Coumestan Inhibits Radical-Induced Oxidation of DNA: Is Hydroxyl a

Article pubs.acs.org/JAFC

Coumestan Inhibits Radical-Induced Oxidation of DNA: Is Hydroxyl a Necessary Functional Group? Gao-Lei Xi and Zai-Qun Liu* Department of Organic Chemistry, College of Chemistry, Jilin University, Changchun 130021, China S Supporting Information *

ABSTRACT: Coumestan is a natural tetracycle with a CC bond shared by a coumarin moiety and a benzofuran moiety. In addition to the function of the hydroxyl group on the antioxidant activity of coumestan, it is worth exploring the influence of the oxygen-abundant scaffold on the antioxidant activity as well. In this work, seven coumestans containing electron-withdrawing and electron-donating groups were synthesized to evaluate the abilities to trap 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS•+), 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH), and galvinoxyl radical, respectively, and to inhibit the oxidations of DNA mediated by •OH, Cu2+/glutathione (GSH), and 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH), respectively. It was found that all of the coumestans used herein can quench the aforementioned radicals and can inhibit •OH-, Cu2+/GSH-, and AAPH-induced oxidations of DNA. In particular, substituent-free coumestan exhibits higher ability to quench DPPH and to inhibit AAPH-induced oxidation of DNA than Trolox. In addition, nonsubstituted coumestan shows a similar ability to inhibit •OH- and Cu2+/GSH-induced oxidations of DNA relative to that of Trolox. The antioxidant effectiveness of the coumestan can be attributed to the lactone in the coumarin moiety and, therefore, a hydroxyl group may not be a necessary functional group for coumestan to be an antioxidant. KEYWORDS: coumestan, antioxidant, free radical, DNA oxidation

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INTRODUCTION

especially substituent-free coumestans, are not usually reported.5 In previously published studies, the pharmacological activities of coumestans are regarded as the contributions from hydroxyl groups.6 In particular, the hydroxyl group attached to the benzofuran (ring D) mainly controls the redox state7 and thus plays the major role in the biological activities for coumestans.8 However, the influence of substituents at ring D on the antioxidant effectiveness remains unclear. In particular, it is worth clarifying the function of the coumestan scaffold itself (without any substituents attached) on the antioxidant effectiveness. These backgrounds encourage us to investigate the antioxidant effectiveness of coumestan with and without the substituent attached to ring D and to clarify whether the hydroxyl group is a necessary substituent for generating the antioxidant effectiveness. The radical-induced oxidation of DNA is a suitable experimental system for estimating the antioxidant effectiveness of natural or synthetic compounds.9 •OH, Cu2+/glutathione (GSH), and 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH) are usually used as radical initiators because they are able to mimic DNA undergoing oxidation caused by hydroxyl, glutathione, and peroxyl radicals. In addition, as the stable radicals at room temperature, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS•+), 2,2′-diphenyl-1picrylhydrazyl radical (DPPH), and galvinoxyl radical are applied to evaluate the ability of an antioxidant to donate its hydrogen atom to N- and O-centered radicals. Therefore,

Coumestan is a naturally occurring skeleton with a coumarin moiety and a benzofuran moiety sharing a CC bond (structure in Scheme 1). 2-Methoxy-3,8,9-trihydroxy coumestan, also called as wedelolactone, is the typically natural coumestan and exhibits various biological activities1 such as antifibrotic,2 antiadipogenesis,3 and immunosuppressive effects.4 However, the antioxidant effects of coumestans, Scheme 1. Coumestans Synthesized in This Work

Received: January 2, 2014 Revised: June 1, 2014 Accepted: June 2, 2014

© XXXX American Chemical Society

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dx.doi.org/10.1021/jf500013v | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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yield 86%; mp 213−215 °C; 1H NMR (400 MHz, CDCl3) δ 7.97 (d, J = 7.6 Hz, 1H), 7.48−7.59 (m, 3H), 7.40 (t, J = 7.6 Hz, 1H), 7.21 (s, 1H), 4.01 (s, 3H), 4.00 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 158.7, 158.3, 152.9, 150.2, 149.5, 148.1, 131.0, 124.5, 121.2, 117.3, 115.2, 112.8, 106.1, 102.1, 95.4, 56.4, 56.3. 9-Chloro-6H-benzofuro[3,2-c]chromen-6-one (4): Rf = 0.32 (ethyl acetate/petroleum ether = 1:19, v/v), 0.59 g of white product, yield 87%; mp 242−244 °C; 1H NMR (400 MHz, CDCl3) δ 8.01−8.06 (m, 2H), 7.69 (s, 1H), 7.63 (t, J = 8.0 Hz, 1H), 7.50−7.53 (m, 1H), 7.41− 7.47 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 160.5, 157.7, 155.5, 153.8, 132.7, 132.3, 130.7, 128.8, 126.1, 124.8, 122.4, 121.9, 117.6, 112.5, 112.4. 9-Bromo-6H-benzofuro[3,2-c]chromen-6-one (5): Rf = 0.39 (ethyl acetate/petroleum ether = 1:9, v/v), 0.42 g of white product, yield 53%; mp 213−215 °C; 1H NMR (400 MHz, CDCl3) δ 8.03 (t, J = 8.0 Hz, 2H), 7.87 (d, J = 1.2 Hz, 1H), 7.61−7.68 (m, 2H), 7.53−7.55 (m, 1H), 7.45 (t, J = 7.6 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 160.3, 157.7, 155.6, 153.8, 132.3, 128.8, 124.8, 122.7, 122.6, 121.9, 120.1, 117.6, 115.4, 112.3, 105.6. 9-Hydroxy-6H-benzofuro[3,2-c]chromen-6-one (6): Rf = 0.29 (ethyl acetate/petroleum ether = 1:2, v/v), 0.24 g of white product, yield 96%; mp 291−293 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.16 (s, 1H), 8.03 (s, 1H), 7.78 (s, 1H), 7.69 (s, 1H), 7.60 (m, 1H), 7.49 (s, 1H), 7.22 (s, 1H), 7.00 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 158.1, 157.7, 157.1, 156.4, 152.5, 131.5, 124.9, 121.2, 121.1, 117.0, 114.4, 114.3, 112.1, 105.3, 98.6. 8,9-Dihydroxy-6H-benzofuro[3,2-c]chromen-6-one (7): Rf = 0.18 (ethyl acetate/petroleum ether = 1:2, v/v), 0.24 g of white product, yield 91%; mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.65 (s, 1H), 9.53 (s, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.64−7.68 (m, 1H), 7.57− 7.59 (m, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.30 (s, 1H), 7.24 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 158.2, 157.9, 152.8, 149.9, 147.0, 145.2, 131.7, 125.4, 121.6, 117.5, 114.3, 112.9, 106.0, 105.4, 99.4. Scavenging ABTS•+, DPPH, and Galvinoxyl Radicals. A 2.0 mL of aqueous solution containing 4.0 mM ABTS and 1.41 mM K2S2O8 was kept for 20 h to form ABTS•+ and then diluted by 100 mL of ethanol. The absorbance of ABTS•+ was around 1.00 at 734 nm (εABTS•+ = 1.6 × 104 M−1 cm−1). DPPH and galvinoxyl radicals were dissolved in ethanol directly. The absorbances of DPPH and galvinoxyl radicals were around 1.00 at 517 nm (εDPPH = 4.09 × 103 M−1 cm−1) and 428 nm (εgalvinoxyl = 1.4 × 105 M−1 cm−1), respectively. A certain concentration of DMSO solution of a coumestan (0.1 mL) was added to 1.9 mL of ABTS•+, DPPH, and galvinoxyl radical solutions, respectively. The decreases of the absorbance of these radicals were recorded at 25 °C at a certain time interval. Cu2+/GSH- and •OH-Induced Oxidations of DNA. Cu2+/GSHinduced oxidation of DNA was carried out according to the following description. Briefly, DNA, CuSO4, and GSH were dissolved in phosphate-buffered solution (PBS1: 6.1 mM Na2HPO4, 3.9 mM NaH2PO4), and coumestans were dissolved in dimethyl sulfoxide (DMSO) as the stock solution. Then, a solution of 2.0 mg/mL DNA, 5.0 mM Cu2+, 3.0 mM GSH, and 0.2 mM coumestan was poured into test tubes with each containing 2.0 mL. The test tubes were incubated at 37 °C for initiating the oxidation of DNA. Three test tubes were taken out at 90 min and cooled immediately. PBS1 solution of EDTA (1.0 mL, 30.0 mM) was added to chelate Cu2+, followed by adding 1.0 mL of thiobarbituric acid (TBA) solution (1.00 g of TBA and 0.40 g of NaOH dissolved in 100 mL of PBS1) and 1.0 mL of 3.0% trichloroacetic acid aqueous solution. The test tubes were heated in boiling water for 30 min and cooled to room temperature, and 1.5 mL of n-butanol was added and shaken vigorously to extract thiobarbituric acid reactive substance (TBARS) for measuring the absorbance at 535 nm. • OH can be generated in the mixture of H2O2 and tetrachlorohydroquinone (TCHQ, dissolved in DMSO as the stock solution). DNA and H2O2 were dissolved in phosphate-buffered solution (PBS2: 8.1 mM Na2HPO4, 1.9 mM NaH2PO4, 10.0 μM EDTA). A solution of 2.0 mg/mL DNA, 4.0 mM TCHQ (dissolved in DMSO as the stock solution), 2.0 mM H2O2, and 0.2 mM coumestan (dissolved in DMSO as the stock solution) was poured into test tubes with each containing

presented herein is a study on the antioxidant effects of seven coumestans with electron-withdrawing groups (−Cl, −Br) and electron-donating groups (−OCH3 and −OH). The abilities to quench ABTS•+, DPPH, and galvinoxyl radical, respectively, and to inhibit the oxidations of DNA caused by •OH, Cu2+/ GSH, and AAPH, respectively, are estimated to reveal the role of the coumestan scaffold in the generation of antioxidant effectiveness.

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MATERIALS AND METHODS

Materials and Instrumentation. ABTS, DPPH, and galvinoxyl radicals were purchased from Fluka Chemie GmbH, Switzerland, and AAPH and naked DNA sodium salt were purchased from Acros Organics, Belgium. Other agents were of analytical grade and used directly. The structures of products were identified by 1H and 13C NMR (Bruker Avance III 400 MHz sprctrometer), and the spectra are included in the Supporting Information. Synthesis and Identification of Coumestan Structure. Esterification of Methyl Salicylate. To a mixture of methyl salicylate (1.52 g, 10 mmol) and the corresponding phenylacetic acid (11 mmol) in 10 mL of pyridine at 0−10 °C was added dropwise POCl3 (1.1 mL, 12 mmol) within 0.5 h under stirring. After the above mixture was stirred for 3 h at 0−10 °C, the concentrated HCl was added to adjust pH 5−6 at the same temperature. The mixture was extracted by ethyl acetate at room temperature, and the organic phase was washed by brine and dried over Na2SO4. After the solvent was evaporated under vacuum, the obtained crude product was purified by silica chromatography with ethyl acetate and petroleum ether being eluents to afford a white product with a yield >70%. Ester Condensation for the Formation of Coumarins. The phenylacetic ester of methyl salicylate (5 mmol) was mixed with anhydrous pyridine (5 mL), followed by the addition of KOH powder (0.7 g, 12.5 mmol) at room temperature. After the mixture was stirred for 6 h in a nitrogen atmosphere, pyridine was evaporated under vacuum, and 15 mL of NaOH (2.0 M) was added. The water phase was washed with ethyl acetate and then acidized by concentrated HCl to adjust the pH to 4−5. The aqueous solution was cooled to afford the crude product, a white solid with a yield >55%. Oxidative Annulation for the Formation of Coumestans. To 30 mL of dried 1,2-dichloroethane solution of the ester condensation products (2.5 mmol) were added anhydrous FeCl3 (1.625 g, 10 mmol) and silica powder (1.625 g), and the mixture was refluxed for 10 h. The solvent was then evaporated under vacuum, and the residue was purified by silica chromatography with ethyl acetate and petroleum ether being eluents to afford white coumestans with yields >60%. To obtain hydroxyl-substituted coumestan, the methoxyl group should be converted into a hydroxyl group by demethylation. In brief, methoxyl-substituted coumestan (1.0 mmol, dissolved in 10 mL of anhydrous CH2Cl2) was added dropwise to 10 mL of CH2Cl2 solution of BBr3 (1.0 M) within 30 min at 0 °C and stirred overnight at room temperature. The reaction mixture was hydrolyzed at 0 °C. The obtained precipitate was purified by silica chromatography with ethyl acetate and petroleum ether (1:2, v/v) being eluent to afford a white product with a yield >90%. 6H-Benzofuro[3,2-c]chromen-6-one (1): Rf = 0.40 (ethyl acetate/ petroleum ether = 1:9, v/v), 0.38 g of white product, yield 64%; mp 177−179 °C; 1H NMR (400 MHz, CDCl3) δ 8.15 (d, J = 4.8 Hz, 1H), 8.04 (d, J = 7.2 Hz, 1H), 7.60−7.68 (m, 2H), 7.42−7.52 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 159.9, 157.9, 155.4, 153.6, 131.9, 126.7, 125.2, 124.6, 123.4, 121.8, 121.8, 117.4, 112.5, 111.7, 105.8. 9-Methoxy-6H-benzofuro[3,2-c]chromen-6-one (2): Rf = 0.31 (ethyl acetate/petroleum ether = 1:9, v/v), 0.59 g of white product, yield 89%; mp 217−219 °C; 1H NMR (400 MHz, CDCl3) δ 7.98 (s, 2H), 7.57 (s, 1H), 7.50 (s, 1H), 7.40 (s, 1H), 7.19 (s, 1H), 7.07 (s, 1H), 3.91 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 159.7, 159.3, 158.2, 156.8, 153.2, 131.3, 124.6, 122.0, 121.5, 117.4, 116.5, 113.6, 112.9, 106.1, 96.8, 55.9. 8,9-Dimethoxy-6H-benzofuro[3,2-c]chromen-6-one (3): Rf = 0.46 (ethyl acetate/petroleum ether = 3:7, v/v), 0.64 g of white product, B



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2.0 mL. The test tubes were incubated at 37 °C for initiating the oxidation of DNA. Three test tubes were taken out at 30 min and cooled immediately. Then, 1.0 mL of TBA solution (1.00 g of TBA and 0.40 g of NaOH dissolved in 100 mL of PBS2) and 1.0 mL of 3.0% trichloroacetic acid aqueous solution were added and heated in boiling water for 30 min. After the test tubes were cooled to room temperature, 1.5 mL of n-butanol was added and shaken vigorously to extract TBARS for measuring the absorbance at 535 nm. In the aforementioned two tests, the absorbances in the control experiment and in the presence of coumestans were assigned as A0 and Adetect, respectively. The effects of coumestans on the oxidation of DNA were expressed by Adetect/A0 × 100. AAPH-Induced Oxidation of DNA Test. A solution of 2.0 mg/ mL DNA (dissolved in PBS2), 40 mM AAPH (dissolved in PBS2), and a certain concentration of a coumestan (dissolved in DMSO as the stock solution) was poured into test tubes with each containing 2.0 mL. The test tubes were incubated at 37 °C for initiating the oxidation of DNA. Three of them were taken out every 2 h and cooled immediately. Then, 1.0 mL of TBA solution (1.00 g of TBA and 0.40 g of NaOH dissolved in 100 mL of PBS2) and 1.0 mL of 3.0% trichloroacetic acid aqueous solution were added and heated in boiling water for 15 min. After the test tubes were cooled to room temperature, 1.5 mL of n-butanol was added and shaken vigorously to extract TBARS for measuring the absorbance at 535 nm. The absorbance of TBARS was plotted versus the reaction period. Statistical Analysis. All of the data were the average value from at least three independent measurements with the experimental error within 10%. The equations were analyzed by one-way ANOVA in Origin 6.0 professional software, and p < 0.001 indicated a significance difference.

should be allocated at phenylacetic acid and methyl salicylate in advance. In this work, chloro-, bromo-, and single- or doublemethoxyl-substituted phenylacetic acids are used as the reagents, and the substituents locate at the benzofuran ring of coumestans. We herein used the results from our previous studies (shown in Scheme 3) to emphasize the importance of the coumestan structure for enhancing the antioxidant effectiveness. Therefore, xanthotoxol is selected as the model for a multiheterocycle containing a single hydroxyl group, whereas compounds MPCP and 4a are chosen to be the model for a catechol moiety linking with coumarin via a single C−C bond. The antioxidant effect of the hydroxyl group in xanthotoxol, a furan-substituted coumarin, is not high enough.15 In comparison with the high antioxidant effectives of coumestan, it can be concluded that a hydroxyl group directly attaching to the coumarin scaffold cannot exhibit high antioxidant effectiveness, but the coumarin scaffold can increase the antioxidant effectiveness of the hydroxyl group attaching to coumestan. Moreover, our previous research indicates that the hydroxyl group at coumarin in compound 4a (see Scheme 3, the stoichiometric factor, n, is 6.15, for the physical meaning, see below) cannot improve the antioxidant effectivess of catechol moiety. Contrarily, a hydroxyl-free coumarin moiety enhances the antioxidant effectiveness of compound MPCP (see Scheme 3, the stoichiometric factor, n, is 6.60).16 By comparing the stoichiometric factors (n) of compound 4a and MPCP, it can be concluded that coumarin cannot remarkably influence the antioxidant effectiveness of the catechol moiety linking coumarin with a single C−C bond. Therefore, the result presented herein reveals that the benzofuran in combination with the coumarin scaffold is able to generate high antioxidant effectiveness even without an attached hydroxyl group. Scavenging radicals is regarded as the primary property of an antioxidant, and ABTS•+, DPPH, and galvinoxyl radicals are stable radicals at ambient temperature for evaluating the abilities of the antioxidant to reduce and to donate its hydrogen atom to N- and O-centered radicals, respectively.17 Figure 1S in the Supporting Information outlines rapid decays of the concentrations of these radicals in the presence of coumestans. The coumestans are able to quench these radicals even in the absence of a hydroxyl group, indicating that the hydroxyl group is not a necessary functional group for coumestans to quench radicals. In addition to some thermodynamic methods for expressing the radical-scavenging ability of the antioxidant,18 kinetic parameters such as the rate constant (k) can reveal the reaction rate between an antioxidant and a radical. We herein apply the rate constant to characterize the radical-scavenging abilities of coumestans. It has been proved that the reaction between an antioxidant and DPPH follows second-order kinetics as shown in eq 1.19

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RESULTS AND DISCUSSION Synthesis of Coumestans and Scavenging Radicals. As a naturally occurring oxygen-abundant tetracycle, the structure of coumestan contains a coumarin and a benzofuran with a C C shared by the lactone of coumarin and the furan ring. In the past decade, efforts contributing to the synthesis of coumestan have mainly focuseed on constructing the lactone ring.10,11 The applications of some reactions such as Sonogashira and Suzuki coupling conditions simplify the ring closure of lactone in coumarin,12,13 but the difficulty in the preparation of catalysts still limits the widespread use of the aforementioned method in the synthesis of coumestans. As shown in Scheme 2, we herein follow another paper, in which furan ring closure acts as the key step catalyzed by FeCl3.14 FeCl3 drives the dehydrogenation from the aromatic ring (D) and hydroxyl group (attaching to ring B) to form a furan ring in the refluxing 1,2-dichloroethane. An intramolecular ester condensation in phenylacetate of methyl salicylate leads to the formation of lactone in coumarin before the final step. Therefore, it can be found that substituents in coumestan Scheme 2. Synthetic Routines of Coumestans

−

d[DPPH] = r = k[DPPH][antioxidant] dt

(1)

The above equation expresses the relationship among the concentrations of two reagents and the reaction rate (r) during the whole reaction period. Thus, eq 1 is also available when the reaction time is 0, and [DPPH]t=0 and [antioxidant]t=0 represent the concentrations of DPPH and the antioxidant at the beginning of the reaction. If the reaction rate at the beginning of the reaction (r0) is measured according to the method in a previous paper20 (data in Table 1S), the rate constant (k) can be calculated by using eq 2: C



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Scheme 3. Contributions of Coumarin and Furan Moieties to the Antioxidant Effects

r0 = k[DPPH]t = 0 [antioxidant]t = 0

0.6 to 1.0 mM−1 s−1. The reaction between an antioxidant and DPPH shows the ability of the antioxidant to donate its hydrogen atom or electron to N-centered radicals, so the hydrogen atom of the hydroxyl group in coumestan still plays the major role in quenching N-centered radicals, and the coumestan scaffold (without the hydroxyl group attached) can also provide the electron for N-centered radicals. This is in agreement with a previous study on coumestan with electrontransferring efficiency higher than that of other phenols.22 In this work,22 coumestan was applied to modify an electrode for electro-oxidizing hydrazine with the anodic peak potential being 320 mV, whereas the oxidation cannot take place until 1100 mV on an unmodified electrode, demonstrating that coumestan plays the electron-transferring role in the oxidation at the electrode. This can be understood by eq 3 (see below), in which the cleavage of the C−O bond generates double single electrons for accepting electrons from other species. However, the electron-donating activity of coumestan also depends upon the electron receptor because, as can be seen from the data in the fourth column of Table 1, the coumestans used herein exhibit different properties in reacting with DPPH and galvinoxyl radicals. The hydroxyl-involved coumestans such as compounds 6 and 7 possess higher k values (2.40 and 6.99 mM−1 s−1, respectively) than Trolox (1.70 mM−1 s−1) in quenching the galvinoxyl radical, whereas the k values for compounds 1−5 (ranging from 0.1 to 0.5 mM−1 s−1) are lower than that of Trolox. The galvinoxyl radical as an O-centered radical accepts the hydrogen atom from the hydroxyl group of coumestans readily but shows difficulty in accepting the electron from the coumestan scaffold. The hydroxyl group other than the coumestan scaffold plays the major role in quenching the O-centered radical. Thus, the coumestan scaffold can also be an electron donator for quenching radicals. This finding encourages us to test whether the radical-scavenging property of coumestans can also be applied to inhibit radicalmediated oxidation of biological species. Inhibiting Cu2+/GSH and •OH-Induced Oxidations of DNA. GSH can be oxidized by copper ions (Cu+ and Cu2+) to form the glutathione radical (GS•) in vivo,23 and •OH is a wellknown radical generated from the metabolism in the process of in vivo oxidative stress.24 Both GS• and •OH are harmful radicals that can initiate oxidative damage of biological species, in which DNA is one of the susceptible biological species to the aforementioned radicals. The in vitro oxidations of DNA caused by GS• and •OH can be used as the experimental system for evaluating the antioxidant activity because the oxidative products from the oxidation of DNA can be readily detected after reaction with TBARS.25 Thus, the activities of coumestans are characterized by inhibiting Cu2+/GSH- and • OH-induced oxidations of DNA. The absorbances of TBARS

(2)

On the basis of the above method,20 the rate constants (k) of coumestans in trapping ABTS•+, DPPH, and galvinoxyl radicals are obtained and listed in Table 1. It is worth noting that the Table 1. Rate Constant (k) for Coumestans and Trolox in Scavenging ABTS•+, DPPH, and Galvinoxyl Radicals rate constant for coumestans and Trolox in scavenging radicals, k (mM−1 s−1) compd

ABTS•+

DPPH

galvinoxyl radical

1 2 3 4 5 6 7 Trolox

0.14 0.19 0.14 0.23 0.25 3.06 8.95 29.2

0.73 0.65 1.02 0.79 0.79 7.20 14.81 0.353

0.17 0.34 0.14 0.34 0.44 2.40 6.99 1.70

reaction between an antioxidant and a radical behaves as firstorder when the concentration of the antioxidant is in excess of the radical, but the concentration of the antioxidant should exceed that of the radical for ∼100-fold to keep the concentration of the antioxidant almost constant during the reaction. As listed in the footnote of Table 1S, the antioxidant concentrations used herein are generally in excess of that of radicals by 5−10-fold, and thus, the reaction rate cannot be readily obtained following the first-order kinetics. As pointed out in eq 1, the reaction rate is simultaneously related to the concentrations of the antioxidant and the radical. Hence, a double-exponential function is selected to express the correlation of the reaction rate with the concentrations of two reagents to precisely estimate the reaction rate as shown in Table 1S, of which the important data are collected in Table 1. The data in the second column of Table 1 show that the k values of coumestans in trapping ABTS•+ are lower than that of Trolox. The reaction of an antioxidant with ABTS•+ shows the ability of the antioxidant to reduce the radical, and the coumestans possess low abilities to reduce the radical. This can be understood from a previous paper,21 in which the oxidative potential of compound 7 (591.4 mV), a hydroxyl-substituted coumestan, is higher than that of general phenolic antioxidants. Therefore, the reductive ability of the coumestan is still dependent upon the phenolic hydroxyl group. On the other hand, the data in the third column of Table 1 show that the k values of coumestans in trapping DPPH are higher than that of Trolox (0.353 mM−1 s−1). In particular, the k values of compounds 6 and 7 are as high as 7.20 and 14.81 mM−1 s−1, respectively, and the k values of compounds 1−5 range from D



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Figure 1. Percentage of TBARS in the presence of 0.20 mM coumestans when the oxidation of DNA (2.0 mg/mL) is caused by 5.0 mM Cu2+ and 3.0 mM GSH for 90 min (right panel) or 4.0 mM H2O2 and 2.0 mM tetrachlorohydroquinone (TCHQ) for 30 min (left panel).

when DNA undergoes Cu2+/GSH-induced oxidation for 90 min and •OH-induced oxidation for 30 min are assigned to be 100% as the control. The absorbances of TBARS in the presence of 0.20 mM various coumestans and Trolox are compared with the control and shown in Figure 1, in which a low percentage of TBARS means a high antioxidant effectiveness. The data in Figure 1 show that all of the coumestans used herein can inhibit both Cu2+/GSH- and •OH-induced oxidations of DNA, and thus coumestans either containing a hydroxyl group or not behave as antioxidants to protect DNA against the aforementioned oxidative damage. Especially, the TBARS percentage of Trolox in inhibiting •OH-induced oxidation of DNA (71.0%) is similar to that of compound 1 (71.4%), revealing that the coumestan scaffold is able to protect DNA against •OH-induced oxidation. Compound 7 generates a low TBARS percentage (65.2%), because it contains double hydroxyl groups at adjacent positions, a classic structure for antioxidant. The TBARS percentages in the presence of other coumestans (compounds 2−6) are higher than that of compound 1, indicating that substituents are not beneficial for enhancing the antioxidant abilities of coumestans in this case. In the experimental system of Cu2+/GSH-induced oxidation of DNA, the TBARS percentage in the presence of compound 1 (86.7%) also approaches that of Trolox (88.3%), indicating that the unsubstituted coumestan can also protect DNA against Cu2+/GSH-induced oxidation. Moreover, as reported in our previous work, xanthotoxol (structure in Scheme 3) decreases the TBARS percentage only to 85%,15 similar to that of the coumestan (86.7%). Therefore, one hydroxyl group attaching to the coumarin moiety (as xanthotoxol) results in the same antioxidative efficiency as the coumestan scaffold (without any substituents attached). Moreover, a hydroxyl group attaching to coumestan as in compound 6 decreases the TBARS percentage to 62.2%, indicating that one hydroxyl group attached to the benzofuran moiety leads to a high activity for coumestan to inhibit Cu2+/ GSH-induced oxidation of DNA. The TBARS percentages of other coumestans (compounds 2−5 and 7) are around that of coumestan (86.7 ± 10%), implying that these substituents do not influence the inhibiting effects of coumestans on Cu2+/ GSH-induced oxidation of DNA markedly. The antioxidant

mechanism of hydroxyl-substituted coumestan is undoubtedly attributed to the hydroxyl group providing a hydrogen atom to quench radicals. •OH is so reactive that it can attack any part of the molecule, including the aromatic rings. However, in a previous study the lactone of the coumarin moiety is hypothesized to be the receptor for the nucleophilic addition.26 This may be a mechanism for coumestan to scavenge radicals. As shown in eq 3, the homolytic cleavage of the ester bond in the lactone (the only bond not participating in the aromatic system of coumestan) generates two single electrons, which can recombine with other radicals. Therefore, it is necessary to measure how many radicals can be trapped by one molecule of coumestan.

Inhibiting AAPH-Induced Oxidation of DNA. The AAPH-induced oxidation of DNA is selected to be the experimental system27 because the number of radicals trapped by an antioxidant in this case can be calculated according to chemical kinetic deduction,28 and the oxidative products from DNA can be followed by measuring TBARS as well. We have measured the numbers of radicals trapped by some antioxidants in this experimental system,29 and here we also adopt the same experimental system for quantitatively comparing the antioxidant effectiveness of coumestans. Therefore, as shown in Figure 2S, an increase of the absorbance means that much TBARS is generated the longer the reaction time in the blank experiment. However, the addition of coumestan hinders the formation of the TBARS for a period, and then the TBARS are produced as in the blank experiment. The inhibition period (tinh) can be measured from the beginning of the reaction to the cross point of tangents for the inhibition and the increasing period in the absorbance line. The relationships between tinh and the concentrations of coumestans are outlined in Figure 3S E



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Figure 2. n values of coumestans in inhibiting 40 mM AAPH-induced oxidation of 2.0 mg/mL DNA at 37 °C.

and expressed by the equation of tinh ∼ [coumestans] (listed in Table 2S). On the basis of the equation tinh ∼ [coumestans], the number of radicals trapped by coumestans can be calculated following chemical kinetics. In view of chemical kinetics, the tinh correlates proportionally with the concentration of an antioxidant as shown as in eq 4:30 t inh = (n/R i)[antioxidant]

and 3 (0.91 and 0.87, respectively) are lower than those of compounds 4 and 5 (1.01 and 1.21, respectively), indicating that an electron-withdrawing group such as −Cl or −Br can improve the antioxidant effect more than the electron-donating group such as −OCH3 and double −OCH3. This fact can be understood from eq 3. The formation of a radical by the cleavage of lactone is the key step for coumestan to accommodate the single electron from other radicals. An electron-withdrawing group is beneficial for coumestan to dispense electrons around the benzofuran and phenolic rings. Furthermore, we have also detected the effects of Trolox and xanthotoxol on AAPH-induced oxidation of DNA.15 It is found that Trolox and xanthotoxol just affect the increasing rate of TBARS and cannot generate tinh, leading to the n values of Trolox, and xanthotoxol cannot be obtained. Thus, coumestan (compound 1 with a 0.74 n value) possesses a higher antioxidant effect than Trolox and xanthotoxol. In conclusion, as a naturally occurring structure, coumestans exhibit wide antioxidant properties, which are attributed not only to the hydroxyl group but to the coumestan scaffold donating or accommodating a single electron. On the contrary, substituents are not beneficial for enhancing antioxidant effectiveness when coumestans are applied to inhibit Cu2+/ GSH- and •OH-induced oxidations of DNA. In addition, the coumestan scaffold is more active in inhibiting AAPH-induced oxidation than Trolox and xanthotoxol. Therefore, the hydroxyl group may not be a necessary factor in inhibiting radicalmediated oxidation of DNA, and the lactone shared with other heterocycles may be a novel structural feature in antioxidants.

(4)

Ri refers to the initiation rate of the radical-induced reaction. Both AAPH and the sodium salt of DNA are water-soluble compounds, and radicals generated from AAPH can attack DNA at the same phase. Ri is thereby assumed to be equal to the radical generation rate (Rg, Rg = (1.4 ± 0.2) × 10−6[AAPH] s−130). n stands for stoichiometric factor, which is the number of the radicals trapped by one molecule of the antioxidant. The coefficient in the equation of tinh ∼ [coumestan] is equivalent to (n/Ri) in eq 4. Hence, when Ri = Rg = 1.4 × 10−6 × 40 mM s−1 = 3.36 μM min−1 in this experimental system, the n value is the product of the coefficient in the equation of tinh ∼ [coumestan] and Ri. The obtained n values of coumestans (listed in Table 2S) are outlined at different steps in Figure 2, in which the structures of coumestans are involved for revealing the influence of coumestan structure on n values. The data in Figure 2 show that compounds 1−3 can scavenge less than one radical and compounds 4 and 5 can trap about one radical. The coumestans even in the absence of hydroxyl groups or in the presence of electron-withdrawing groups such as −Cl and −Br are still able to inhibit AAPHinduced oxidation of DNA. Interestingly, compounds 6 and 7 can trap 4.74 and 3.39 radicals, respectively, revealing that the phenolic hydroxyl group remarkably improves the antioxidant abilities of coumestans. However, compound 6, a singlehydroxyl-substituted coumestan, can trap more radicals (n = 4.74) than compound 7, a double-hydroxyl-substituted coumestan (n = 3.39), revealing that compound 6 exhibits a higher antioxidant effect than compound 7. This is in agreement with the observation on Cu2+/GSH-induced oxidation of DNA, in which compound 6 is a stronger antioxidant than compound 7. On the other hand, as shown in Scheme 3, MPCP and 4a are coumarin-substituted dihydropyrazoles with 6.60 and 6.15 n values, respectively,16 higher than that of coumestans used herein. Therefore, the coumarin moiety as a single substituent can improve the antioxidant effect of the catechol moiety within the same molecule. On the contrary, the coumarin moiety as a part of the tetracycle cannot markedly ameliorate the antioxidant effect of the catechol moiety. Figure 2 also shows that the n values of compounds 2

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ASSOCIATED CONTENT

S Supporting Information *

Additional experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(Z.-Q. L.) Mail: Department of Organic Chemistry, College of Chemistry, Jilin University, No. 2519 Jiefang Road, Changchun 130021, China. E-mail: [email protected]. Funding

Financial support from the Jilin Provincial Science and Technology Department, China, is acknowledged gratefully (20130206075GX). Notes

The authors declare no competing financial interest. F



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Article

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Coumestan Inhibits Radical-Induced Oxidation of DNA: Is Hydroxyl a

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