Radical Dynamics of Puerarin as Revealed by Laser Flash Photolysis

Jan 31, 2008 - Dan-Dan Li , Rui-Min Han , Ran Liang , Chang-Hui Chen , Wenzhen Lai , Jian-Ping Zhang , and Leif H. Skibsted. The Journal of Physical ...
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J. Phys. Chem. B 2008, 112, 2273-2280

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Radical Dynamics of Puerarin as Revealed by Laser Flash Photolysis and Spin Density Analysis Yu-Xi Tian,†,‡ Rui-Min Han,*,‡ Li-Min Fu,‡ Jian-Ping Zhang,*,†,‡ and Leif H. Skibsted§ Beijing National Laboratory for Molecular Science (BNLMS), State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, China, Department of Chemistry, Renmin UniVersity of China, Beijing 100872, China, and Food Chemistry, Department of Food Science, Faculty of Life Sciences, UniVersity of Copenhagen, RolighedsVej 30, DK-1058 Frederiksberg C, Denmark ReceiVed: October 1, 2007; In Final Form: NoVember 28, 2007

Puerarin, a C-glycoside of daidzein, forms upon direct photoexcitation in acetonitrile an excited-state with a lifetime of 4.2 µs assigned by oxygen quenching and sensitized formation of triplet zeaxanthin as a triplet and phenoxyl radicals of ms lifetime insensitive to oxygen and with spin density delocalized over the ACB isoflavonoid ring system, [ACB]•, as shown by laser flash photolysis and theoretical spin density calculations. Photoexcitation of A-ring 7-phenolate puerarin yields a [AC]• radical, which converts into the [ACB]• radical with a rate constant of 3.6 × 105 s-1 in 5% methanolic acetonitrile in a process triggered by B-ring deprotonation (4′-phenol). For the 7-phenolate with the 4′-phenol derivatized to yield a propyl anisole, no rearrangement of the initially formed [AC]• radical was observed. With the A-ring phenol derivatized, the 7-propyl-4′-phenolate forms a radical with spin density delocalized over the CB ring system, [CB]•, together with a minor fraction of [ACB]• due to propyl radical dissociations confirmed by BDE-calculations. Dianionic puerarin forms initially the [ACB]• radical, which is converted into the [CB]• radical in a slower process (1.6 × 104 s-1) assigned to 7-methylation. The radical dynamics is discussed in relation to puerarin/carotenoid antioxidant synergism at water/lipid interphases.

1. Introduction Flavonoids are widely present in fresh fruits, green vegetables, nuts, and grains and are accordingly part of what are considered to be healthy human diets. Flavonoids are in Vitro antioxidants, and the anti-inflammatory and anticarcinogenic effects seen in epidemiological studies are often assigned to in ViVo antioxidant activity.1 Although the importance of antioxidant activity in the human body has been questioned due to the low bioavailability of some flavonoids,2 the pro- and antioxidative properties of flavonoids and also the less bioavailable of the flavonoids are still of relevance for processes in the digestive tract. Besides anti- and prooxidative effects, flavonoids may further function as messengers in signal transduction of cells.3 Isoflavonoids, which are present mainly in leguminous plants and known as plant-protective phytoalexins,4 function like flavonoids as antioxidants and have been found to protect against cataracts, inflammations, and allergies.5-8 Clinical evidence shows that intake of isoflavonoids reduces the risk for certain hormone-dependent diseases such as breast and prostate cancers, osteoporosis, and cardiovascular disease.9-12 The backbone of isoflavonoids consists of a phenyl ring (Aring) associated to a six-membered heterocycle (C-ring) and another phenyl ring (B-ring) at the C3 position of the C-ring, * To whom correspondence should be addressed. Doctor Rui-Min Han and Professor Jian-Ping Zhang, Department of Chemistry, Renmin University of China, No. 59, ZhongGuanCun Street, Beijing 100872, China. E-mail: [email protected] (J.-P. Zhang). Tel: +86-10-6251-6604. Fax: +86-10-6251-6444. † Chinese Academy of Sciences. ‡ Renmin University of China. § University of Copenhagen.

whereas the B-ring in flavonoids is substituted at the C2 position. Despite the subtle structural difference, the antioxidative activity of 4′,5,7-trihydroxy isoflavone genistein is twice as that of the isomeric flavone apigenin.13 Other isoflavonoids such as 6- and 3′-hydroxydaidzein, 4′,7,8-trihydroxyisoflavan, and equol have been found to be even more active antioxidants than quercetin, otherwise known as the most potent antioxidant in the flavonoid family.14,15 For flavonoids, it has been established that the C2d C3 double bond, the C4 keto group, the C3 and C5 hydroxyl groups, and the ortho-diphenolic structure in the B-ring are closely relevant to the antioxidation activity.16,17 Yet, the structure-activity relationship for isoflavonoids, which are indispensable in understanding the antioxidation potency, remains to be explored. Puerarin (P) is a water-soluble isoflavone C-glycoside from the root of the traditional Chinese medical herb pueraria lobata (Scheme 1), which has been shown to yield cardiac protection.18,19 We have recently examined antioxidative activities of P and the propyl anisole derivatives, 7-C3H7-P and 4′-C3H7P, in aqueous solution. Whereas both derivatives in aqueous solution at pH ∼7 were found to be poorer antioxidants than P, the neutral or anionic forms of 4′-hydroxyl were proven to be more active in radical scavenging than 7-hydroxyl.20 The monoand dianionic forms of P were moreover found to show antioxidant synergism with a carotenoid in liposome. As the molecular and electronic structures as well as the fate of radical species are important for understanding anti- or pro-oxidative properties of potential antioxidants, the present work will focus on the radical and the excited-state dynamics of P and the propyl derivatives as studied by laser flash photolysis and theoretical spin density analysis. Our results have revealed the presence

10.1021/jp709579s CCC: $40.75 © 2008 American Chemical Society Published on Web 01/31/2008

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SCHEME 1: Molecular Structures of Puerarin and Derivativesa

a

The backbone of isoflavonid is highlighted with thicker solid lines.

of short-lived radical precursors of the stabilized radical of P surviving longer than milliseconds and the inertness of P radicals toward oxygen and zeaxanthin, which may be helpful in understanding the antioxidative properties of P and other isoflavonoids and their interaction with carotenoids. 2. Materials and Methods 2.1. Sample Preparation. The crude product of puerarin (Huike Plant Exploitation Inc., Shanxi, China) was twice recrystallized in a binary solvent of acetic acid (>99.5%, dried and refluxed by the use of P2O5 and KMnO4, respectively, Beijing Chemical Plant, Beijing, China) and methanol (>99.5%, Beijing Chemical Plant, Beijing, China) with a 1:1 volume ratio. The purity of puerarin (>98%) was then confirmed by high performance liquid chromatograph (HPLC) analysis using a C-18 reversed-phase column with methanol/water ) 3:7 (v/v) as the elute (L-7100 pump + L-7455 detector, Hitachi Ltd., Japan). Carotenoid zeaxanthin (Z) sealed in ampoules under argon (Roche A/S, Hvidovre, Denmark) was from the source as previously used.21 HPLC grade acetonitrile (Caledon Laboratories, Georgetown, Ont., Canada) was used as received. Derivatives of P, 7-C3H7-P, and 4′-C3H7-P were prepared as previously described.22 Solutions of phenolate of P (PO-) were obtained by adding 1 equiv of tetramethylammonium hydroxide ((CH3)4N+OH-, 97%, Sigma, St. Louis, MO) into 5% methanolic acetonitrile solution of P at 4.0 × 10-4 M, whereas solutions of the phenolates PO2-, 7-C3H7-PO-, and 4′-C3H7PO- at the same molar concentration were prepared by adding excess amount of (CH3)4N+OH-. Neutral P in acetonitrile was prepared at a concentration of 2.0 × 10-4 M. Figure 1 shows the absorption spectra of neutral and anionic P solutions.

Figure 1. Steady-state absorption spectra of neutral in acetonitrile and anionic puerarins in 5% methanolic acetonitrile. For flash photolysis, laser pulses at 266 and 355 nm (arrows) were used to excite neutral and anionic puerarins, respectively.

2.2. Laser Flash Photolysis. The laser flash photolysis apparatus had been described elsewhere.23 Briefly, nanosecond laser pulses for excitation at 266 nm or 355 nm were supplied by a Nd3+:YAG laser (Tempest 300, New Wave Research, U.S.A.), and the excitation energy was ∼3 mJ/pulse. The optical path length of a flowing-type quartz cell was 5 mm, and the sample absorbance was adjusted to 0.5 at the excitation wavelength. Anaerobic condition was achieved by bubbling the solution with high-purity argon for 1 h. To avoid the photodegradation effect, a sample volume as large as 30 mL was circulated between an ice-cooled reservoir and the quartz cell. All of the spectroscopic measurements were carried out at room temperature. 2.3. Quantum Chemical Calculations of Spin Densities and Bond Dissociation Energy. For neutral radical with an oxidized 7-hydroxyl, the anionic radical with a deprotonated 7-hydroxyl

Figure 2. Time-resolved absorption spectra of (a) puerarin (2.0 × 10-4 M), (b) puerarin (2.0 × 10-4 M) and zeaxanthin (2.0 × 10-5 M) in acetonitrile under anaerobic condition. The excitation wavelength was 266 nm. The gray line in (a) for comparison is obtained by subtracting the 50-µs bleaching spectrum of zeaxanthin alone (BLC-Z, gray line in (b)) from the 50-µs spectrum in (b).

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Figure 3. Time evolution profiles at indicated probing wavelengths plotted from the time-resolved spectra in Figure 2 for (a) puerarin and for (b) puerarin and zeaxanthin in acetonitrile under anaerobic condition. Inset in (a) shows the kinetics for aerobic puerarin solution. The triplet excitedstate of puerarin (O) and zeaxanthin (0) were probed at 505 and 600 nm, respectively. Solid lines are derived from curve fitting (see Table 1 for time constants).

Figure 4. Dependence of ∆OD amplitude of puerarin radical on excitation energy Eex for the anaerobic acetonitrile solution of puerarin (2.0 × 10-4 M). Probing wavelength, 400 nm; excitation wavelength, 266 nm; delay time, 50 µs. The solid line is for guiding your eyes.

and an oxidized 4′-hydroxyl, and the neutral radicals of propyl derivatives with an oxidized 7-hydroxyl or 4′-hydroxyl, quantum chemical calculations were performed using the Gaussian 03 package.24 Following refs 25 and 26, the geometries of the four different radicals were optimized by the use of UB3LYP27,28 density functions in conjunction with the 6-31G* basis set,29,30 based on which spin densities were calculated using the 6-311++G** basis set. This approach has been proven to be of high accuracy in calculating the spin density of relevant biomolecules.25,26,31 The bond dissociation energies (BDE) were calculated as the gas-phase enthalpy difference for the reaction: ArOH f ArO• + H• using 6-311G** basis sets. 3. Results Selection of excitation wavelength of 266 nm for neutral solution and of 355 nm for alkaline solutions, respectively, for P and derivatives, was based on the absorption spectra shown in Figure 1. It is seen from Figure 1 that the absorption spectra of anionic PO- and PO2- exhibit intensive absorption to the longer-wavelength side of the spectrum of neutral P, which may originate from the absorptive transition with intramolecular charge-transfer character owing to the electron donating properties of phenolates, and which allow selective excitation of anionic Ps to generate the corresponding phenoxyl radicals. 3.1. Characterization of Neutral Puerarin upon 266 nm Flash Photolysis. Figure 2a and b shows the time-resolved absorption spectra of P in anaerobic acetonitrile solutions in the absence and presence of Z, respectively, and the corre-

Figure 5. Transient absorption spectrum averaged over delay times of 20∼50 µs for dipropylpuerarin in anaerobic acetonitrile (2.0 × 10-4 M). Excitation wavelength, 266 nm.

sponding kinetics at selected probing wavelengths are shown in Figure 3a and b. The 20 ns transients in either panels of Figure 2 exhibit excited-state absorption (ESA) peaks at 363 and 545 nm; both are ascribed to the lowest triplet excited-state 3P* based on the following evidences: (i) The kinetics at 363 and 545 nm decayed with the same time constants of 95 ns and 4.2 µs in the presence and in the absence of oxygen, respectively, indicating the same origin of the two ESA bands; (ii) The transient species generated from P was quenched by oxygen, i.e., on changing from anaerobic to aerobic conditions, its deactivation was drastically accelerated (4.2 µs to 95 ns; Figure 3a); (iii) The transient species behaved as a sensitizer of triplet excited-state Z (3Z*) with characteristic ESA peaked at 505 nm (Figure 2b), as also verified by a tight correlation between the decay of 3P* and the rise of 3Z* (both 2.3 µs; Figure 3b), i.e., the P-to-Z triplet excitation energy transfer. It is known that 3Z* without sensitization would not be detected because of its extremely low quantum yield upon direct photoexcitation (ΦISC ≈ 0.001). In this context, Brede et al. had recently reported considerably large ΦISC ≈ 0.5 for phenol (ArOH) whose hydroxyl was free from steric hindrance and observed efficient phenol-to-β-carotene (β-Car) triplet excitation energy transfer.32 In Figure 2a, the 50 µs transient with clear features at 400 and 520 nm is distinctly different from the ESA of 3P* at 20 ns. It is known that the 266 nm excitation of phenol induces photoionization or O-H dissociation leading to cationic or neutral radicals, respectively, and that the quantum yields of these reactions are solvent- and/or pH-dependent.33 Furthermore, it has been proven that biphotonic ionization of phenols

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Figure 6. Results of unrestricted spin density calculations for free radicals: (a) [AC]•, 7-hydroxyl oxidized, (b) [ACB]•, 7-hydroxyl deprotonated and 4′-hydroxyl oxidized, (c) [AC]•, 7-hydroxyl oxidized, and (d) [CB]•, 4′-hydroxyl oxidized. Numerals indicate spin densities, those absolute values above 0.010 are shown in bold face, and those below 0.001 are not displayed.

generates cationic radicals undergone subsequent deprotonation in the presence of water.34,35 We have examined the excitationenergy dependence of the absorption intensity at 400 nm for the 50 µs spectrum (Figure 4), and the possibility of biphotonic ionization is excluded by the observed linear relation. Therefore, the transient spectrum at 50 µs can be ascribed to a P radical, generated from singlet excited-state P (1P*) via O-H dissociation as is commonly seen for simple phenols under conditions similar to the present work.36 The mechanism in forming P radical from 1P* in competition with intersystem crossing to 3P* rather than from 3P* is supported by the similarity between the transient spectra in the presence and absence of oxygen recorded after the decaying out of 3P* (data not shown), i.e., oxygen quenching of 3P* did not affect the yield of P radical. More importantly, this result demonstrates the nonreactivity of P radical to oxygen. The weak absorption at later delay time in Figure 2 suggest a rather low quantum yield of O-H dissociation, which was reported for other phenols to be ∼0.05 in forming ArO•.32 The long-lived P radical was also observed in the presence of Z as indicated by the 50-µs spectrum in Figure 2b, which appears different from the 50 µs spectrum in Figure 2a, owing to a superposition of the P radical absorption and the bleaching of ground-state absorption of Z. However, as shown at the bottom of Figure 2a, spectrum obtained by compensating the 50 µs spectrum in Figure 2b with the bleaching of ground-state absorption of Z (BLC-Z) is almost identical to the 50 µs spectrum in Figure 2a. Most importantly, this result implies that Z cannot scavenge the P radical. In this relation, our previous

study showed that monoanionic and dianionic P can regenerate β-Car from its radical cation (β-Car•+).20 In neutral acetonitrile, the dipropyl derivative of P also formed radicals, as may be seen from the ∆OD spectrum in Figure 5 for the stabilized radicals formed upon 266 nm excitation. Here, the formation of dipropyl P radical implies the dissociation of O-C3H7 to be discussed in relation to BDE calculations. 3.2. Spin Densities of Radicals of Puerarin and Derivatives. On laser photolysis the photo-oxidation of phenolates PO-, PO2-, 7-C3H7-PO-, and 4′-C3H7-PO- are expected to form the corresponding radicals (a-d) shown in Figure 6. We shall present the results of spin density analyses on the four different kinds of radicals to support the assignments of the transient spectra to be made subsequently. In addition, the electronic structures of radicals of Ps may be helpful to understand the relative efficiencies of P and the derivatives as antioxidants. Geometry optimization yielded the AC-to-B dihedral angels of 36.6, 25.7, 36.9, and 30.5° for the radicals shown in Figure 6a-d, respectively, and the distribution of spin densities are accordingly shown in numerals, which can be categorized into three different types. (i) [AC]•: The molecular structures shown in Figure 6a and c have similar AC-to-B dihedral angels and nearly identical profiles of spin density, the unpaired electron mainly delocalizes on the coplanar and conjugated A-C rings with a short extension to the oxygen in the adjacent glucoside. (ii) [ACB]•: The molecular structure in Figure 6b is more planar than the others, and the spin density concentrates on the B-ring and delocalizeds significantly to the A-ring phenolate. (iii) [CB]•: The spin density mainly distributes over the B-ring

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Figure 7. Transient spectra at indicated delay times for (a) puerarin with one-equivalent of (CH3)4N+OH-, (b) puerarin with excess of (CH3)4N+OH-, (c) 4′-propylpuerarin with excess of (CH3)4N+OH-, and (d) 7-propylpuerarin with excess of (CH3)4N+OH-, all in anaerobic 5% methanolic acetonitrile with concentration of 4.0 × 10-4 M for puerarin or derivatives. Excitation wavelength, 355 nm.

along with the proximate C2dC3 moiety in the C-ring (Figure 6d). The above three different types of electronic structures all show delocalization of unpaired electron on the isoflavone backbone. These electronic structures of P radicals serve as the starting points of phenolate photodissociation for the subsequent temporal evolution. 3.3. Spectral Dynamics of Anionic Puerarin and Monoderivatives upon 355 nm Flash Photolysis. Photoexcitation of phenols in alkaline solution is known to produce phenoxyl radicals ArO- + hV f ArO• + esolv, and the quantum yield of electron detachment or radical formation can be as high as unity.37 Figure 7a presents the transient spectra recorded for PO- in 5% methanolic acetonitrile alkaline solution (methanol added for increasing the solubility of (CH3)4N+OH-), and Figure 8a displays the corresponding time evolution profiles at indicated probing wavelengths. Previous investigation had shown that ∼90% of P was deprotonated at 7-phenoxyl in the presence of 1 equiv of (CH3)4N+OH-,20 and the deprotonation product POpossesses a resonant structure between 7-phenolate and 4-keto via the A-C conjugation, a case that is analogues to the flavone derivatives with dipolar benzopyrone structure.38 Accordingly, the 100 ns transient in Figure 7a with a broad ESA band can be ascribed to [AC]• formed via photoinduced electron detachment. The involvement of triplet excited-state species can be excluded because, upon photo-oxidation of PO-, the 440 nm kinetics is insensitive to oxygen as shown in Figure 8a. Similar absorption spectrum was also reported for the free radical of galangin, a flavone without any hydroxyl substituent in the B-ring.39 The accompanying negative feature to the shorterwavelength side is obviously the bleaching of ground-state absorption. In Figure 8a, the kinetics at probing wavelengths of 360, 440, and 535 nm could be simultaneously fit to a threeexponential model function, and the resulted time constants are listed in Table 1. The 360 nm trace clearly shows a rise phase (τr ≈ 2.8 µs), whereas the other two traces follow biphasic decay

with time constants of τd1 ≈ 0.1 µs and τd2 ≈ 2.9 µs. Here, a tight decay-to-rise correlation can be established, i.e., the transient spectrum of [AC]• at 100 ns evolved with a time scale of ∼3 µs into a distinct dual-band feature peaked at 380 and 535 nm at 1 ms as shown in Figure 7a. These two absorption bands share the same decay kinetics and, therefore, may originate from the same transient species. In Figure 7b, the 100 ns transient recorded following optical excitation of dianionic PO2- is characterized by a dual-band feature peaked at 380 and 535 nm, which can be assigned to the electronic structure [ACB]• induced by electron detachment of the B-ring phenolate. Indeed the reduction potential of B-ring phenolate (905 mV vs Cp2Fe/Cp2Fe+) is lower than that of the A-ring phenolate (1064 mV vs Cp2Fe/Cp2Fe+). The involvement of triplet excited-state species is precluded because the 460 nm kinetics behavior of the radicals initiated from PO2- in Figure 8b was also found to be insensitive to oxygen. Here again, a decay-to-rise correlation can be established, i.e., the dual-band transient of [ACB]• at 100 ns evolved with a time scale of ∼60 µs into a single-band feature at 460 nm at 1 ms (Figure 8b, Table 1). Figure 7c illustrates the transient spectra recorded following pulsed excitation of 4′-C3H7-PO-. These broad spectra are similar to the 100 ns transient in Figure 7a and are definitively ascribed to [AC]•, because the 4′-propyl substitution precludes B-ring as a radical site. The transient decays monotonically without changing spectral shape with apparent decay time constants of ∼1 µs, ∼20 µs, and ∼1 ms (Figure 8c, Table 1). Figure 7d shows the results of photo-oxidation of 7-C3H7PO-, the initial broad-band absorption decays rapidly with time constants of 0.1 and 1.9 µs and, subsequently, an absorption peaked at 460 nm rise up with a time constant of 12 µs (Figure 8d, Table 1). Different from the above two cases, no kinetics correlation is found between the 100 ns and the 40 µs spectra; however, significant contribution of the 40 µs spectrum can be

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Figure 8. Time evolution profiles in anaerobic (O) and aerobic (b) condition at indicated probing wavelengths plotted from the time-resolved spectra in Figure 7. (a) Puerarin with one-equivalent (CH3)4N+OH-, (b) puerarin with excess of (CH3)4N+OH-, (c) 4′-propylpuerarin with excess of (CH3)4N+OH-, and (d) 7-propylpuerarin with excess of (CH3)4N+OH-. The concentrations of puerarin or derivatives were all 4.0 × 10-4 M in 5% methanolic acetonitrile. Solid lines are derived from curve fitting (see Table 1 for time constants).

TABLE 1: Rise and Decay Time Constants (τr and τd) Derived from the Kinetics Curves in Figure 3 and Figure 8 at Selected Probing Wavelengths (λpr) for Samples under Different Excitation Wavelengths (λex) time constant λex/nm 266

355

sample

λpr/nm

P

600

P+Z

505 600 360/440/535b 380/460/535b 460 460

POPO24′-C3H7-P7-C3H7-P-

τr/µs

2.3 ( 0.0 2.8 ( 0.2 61 ( 4 12 ( 1

τd1/µs

τd2/µs

4.2 ( 0.4 95 ( 6 nsa 5.1 ( 0.1 2.3 ( 0.0 0.1 ( 0.0 0.9 ( 0.1 1.1 ( 0.0 0.1 ( 0.0

2.9 ( 0.2 63 ( 4 22 ( 12 1.9 ( 0.2

τd3/ms

∼1

a Case of aerobic sample solution, all the other cases are anaerobic. b Kinetics curves at three probing wavelengths were simultaneously fit to a three-exponential model function.

seen in the 100 ns spectrum (Figure 7d). We assigned the 40 µs spectrum to [CB]•, which is almost identical to the 1 ms spectrum in Figure 7b. Each kinetics curve in Figure 8 exhibits a fast component decaying in hundreds ns and slower ones rising or decaying in a few tens of µs to ms. For 4′-C3H7-PO- and 7-C3H7-PO- in Figure 8c and d, respectively, the fast-decay components can be attributed to germinate recombination between the photogenerated radicals and the solvated electrons, whereas those for PO- and PO2- in Figure 8a and b, respectively, may be caused, besides the germinate recombination, by the transformation of the molecular and/or electronic structures of the photoinduced radicals as evidenced by the decay-to-rise correlations. Impor-

tantly, the radical dynamics were found to be independent of the P concentration (data not shown); therefore, the conversion between different radical forms is considered as intramolecular electron-transfer process. In the case of epigallocaxtechin, Jovanovic et al. had reported the transformation between different forms of free radicals as a result of intramolecular electron-transfer taking place in a few to a hundred µs and showing pH dependence.40 4. Discussion We have carried out laser flash photolysis of the neutral, monoanionic, and dianionic forms of P (P, PO-, and PO2-) and spin density analyses on the corresponding photoinduced

Radical Dynamics of Puerarin free radicals in an attempt to understand the dynamics and the electronic structures of P radicals, and for this purpose, the anionic propyl derivatives, 4′-C3H7-PO-, 7-C3H7-PO-, and the neutral dipropyl derivative were also studied. Hereafter we discuss, on the basis of the experimental and theoretical results, the dynamics and the electronic structures of P radicals as well as their interaction with the carotenoid Z and oxygen. Photo-oxidation of 4′-C3H7-PO- showed rather simple spectral dynamics, i.e., the transient absorption decayed monotonically without changing spectral shape (Figures 7c, 8c). The electronic structure of the initial radical is denoted as [AC]• in a sense that the unpaired electron mainly delocalizes on the conjugated AC rings (Figure 6c), which remains unchanged during the temporal evolution. The radical generated from PO- by photo-oxidation also holds the [AC]• structure, because the early transient (100 ns, Figure 7a) closely resembles those of the radical formed from 4′-C3H7PO- (Figure 7c) with almost identical spin density profiles (Figure 6a and c). This is not surprising because the molecular structure of P differs from that of 4′-C3H7-P merely by the C3H7- substituent in the B-ring that is separated from the conjugated A-C rings. Interestingly, the initial [AC]• radical was not stable, i.e., the 100 ns spectrum transformed with a time constant of ∼3 µs into a dual-band feature that stayed up to 1 ms (Figures 7a and 8a). This result strongly suggests that the initial [AC]• radical experienced dynamic change of electronic structure, and transformed into a stabilized radical product. Because protolytic groups of radicals in general are more acidic than their parent compounds, corresponding to a decrease in pKa value of approximately 4-5 units, we suggest that the radical transformation is initiated by proton dissociation to yield the 4′-phenolate form of the 7-phenoxyl radical as outlined in Scheme 2a. Spin density will delocalize in this radical anion and include the full ring system: [ACB]•. As for the spectral dynamics of 7-C3H7-PO- in Figure 7d, photo-oxidation of the B-ring phenolate gave rise to a broad ∆OD spectrum at 100 ns that converged rapidly to a singlepeak band at 460 nm (e.g., the 40-µs one). The lack of correlation between the rise of the 460-nm band (τr ≈ 12 µs) and the decay of the blue- or red-side absorption (τd ≈ 0.1 and 1.9 µs; Table 1) suggests their different origins. Here, the final radical product bears the [CB]• pattern of spin density. The calculated BDE for P and the propyl anisole derivatives shown in Table 2 indicate that the oxygen-carbon bond of the 7-propyl group is in particular weak. We accordingly suggest that, upon photoexcitation, this bond breaks to yield propyl radical as a minor reaction in parallel with the formation of the 4′-phenoxyl radical, which appears as upon photoexcitation to yield a second component in the initial transient spectrum as described above. Compared to 7-C3H7-PO-, photoexcitation of PO2- resulted in a dual-band transient at 100 ns, which converted with a time constant of ∼60 µs into a single-band feature (e.g., the 1-ms transient, Figure 7b). Note that the latter spectrum is almost identical to the characteristic spectrum of the [CB]• radical resulting from 7-C3H7-PO- (at 40 µs, Figure 7d). The initial radical formed from PO2- has the [ACB]• type distribution of unpaired electron, and the change in spectral dynamics implies that the final radical product has the similar electronic structure as the [CB]• radical. However, the detailed mechanism of the [ACB]•-to-[CB]• radical transformation indicated in Scheme 2b remains unclear, but we suggest, that the 7-phenolate is transformed into a methyl anisole under the alkaline conditions with excess of methanol (∼1 M) and (CH3)4N+OH-, which in

J. Phys. Chem. B, Vol. 112, No. 7, 2008 2279 SCHEME 2: Mechanism of Radical Generation via Photoionization of Puerarin Monoanion (a) and Dianion (b), Glc ) glucoside

TABLE 2: Calculated Bond Dissociation Enthalpy (EBDE in kcal/mol) of Puerarin and Derivatives compound

bond

EBDEa

bond

EBDEa

puerarin 4′-propylpuerarin 7-propylpuerarin 7,4′-dipropylpuerarin

7-O-H 7-O-H 7-O-C3H7 7-O-C3H7

88.11 87.97 64.79 64.50

4′-O-H 4′-O-C3H7 4′-O-H 4′-O-C3H7

87.39 65.12 87.13 64.71

a

elec elec Estimated as Eelec phenoxyl radical+ EH-atom - Ephenoxyl.

effect hampers the extension of the resonance to the A-ring. This suggestion draws some support from the slower [ACB]•to-[CB]• transformation compared to the [AC]•-to-[ACB]• one. For neutral P, it has been established that the singlet excitedstate P, rather than the triplet excited-state is the precursor of P radicals via photoinitiated hydrogen abstraction. Because the characteristic spectrum of the radical in Figure 2a (the 50 µs spectrum) is almost identical to the 1 ms spectrum in Figure 7a, we conclude that the radical product generated via hydrogen abstraction from neutral P and via photo-oxidation of PO- in their stablilized form possess similar electronic structures, assigned as [ACB]• following deprotonation, c.f. Scheme 2a. Taken together, the above discussion proves that photoexcitation of anionic or neutral Ps leads to completely different dynamics of radical stabilization in forming the final radical products involving proton dissociation of radical species and most likely homolysis of covalent bonds under some conditions. 5. Conclusions We have examined the dynamics of excited states and radicals of Ps by means of laser flash photolysis, as well as the spindensity distribution on the isoflavonoid backbone by theoretical analyses. The facile interaction among the radical resonance structures suggests a significant delocalization of the unpaired

2280 J. Phys. Chem. B, Vol. 112, No. 7, 2008 electron over the isoflavone backbone. Photoexcitation of neutral P in acetonitrile generated both a long-lived triplet excited-state and phenoxyl radicals of P insensitive to oxygen, whereas photoexcitation of anionic Ps resulted merely in formation of radical species. Most significantly, the conjugated A-C-ring of monoanionic P radical was further delocalized to an ACB radical anion with a first-order rate constant of 3.6 × 105 s-1 via intramolecular electron-transfer process triggered by deprotonation. As for the interaction of P radicals with carotenoids, Z was found not to regenerate P from its radicals. In contrast, the reverse reaction, regeneration of carotenoids from carotenoid radical cation by P anions suggested to account for P/β-Car antioxidant synergism observed in phosphatidyl liposome may find an explanation in facile interconversion of P radicals. Acknowledgment. This work has been supported by the grants-in-aid from the Natural Science Foundation of China (#20673144 and #20433010) and from the Ministry of Science and Technology of China (#2006BAI08B04-06). The continuing support by LMC, Centre for Advanced Food Studies to the Food Chemistry group at University of Copenhagen is acknowledged. References and Notes (1) Pietta, P. G. J. Nat. Prod. 2000, 63, 1035. (2) Lotito, S. B.; Frei, B. Free Rad. Biol. Med. 2006, 41, 1727. (3) Williams, R. J.; Spencer, J. P. E.; Rice-Evans, C. Free Rad. Biol. Med. 2004, 36, 838. (4) Harborne, J. B. The flaVonoids-adVances in research since 1986; Chapman and Hall: London, 1994; p 589. (5) Jha, H. C.; von Recklinghausen, G.; Zilliken, F. Biochem. Pharmacol. 1985, 34, 1367. (6) Moosmann, B.; Behl, C. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 8867. (7) Voss, C.; Sepulveda-Boza, S.; Zilliken, F. W. Biochem. Pharmacol. 1992, 44, 157. (8) Akiyama, T.; Ishida, J.; Nakagawa, S.; Ogawara, H.; Watanabe, S.; Itoh, N.; Shibuya, M.; Fukami, Y. J. Biol. Chem. 1987, 262, 5592. (9) Booth, C.; Hargreaves, D. F.; Hadfield, J. A.; McGown, A. T.; Potten, C. S. Brit. J. Cancer 1999, 80, 1550. (10) Heruth, D. P.; Wetmore, L. A.; Leyva, A.; Rothberg, P. G. J. Cell Biochem. 1995, 58, 83. (11) Hoffman, R. Biochem. Biophys. Res. Commun. 1995, 211, 600. (12) Messina, M. J.; Persky, V.; Setchell, K. D.; Barnes, S. Nutr. Cancer 1994, 21, 113. (13) Ruiz-Larrea, M. B.; Mohan, A. R.; Paganga, G.; Miller, N. J.; Bolwell, G. P.; Rice-Evans, C. A. Free Rad. Res. 1996, 26, 63. (14) Briviba, K.; Sies, H.; Sepulveda-Boza, S.; Zilliken, F. W. FlaVonoids in Health and Disease; Rice-Evans, C. A., Packer, L., Eds.; Marcel Dekker: New York, 1998; p 299. (15) Ru¨fer, C. E.; Kulling, S. E. J. Agric. Food Chem. 2006, 54, 2926. (16) Dugas, A. J., Jr.; Castan˜eda-Acosta, J.; Bonin, G. C.; Price, K. L.; Fischer, N. H.; Winston, G. W. J. Nat. Prod. 2000, 63, 327.

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