyloxy-coumarin) [(90), refer Table 1] isolated from the peel of citrus fruit

(Citrus natsudaidai Hayata) has been reported to have chemopre-ventive

effects on chemically induced carcinogenesis [206]. The

findings suggested that auraptene may exert a part of its cancer

chemopreventive activity through enhancement of immune func-

tion. Further, auraptene isolated from the citrus fruit has demon-

strated its anti-tumor promoting effect in mouse skin and anti-

carcinogenesis activities in the rat tongue, esophagus and colon

[207]. The first report on the occurrence of auraptene, imperatorin

(91) and 7-prenyloxycoumarin (92) in the genus Zosima appeared recently [208]. Zosima absinthifolia belongs to the Apiaceae family and is found in Iran, Turkey, Iraq and different countries of the

OO O

OMe

80

O OO

81

OO O

OMe

79

OO O

OMe82

O

O

O

83

O O

84

O OOO

O

O

OO O

85

O OO

O

OH

CH3

HOH2C

86

H3CO

87

O O

O

HO OH

OMe

OMe

O

OMe

OO

OMe

88

O

O

OH

OMeMeO

OH


Caucasus, Middle East and Central Asia. The fruits are used as food flavoring and as a food spice in Iran. The results [208] indicated that auraptene, imperatorin and 7-prenyloxycoumarin, especially imperatorin exhibited fungi toxic activity against Sclerotinia scle-rotiorum, a common plant pathogen.

Naturally occurring coumarins possess anti-carcinogenic activi-ties in part by inducing carcinogen-detoxifying enzymes, viz glutathione S-transferase (GST) and/or NAD(P)H quinone oxido-reductase (NQO1). Prince et al. [209] examined a broader range of naturally occurring citrus coumarins for their abilities to activate the antioxidant response element (ARE) and to induce GST and NQO1. Oltipraz, imperatorin, isopimpinellin (93), and auraptene all significantly increased liver cytosolic GST activities in Nrf2 heterozygous mice. Of these compounds, only isopimpinellin significantly increased liver cytosolic NQO1 activities, and this effect was not attenuated in Nrf2( / ) mice. These results strongly suggested that imperatorin and auraptene induce murine liver cytosolic GST activities via the Nrf2/ARE mechanism.

Structurally diverse compounds can confer resistance to afla-toxin B1

hepatocarcinogenesis. It has been reported that feeding

with a diet containing coumarin can prevent the initiation of Afla-toxin-B1 (AFB1) hepatocarcinogenesis in association with the induction of AFB1-aldehyde reductase (AFAR) and m-class glutathione transferase GSTA5, p-class GSTP1 and NAD(P)H quinine oxido-reductase in rat liver [210,211]. The study

showed

that coumarin is highly effective at inducing not only AFAR and

GSTA5, but also certain other drug-metabolizing enzymes. On the

basis of this information, the hypothesis that enzyme induction by

coumarin would confer resistance to aflatoxin B1 tumorigenesis

was

tested in the rat. The results from dietary intervention showed that

coumarin consumption does indeed provide protection against ini-

tiation of aflatoxin B1 hepatocarcinogenesis. The data presented

[210] also reveal the ability of different phytochemicals and syn-

thetic drugs to induce different enzymes in the liver in zone- and

sex-specific fashions. Therefore, one of the effective methods to overcome the toxic and carcinogenic effects of AFB1 is to enhance AFB1 metabolism toward its detoxification in humans or animals. There have been some trials of the intervention of chemo-protective agents against AFB1-induced DNA damage and carcinogenesis by inducing class I and II enzymes [212]. Recently, to clarify whether enzymes involved in AFB1 metabolism in pigs respond to

Table 1. Structures of Coumarins Used as Antioxidants

Compound R3 R4 R5 R6 R7 R8

1 Coumarin H H H H H H

2 7-hydroxycoumarin (Umbelliferone) H H H H OH H

3 6,7-dihydroxycoumarin (Esculetin) H H H OH OH H

4 Esculin H H H OGl OH H

5 7,8-Dihydroxycoumarin H H H H OH OH

6 7,8-dihydroxy-4-methylcoumarin H Me H H OH OH

10 4-methyl-7-hydroxycoumarin (Mendiaxon) H Me H H OH H

12 4-methyl-6,7-dihydroxycoumarin H Me H OH OH H

13 7,8-dihydroxy-6-methoxycoumarin (Fraxetin) H H H OMe OH OH

16 4-methylcoumarin H Me H H H H

17 Scopoletin H H H OMe OH H

18 7,8-diacetoxy-4-methylcoumarin H Me H H acetoxy acetoxy

23 3-hydroxycoumarin OH H H H H H

24 3-aminocoumarin NH2 H H H H H

25 3-acetylaminocoumarin acetylamino H H H H H

26 Coumarin-3-carboxylic acid COOH H H H H H

27 3-hydroxyscopoletin OH H H OMe OH H

28 3-hydroxyisoscopoletin OH H H OH OMe H

29 3,7-dihydroxycoumarin (3-hydroxyumbelliferone) OH H H H OH H

31 4-hydroxycoumarin H OH H H H H

89 6,7-dihydroxy-4,4-dimethylhydrocoumarin 2H 2Me H OH OH H

90 Auraptene H H H H OGer H

O O

O

O

91 92

OO

OOO

O

O93

O


antioxidant agents, the effect of feeding piglets with diets containing green tea extracts (sunphenon) or coumarin on in vitro AFB1 metabolism by their liver and intestinal tissues was studied [213]. It was concluded that coumarin may affect AFB1 metabolism towards the enhancement of detoxification through the suppression of P450 enzymes in the liver and intestine, and through the enhancement of GST toward AFB1 in the intestine. The effect of the green tea extracts (sunphenon) was not as prominent as that of coumarins.

CONCLUSION

Coumarins occupy an important place in the realm of natural products and synthetic organic chemistry. As substitutions can occur at any of the six available sites of their basic molecular moiety, these compounds are extremely variable in structure and activity. This structural diversity leads to coumarins displaying multiple biological properties that promote human health and help reducing the risk of diseases. Of the many actions of coumarins, antioxidant and antiproliferative effects stand out.

A large number

of structurally novel coumarin derivatives have been reported to show substantial antioxidant and cytotoxic activity in vitro and in vivo. Moreover, the inhibitory action on inflammatory cells appears to surpass any other clinically available

compounds. A number of

coumarins were found to affect the formation and scavenging of reactive oxygen species, and reactive nitrogen species, exhibiting tissue-protective antioxidant properties, which may include numerous different molecular mechanisms and are probably related to their structural analogy with flavonoids and benzophenones. Such studies are of importance in view of the presence in the human diet of many coumarins and other plant polyphenolics, some of which are attracting interest on account of their antioxidant properties and possible roles in therapy and for food preservation. Given that certain substituents are known to be required

or increase

their actions, the therapeutic potential of select coumarins

is fairly

obvious. The recognition of key structural features within coumarin family is crucial for the design and development of new analogues with improved activity and for the characterization of their mechanism of action and potential side effects. Although some coumarins have already been characterized to evoke a particular biological activity, the challenge would be the design and synthesis of new derivatives with high specific activity and define their mechanism of action to achieve new therapeutic drugs. The present review highligts the current progress in the development of coumarin scaffolds for drug discovery as novel antioxidant agents. The major challenges about coumarins include the translation of current knowledge into new potential lead compounds and the repositioning of known compounds for the medical treatment.

ABBREVIATIONS

RS = reactive species

OS = oxidative stress

DPPH = 1,1-diphenyl-2-picryl-hydrazyl

ODC = ornithine decarboxylase

TBHQ = tert-butylhydroquinone

DPH-PA = 3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid

t-BHP = t-butyl hydroperoxide

LDH = lactate dehydrogenase

ALT = alanine transaminase

MDA = malondialdehyde

TAC = total antioxidant capacity

PBL = peripheral blood lymphocytes

LDL = low-density lipoprotein

AAPH = 2,2'-azobis(2-amidinopropane hydrochloride)

BP = benzophenone

DSBP = 3,3'-disulfobenzophenonate

TOH = alpha-tocopherol

SECCA = the succinimidyl ester of coumarin-3-carboxylic acid

KHB = Krebs-Henseleit bicarbonate

NK = natural killer cells

GST = glutathione S-transferase

AFAR = Aflatoxin B1 aldehyde

reductase

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Received: April 26, 2011 Revised: July 19, 2011 Accepted: July 21, 2011

coumarins as antioxidants

Documents

natural coumarins

reactive oxygen species

development of coumarins

lot of coumarins

introduction free radicals

reactive nitrogen species

reactive sulphur species

ros damage