Oxidation of naringenin by gamma-radiation

ARTICLE IN PRESS

Radiation Physics and Chemistry 77 (2008) 728–733 www.elsevier.com/locate/radphyschem

Oxidation of naringenin by gamma-radiation Tristan O. Nagya, Karin Ledolterb, Sonja Solara, a

Department of Nutritional Sciences, Section Radiation Biology, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria Department of Biomolecular Structural Chemistry, University of Vienna, Campus-Vienna-Biocenter 5, A-1030 Vienna, Austria

b

Received 10 August 2007; accepted 9 October 2007

Abstract The reaction of dOH with naringenin (40 ,5,7-trihydroxyflavanone) in the presence of air induced the formation of the hydroxylation product eriodictyol (30 ,40 ,5,7-tetrahydroxyflavanone). Its yield was dependent on pH. The initial degradation yield of naringenin was Gi(-Nar) ¼ (2.570.2)  107 mol dm3 J1. For the reaction with dOH, a rate constant k (dOH+naringenin) ¼ (7.270.7)  109 M1 s1 was determined. In the presence of N2O and NaN3/N2O, no eriodyctiol was formed. Apigenin (40 ,5,7-trihydroxyflavon) was detected as decay product of the naringenin phenoxyl radicals. In Ar-saturated solutions, naringenin exhibited a pronounced radiation resistance, G(-naringenin) 0.3  107 mol dm3 J1. r 2007 Elsevier Ltd. All rights reserved. Keywords: Naringenin; Eriodictyol; Flavanoids; OH-radicals; Gamma radiolysis; HPLC; Naringenin phenoxyl radicals

1. Introduction Naringenin (40 ,5,7-trihydroxyflavanone), mainly present in citrus fruits and tomatoes (Krause and Galensa, 1992; Heo et al., 2004; USDA Database, 2007), is a member of the flavonoid family. These compounds are frequently components of the human diet and they have gained increasing interest because of their positive health effects, which are attributed to their antioxidative activity. They protect the cells against the damaging effects of reactive oxygen species (e.g. superoxide anion radicals, hydroxyl radicals, peroxyl radicals) and inactivate free radicals by hydrogen/electron transfer or metal chelation (Jovanovic et al., 1994; van Acker et al., 1996; Rice-Evans et al., 1996; Gaziano, 2000; Madsen et al., 2000; Butkovic et al., 2004). Naringenin phenoxyl radicals have been found to act as prooxidants (Galati et al., 2001). Investigating the effect of gamma irradiation on the phenolic compounds in tomatoes, eriodictyol (30 ,40 ,5,7-tetrahydroxyflavanone) was found as irradiation product of naringenin (Schindler et al., 2005). To get a better insight into the reaction Corresponding author. Tel.: +43 1 4277 54970; fax: +43 1 4277 54964.

E-mail address: [email protected] (S. Solar). 0969-806X/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2007.10.007

mechanism of the formation of this product, the present study was focused on the reactions of OH-radicals with naringenin under different conditions (pH, oxygen, N2O). In addition, the reactions of naringenin phenoxyl radicals were investigated.

2. Materials and methods 2.1. Chemicals Naringenin (Nar) (40 ,5,7-trihydroxyflavanone) (495%), eriodictyol (Eri) (30 ,40 ,5,7-tetrahydroxyflavanone) (Rotichroms HPLC), apigenin (Api) (40 ,5,7-trihydroxyflavon) (Rotichroms HPLC) and methanol (Rotisolvs HPLC Ultra Gradient Grade) were purchased from ROTH, Karlsruhe. All other chemicals (NaN3, NaOH, HClO4, HCOOH, N,N-dimethyl-4-nitrosoaniline) were of highest purity grade available (Merck, Fluka, Sigma-Aldrich). The pH was adjusted with HClO4 and NaOH, respectively. The solutions were freshly prepared with triply distilled water. To obtain airfree conditions, the solutions were saturated with Ar (5.0) or N2O (Stickoxydul p.n.), both Messer Austria, for about 40 min before irradiation.

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Irradiations were carried out with a Co-60-gammasource, ‘‘Gammacell 220’’, Nordion International Inc., Kanata, Ont., Canada. The dose rate was 55–65 Gy min1. 2.3. Product analysis The degradation of naringenin and the formation of products were determined by reverse-phase liquid chromatography (Hewlett-Packard series 1050 and 1100; column: Hypersil ODS (250  4 mm; 5 mm) fitted with a Hypersil guard column (4  4 mm; 5mm), (Agilent Technologies, Bo¨blingen, Germany), temperature: 30 1C; injection volume: 50 ml; flow rate: 1 ml/min; eluent: H2O (2.5% (w/v) formic acid)/CH3OH ¼ 50/50 (v/v). For detection a diode array detector was used; the absorptions were measured at 220, 280 and 340 nm. Standards were used for peak identification and quantifications. Peak identification was based on both UV–vis spectra (range 210–600 nm), recorded during HPLC analysis, and retention times (Rt). Concentrations were determined from calibration curves of standard solutions. Absorptions were also determined by a UV–vis spectrometer, Perkin-Elmer, Lambda 650. 3. Results and discussion For the dissociation of the 40 OH group of Nar (Scheme 1), a pK 9.1 was reported (Jovanovic et al., 1994). 3.1. Radiolysis in the presence of air and different pH-values Exposure of water or dilute aqueous solutions to d ionizing radiation leads to the primary species e aq , OH, d d H , H2 and H2O2. Their yields are: G( OH)ffiG(e aq )ffi 2.9  107 mol dm3 J1, G(Hd) ¼ 0.6  107 mol dm3 J1, G(H2) ¼ 0.5  107 mol dm3 J1 and G(H2O2) ¼ 0.7  107 mol dm3 J1. In the presence of oxygen, the reducing d d radicals Hd and e aq are converted into HO2 and O2 , 10 3 1 1 reactions (1), (2) and (3), k1k2 ¼ 2  10 dm mol s (Buxton et al., 1988). Hd þ O2 ! HO2 d

(1)

d e aq þO2 ! O2

(2)

HO2 d "Hþ þ O2 d ;

pK ¼ 4:8

(3)

In the absence of metal ions, the reactivity of the superoxide radicals HOd2 and Od towards phenols is 2 O

HO A

C

OH

O

B

OH

Scheme 1. Chemical structure of naringenin.

o105 dm3 mol1 s1 (Bielski et al. 1985). Hence they are mainly combining to hydrogen peroxide (Getoff and Prucha, 1983; Bielski et al., 1985; Bielski and Cabelli, 1991; Cabelli 1997). The rate constant of dOH with Nar was determined by competition kinetics using N,N-dimethyl-4-nitrosoaniline (RNO) as standard competitor (Shetiya et al., 1976), k(dOH+RNO) ¼ 1.25  1010 dm3 mol1 s1 (Buxton et al., 1988). Measuring the absorption change of RNO at 440 nm (pH 9.5) induced by OH-radicals and by various concentrations of Nar, a rate constant k (dOH+Nar) ¼ (7.270.7)  109 dm3 mol1 s1 was obtained (Fig. 1). This value is lower than the rate constant for the flavonol quercetin, which has a higher electron density than Nar, k(dOH+quercetin) ¼ 1.5  1010 dm3 mol1 s1 (Wang et al., 1993). Gamma radiolysis of 2  104 mol dm3 Nar in the presence of air (O2 ¼ 0.25  103 mol dm3) was carried out at different pH values. The decomposition of Nar and the formation of Eri at pH 8.8 are presented as a function of dose in Fig. 2. The degradation of Nar is linear up to a dose of about 150 Gy. From this linear part of the yield/ dose plot, an initial degradation yield Gi(-Nar) ¼ (2.570.2)  107 mol dm3 J1 was calculated, which is only slightly less than the dOH concentration. For the formation of Eri, a Gi(Eri) ¼ (0.4570.05)  107 mol dm3 J1 was obtained which corresponds to 1870.2% of the decomposed substrate (Fig. 2). At pH 9.6, the initial degradation yield of Nar remained similar, Eri, however, was formed with a much lower yield, Gi(Eri) ¼ (0.1770.02)  107 mol dm3 J1, and at pH 10.1 it dropped to only Gi(Eri) ¼ (0.0770.005)  107 mol dm3 J1. Since the B-ring is electron richer than the A-ring of flavonoids, it is an apparent target of any oxidant

2.0 1.8

ΔOD0 / ΔODn

2.2. Irradiation source

729

1.6 1.4 1.2 1.0 0.8 0.0

0.2

0.4

0.6 0.8 1.0 [Nar] / [RNO]

1.2

1.4

1.6

Fig. 1. Competition plot to determine the rate constant of dOH radicals with naringenin. DOD0: difference in optical density between irradiated (0.1 kGy) and unirradiated aqueous solution of N,N-dimethyl-4-nitrosoaniline (RNO). DODn: difference in optical density between irradiated and unirradiated aqueous solution of RNO containing various concentration of Nar; [Nar] ¼ (1–7)  105 mol dm3, [RNO] ¼ 5  105 mol dm3. The given measuring points represent mean values of four measurements.

ARTICLE IN PRESS T.O. Nagy et al. / Radiation Physics and Chemistry 77 (2008) 728–733

730

30 200 25

100

20 μmol

Naringenin

μmol

Eriodictyol

150

15 10

50 5 0

0 0

200

400

600

800

Gy

3.2. Radiolysis in the presence of N2O

Fig. 2. Decomposition of Nar (left scale) and formation of Eri (right scale) as a function of dose. Solution: 2  104 mol dm3 Nar, pH 8.8, air. The given measuring points represent mean values of six measurements.

(Jovanovic et al., 1994). Therefore, it can be assumed that the main OH-attack on naringenin takes place on the B-ring, whereby OH-adducts (Nar-OH-adduct), preferentially adjacent to the 40 OH group, are formed (reaction (4)). In the presence of air peroxyl radicals on different positions of the ring are generated. The rate constant for O2 addition to carbon-centered radicals is usually close to diffusion-controlled, k(Rd+O2) 1  109 dm3 mol1 s1, for a compilation of rate constants (see Neta et al., 1990). Cyclohexadienylperoxyl radicals can undergo HOd2 elimination, especially those that carry the peroxyl radical function in an ortho-position to the H atom to be eliminated (von Sonntag and Schuchmann, 1997). By such an elimination, the hydroxylation product of the parent compound is formed, which in the case of naringenin is eriodictyol (reaction (5)). OH

OH

OH

O

HO

O

HO

Beside addition of O2, OH-adducts of phenolic compounds can also split water under formation of the corresponding phenoxyl radicals (reaction (6)). Water elimination is acid–base catalysed, whereby the OHcatalysed dehydration reaction of the OH-adduct radicals of phenol was reported to be faster than the H+-catalysed reaction (Mvula et al., 2001). This fact is reflected in the results for Nar. The decreasing yield of Eri with increasing pH can be ascribed to the favor of reaction (6) over reaction (5) in basic medium. Similar pH dependences were observed for the OH-adducts of 4-hydroxybenzoic acid (Swoboda and Solar, 1999).

H

OH

To avoid the influence of oxygen and to enhance the OH radical yield radiolysis in the presence of N2O was performed. Under this conditions, the hydrated electrons d e aq are converted into OH radicals (reaction (7)), k7 ¼ 9 3 1 1 9.1  10 dm mol s (Buxton et al., 1988); G(dOH)ffi 5.8  107 mol dm3 J1.

d

d  e aq þ N2 O ! OH þ OH þ N2

(7) 4

3

In N2O saturated solutions (2  10 mol dm Nar, pH 8.8–8.9), no hydroxylation product could be detected. Due to the lack of O2, reaction (5) cannot take place. The initial degradation yield of Nar was G(-Nar) ¼ 2.9  107 mol dm3 J1, i.e. only half of the dOH radicals induced degradation, which indicated that the primarily formed OH-adducts undergo reactions leading to reformation of the substrate. Second-order decay of the OHadducts by disproportionation reactions (8) and (9), whereby Nar and Eri are formed, is apparently of minor importance since no eriodictyol was detectable. Therefore, it may be concluded that the first order process, water elimination and phenoxyl radical production, reaction (6), is the dominant reaction pathway. OH

OH

OH

O Nar

O 2 NarOH-adduct

(4)

O

HO Nar-OH-adduct

O2 OH

O

HO

O

OH

OH

OH

+

H 2O + OH

O

OH

O

(8)

OH

OH OO HO H OH - HO2

O

O

HO

Nar-OH-adduct

OH

NarOHadduct þ NarPhOd ! Nar þ Eri

(9)

O

3.3. Radiolysis in the presence of azide/N2O

Eri

(5) O O

HO Nar-OH-adduct

- H2O

(6) OH

O

NarPhO

To study the fate of the NarPhOd radicals in more detail, irradiations in the presence of sodium azide were carried out (2  104 mol dm3 Nar, 2  102 mol dm3 NaN3, pH 9.6, N2O). The dOH radicals formed in N2O saturated solutions are scavenged by azide ions and converted into Nd3 radicals (reaction (10)), G(dOH) ¼ (Nd3 )ffi 5.8  107 mol dm3 J1, k10 ¼ 1.2  1010 dm3 mol1 s1 (Neta et al., 1988). Azide radicals react with phenolic substrates

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selectively by electron transfer to phenoxyl radicals (reaction (11)). The rate constants of one-electron oxidations of flavonoids with azide ions are close to diffusion controlled, k  5  109 dm3 mol1 s1, e.g. for eriodyctiol k ¼ 3.1  109 dm3 mol1 s1 and apigenin 4.8  109 dm3 mol1 s1 (Bors et al., 1990). (10)

N3 d þ Nar ! N3  þ NarPhOd

OH O

Nar +

2NarPhO

OH

HO

0.6 λ 365 nm

0.2

0.0 0.0

0.2

0.6

0.8

1.0

kGy Fig. 4. Increase of absorptions observed after irradiation of 2  104 mol dm3 Nar, 2  102 mol dm3 NaN3, pH 9.6, saturated with N2O. Absorptions were measured using the not irradiated solution as reference.

175 APIGENIN 150 125 100 75 50

OH

O

25

apigenin

(12)

NARINGENIN

0 250

200

300

350

400

450

500

550

nm Fig. 5. UV-spectra of Nar and Api obtained under the HPLCchromatographic conditions: H2O (2.5% (w/v) formic acid)/CH3OH ¼ 50/50 (v/v).

150 μmol Nar

0.4

O

or OH O

λ 260 nm

0.4

(11)

Taking into account the rate constant of dOH with Nar and assuming a rate constant for k(Nd3 +Nar) 3  109 dm3 mol1 s1 it can be expected that reactions (10) and (11) are taking place quantitatively. The initial degradation yield of Nar in the presence of NaN3 was G(-Nar)= (3.670.02)  107 mol dm3 J1, which corresponds to about 62% of the Nd3 concentration (Fig. 3). This indicates that NarPhOd decay partly under reformation of Nar. Eri was not observable. The formation of radiation products as a function of dose was followed spectrophotometrically using the not irradiated solution as reference. The build up of absorptions at 260 and 365 nm exhibited linearity up to 500 Gy (Fig. 4). Phenoxyl radicals are known to decay by combination reactions like dimerization and disoroportionation. Under the low dose-rate conditions of gamma-radiolysis, however, phenoxyl radicals react predominately with products by electron transfer, so that the yields of simple combination products are very low (Ye and Schuler, 1989). In the case of disproportionation of NarPhOd beside Nar, the formation of apigenin or an isomeric quinone could be formed (reaction (12)). HO

0.8

OD/cm

OH þ N3  ! OH þ N3 d

1.0

Norm.

d

731

100

50

0 0

200

400

600

800

1000

Gy Fig. 3. Degradation of Nar in the presence azide ions as a function of dose. Solution: 2  104 mol dm3 Nar, 2  102 mol dm3 NaN3, pH 9.6, N2O. The given measuring points represent mean values of three measurements.

HPLC chromatograms of azide containing solutions are presented in Fig. 6. Apart from NaN3 (Rt 2.1 min) and Nar (Rt 6.7 min) a product with Rt 12.7 min was observable in low concentrations. Based on the UV-spectrum (Fig. 5) and on the Rt of the reference compound, it could be identified as apigenin. This was also confirmed by LC/MS measurements (LCMS-Q-TOF, Bruker Daltonics microTOF-Q coupled to a Dionex Ultimate 3000 micro-HPLCsystem), which demonstrated unequivocally that this compound has the mass spectrum of Api (molecular mass 270 g mol1). It should be mentioned that the mass spectroscopic analysis further proved that whether Eri

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732

340 nm

0 Gy

100

Naringenin

mAU

80 60

2.104 2.167 2.489

20 0

3.251

40

2

4

6

8

10

12

14

60

6.704

min

340 nm

400 Gy Naringenin

mAU

50 40 30

2.163 2.487 2.587 2.866

10

12.677

20 Apigenin

0 2

4

6

8

10

12

14

min Fig. 6. HPLC-chromatograms at 340 nm of unirradiated and with 400 Gy irradiated solutions of 2  104 mol dm3 Nar, 2  102 mol dm3 NaN3, pH 9.6, saturated with N2O. Rt: 2.1 min NaN3, 6.7 min Nar, and 12.7 min Api.

nor 4-hydroxybenzoic acid (a possible fragmentation product) were formed under these reaction conditions.

(reaction (14)). OH HO

3.4. Radiolysis in air free solutions

O

(13)

Nar + e-aq

To investigate the radiation stability of Nar in oxygen d free solution where both, reducing (e aq , H ) and oxidizing d ( OH) species are present, irradiations were performed in Ar saturated solution (2  104 mol dm3 Nar, pH 8.7). The decomposition rate was distinctly reduced. Whereas in the presence of air 10% degradation was achieved with 80 Gy, in the presence of argon a 7.5 times higher dose, 600 Gy, was required. As initial degradation yield Gi(-Nar) ¼ (0.3370.04)  107 mol dm3 J1 was calculated. This exceedingly slow going degradation may be referred to back reactions. Flavonoids were found to react very efficiently with e aq , especially those having a C4-keto group, which is the active site for the e aq attack. For Nar a rate constant k(Nar+ e ) ¼ (1.270.1)  1010 dm3 mol1 s1 was reported (Cai aq et al., 1999). The addition of e aq to a carbonyl group leads to radical anions, which was reported for several unsaturated carbonyl compounds (Lilie and Henglein, 1969) and chlorobenzaldehydes (Solar et al., 1995). The formation of the electron adduct of Nar is given in reaction (13). These species can reduce the phenoxyl radicals, formed by the dOH-attack, whereby Nar is regenerated

. OH OO

OH HO

O

HO

O

H+

+

2 Nar

. OH

O-

OH

O

(14)

4. Conclusion The reactions of dOH with naringenin induce mainly the formation of OH-adducts on the B-ring. In the presence of air, these radicals undergo competitive reactions, water splitting and addition of O2. The latter mainly results in the production of eriodictyol, the hydroxylated form of naringenin. Direct electron transfer from the phenolat of naringenin (pK 9.1) to dOH, i.e. direct NarPhOd

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formation, seems to be of minor importance since eriodictyol is also formed at pH49.5, although in less amounts due to the pH-dependent water splitting reaction. The fact that the degradation of naringenin in the absence of air is extremely low may be important for the use of radiation as conservation method for flavonoid containing food. Acknowledgment The authors like to thank Prof. J. Ko¨nig and S. Haselmaier for the mass spectrometric measurements. References Bielski, B.H.J., Cabelli, D.E., 1991. Review: highlights of current research involving superoxide and perhydroxyl radicals in aqueous solutions. Int. J. Radiat. Biol. 59, 291–319. Bielski, B.H.J., Cabelli, D.E., Arudi, R.L., 1985. Reactivity of HOd2 /Od 2 radicals in aqueous solution. J. Phys. Chem. Ref. Data 14 (4), 1041–1100. Bors, W., Heller, W., Michel, C., Saran, W., 1990. Flavonoids as antioxidants: determination of radical-scavenging efficiencies. Met. Enzymol. 186, 343–355. Butkovic, V., Klasinc, L., Bors, W., 2004. Kinetic study of flavonoid reactions with stable radicals. J. Agric. Food Chem. 52, 518, 523, 521. Buxton, G.V., Greenstock, C.L., Helman, W.P., Ross, A.B., 1988. Critical review of rate constants for reactions of hydrated electrons, hydrogen atoms and hydroxyl radicals in aqueous solution. J. Phys. Chem. Ref. Data 17, 513–886. Cabelli, D.E., 1997. The reactions of HOd2 /Od radicals in aqueous 2 solutions. In: Alfassy, Z. (Ed.), Peroxyl Radicals. Wiley, Chichester, GB, pp. 407–437. Cai, Z., Li, X., Katsumura, Y., 1999. Interaction of hydrated electron with dietary flavonoids and phenolic acids: rate constants and transient spectra studied by pulse radiolysis. Free Radic. Biol. Med. 27 (7/8), 822–829. Galati, G., Moridani, M.Y., Chan, T.S., O‘Brien, P.J., 2001. Peroxidative metabolism of apigenin and naringenin versus luteolin and quercetin: glutathion oxidation and conjugation. Free Radic. Biol. Med. 30 (4), 370–382. Gaziano, J.M., 2000. Dietary antioxidants and cardiovascular disease. Vitamins hormones. Adv. Res. Appl. 58 (1), 299–320. Getoff, N., Prucha, M., 1983. Spectroscopic and kinetic characteristics of HOd2 and Od 2 species studied by pulse radiolysis. Z. Naturforsch. 38A, 589–590. Heo, H.J., Kim, D.-O., Shin, S.C., Kim, M.J., Kim, B.G., Shin, D.-H., 2004. Effect of antioxidant flavanone, naringenin, from Citrus junos on neuroprotection. J. Agric. Food Chem. 52, 1520–1525.

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