Spectrophotometric study on the oxidation of rutin by horseradish peroxidase and characteristics of the oxidized products

Biochimica et Biophysica Acta 882 (1986) 445-451 Elsevier

445

BBA 22355

S p e c t r o p h o t o m e t r i c study on the oxidation of rutin by horseradish p e r o x i d a s e and characteristics of the o x i d i z e d products

Umeo Takahama Department o/Biology, Kyushu Dental College, Kitakyushu 803 (Japan) (Received October 29th, 1985) (Revised manuscript received April 3rd, 1986)

Key words: Peroxidase; Rutin oxidation; Flavonoid; Hydrogen peroxide

Rutin (3',4',5,7-tetrahydroxyflavone-3-rutinoside) was oxidized by a horseradish peroxidase-H zO2 system to an ascorbate-reducible product which had an absorption maximum at about 290 nm and a shoulder at about 440 nm at pH 4. At pH 7.8, ascorbate-reducible compounds and sodium hydrosulfite-reducible and -nonreducible compounds were formed by the oxidation. The ascorbate-reducible compounds consisted of at least two components, the absorption bands of which were at 460-480 nm and about 620 nm. The sodium hydrosulfite-reducible compounds also consisted of two components, and one of the components which had an absorption maximum at about 480 nm seems to be formed from the ascorbate-reducible component of an absorption maximum at the blue region by a nonenzymatic reaction. A mixture of oxidized products of rutin formed by tert-butyl hydroperoxide-dependent oxidation was similar to that formed by the enzymatic reaction. It is discussed that the 3'- and 4'-OH groups of rutin were oxidized by the horseradish peroxidase-H 202 system and that the oxidized product which could be reduced by ascorbate is an o-quinone derivative.

Introduction

The antioxidative function of flavonols such as quercetin has been discussed with respect to their being hydroxyl group-substituted compounds [1] and their localization in plant cells [2]. Hydroxyl groups in ring B (Fig. 1) seem to preferentially participate in their antioxidative function [1,3]. The compounds are oxidized when they function as antioxidants scavenging O f [4], singlet oxygen [5], and lipid radicals [6]. Such a function has also been reported using flavonol glycosides such as rutin, etc. [4,6,7]. In general, flavonoid glycosides are believed to be localized in hydrophilic regions such as vacuoles. As to their physiological roles, being a screen for ultraviolet light [8] and an H202 Xscavenger [7] are proposed because of their stabil-

ity in ultraviolet [9] and blue light [10] and their ability to be potent electron donors to peroxidase [8,11]. However, the oxidation of flavonol glycosides during their antioxidative functions has not been studied. The elucidation of the mechanism

OH

0

Fig. 1. Chemical structure of rutin. G1, glucose; Rha, rhamnose.

0304-4165/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

446

may be important for the determination of their function in plant cells and their pharmacological function in decreasing increased capillary permeability, etc. [12]. This paper deals with rutin (Fig. 1) oxidation by tert-butyl hydroperoxide and a horseradish peroxidase-H202 system.

AA

0

-0.01

-0.02

Materials and Methods

Horseradish peroxidase (grade II) was obtained from Boehringer Mannheim G m b H . Rutin (quercetin-3-rutinoside) and quercetin (3,Y,4',5,7pentahydroxyflavone) were obtained from Wako Pure Chemical Industries Co. Apigenin (Y,5,7-trihydroxyflavone) and kaempferol (3,4',5,7-tetrahydroxyflavone) were from Sigma Chemical Co. Luteolin (3',4',5,7-tetrahydroxyflavone) was from Laboratories Sarget. tert-Butyl hydroperoxide (70% aqueous solution) was obtained from Nakarai Chemicals Ltd. Flavonoid oxidation by horseradish peroxidase was followed at 25°C using a Hitachi 557 doublebeam and dual-wavelength spectrophotometer. Reactions were started by adding H202 to a cuvette of light-path 10 mm. Further details are shown in the figure legends. Flavonoid oxidation by tertbutyl hydroperoxide was also followed spectrophotometrically. Radical reactions were started by adding 70% aqueous solution of tert-butyl hydroperoxide, tert-Butyl hydroperoxide is known to dissociate into hydroxyl radical and tert-butoxyl radical.

- 0.03

I

-0.0~

J

l

I

300

t

I

1

I

J

400

I

I

I

I

]

500 Wctvelength

I

I

I

I

J

I

600 ( nm )

J

I

I

700

I 800

Fig. 2. Difference spectra of tert-butyl hydroperoxide-dependent oxidation of rutin. The reaction mixtures (2.5 ml) contained 4 ~tM rutin, 50 /zM CuSO 4 and 50 m M sodium phosphate (pH 7.8). At zero time, 5 /L1 70% tert-butyl hydroperoxide was added to the sample cell and repetitive scanning (2 n m / s ) was started every 9.6 min. The inset shows absorption spectra of rutin (I) and oxidized products (II).

1.0

~%70~o.1

0.10

\

a82o=rO.OOs 1

2 min

i~x

Off3

';..x x x2

'..3.?k Results

When rutin was oxidized by tert-butyl hydroperoxide, loss of the 400 nm absorbance was accompanied by an absorbance increase in the visible region (Fig. 2). Two broad difference peaks were detected at about 480 and 620 nm. An absorbance increase due to oxidation was also observed at about 300 nm. There were two isosbestic points at about 345 and 460 nm. The oxidized product had a broad peak at about 600 nm and a shoulder at about 370 nm. A peak of the oxidized products (see later) is observed at about 330 nm as a shoulder (Fig. 2, inset) because of the absorption band of tert-butyl hydroperoxide in the near ultraviolet region. Some of the oxidized

t

300

\.~ _ ".,.'.-. .

.

.

.

.

4O0

500 Wavelength

600

700

800

(nm)

Fig. 3. Peroxidase-dependent oxidation of rutin. The reaction mixture (2.5 ml) contained 40 # M rutin, 20 # M H202, 0.8 # g / m l horseradish peroxidase and 50 m M sodium phosphate (pH 7.8). Reactions were started by adding H202. - - , rutin; . . . . . . , 3 min after H202 addition; . . . . . . , mixture of curve ( . . . . . . ) plus 1 m M sodium ascorbate; . . . . . , mixture of curve ( . . . . . . ) plus sodium hydrosulfite. The inset shows time-courses of absorbance changes. An arrow indicates the addition of H202. The reaction mixture contained 0.4 / t g / m l horseradish peroxidase. Other conditions were the same as those used to record absorption spectra.

447

products were reduced by ascorbate. A difference spectrum of ascorbate-reduced minus the oxidized products of rutin had two broad peaks at about 480 and 600 nm. In this experiment, CuSO 4 (50 /tM) was added to stimulate the degradation of tert-butyl hydroperoxide. The addition of CuSO 4 caused a shift of the absorbance band of rutin to a longer wavelength. Because tert-butyl hydroperoxide-dependent oxidation of rutin was slow, rutin oxidation was followed using a horseradish peroxidase-H202 system (Fig. 3) to characterize the oxidized products of rutin depicted in Fig. 2. In the oxidation of rutin by peroxidase, an absorbance increase in the visible region such as that in Fig. 2 was observed; the absorbance increase appeared as two shoulders at around 500 and 600 nm. A difference spectrum of the oxidized products minus rutin had two peaks at 480 nm and 620 nm. In the ultraviolet region, the mixture of the oxidized products had an absorption maximum at about 330 nm (Fig. 3). This peak was observed when the absorption band of rutin disappeared, because of the smaller absorption coefficient of the mixture than that of rutin. The mixture of the oxidized products consisted of at least three components; an ascorbate-reducible component and sodium hydrosulfite-reducible and -nonreducible components. The ascorbate-reducible component (trace 2 minus trace 3) had two peaks at about 480 and 620 nm, but rutin was not formed by the reduction. A difference spectrum of ascorbate-nonreduced minus sodium hydrosulfite-reduced (trace 3 minus trace 4) had a peak at 480 nm and a shoulder at around 620 nm. That of sodium hydrosulfite-nonreduced minus rutin (trace 4 minus trace 1) had a peak at about 520 nm. In the ultraviolet region, absorption characteristics of ascorbate and sodium hydrosulfite affected the absorption spectra of ascorbate- and sodium hydrosulfite-nonreducible components. Time-courses of absorbance changes at 370, 480 and 620 nm are also shown in Fig. 3. Rutin oxidation as judged by the absorbance decrease at 370 nm was complete within 4 min after the addition of H=O=. At 480 nm, an absorbance decrease following an absorbance increase was observed. The absorbance increase was kinetically similar to that of the decrease at 370 nm. The

AA

0.02

\ 0

5 Time (rnin)

10

Fig. 4. Time-courses of production of ascorbate- and sodium hydrosulfite-reducible components. The reaction mixture (2.5 ml) contained 40/~M rutin, 20/~M H202, 0 . 4 / ~ g / m l horseradish peroxidase and 50 m M sodium phosphate (pH 7.8). After definite times of incubation, 1 m M sodium ascorbate and sodium hydrosulfite were added separately and the extent of the absorbance decrease was plotted. • and O, an ascorbatereducible component; • and zx, a sodium hydrosulfite-reducible component. - - , 480 nm; . . . . . . , 6 2 0 nm.

absorbance increase at 620 nm was divided into two phases; a rapid and a slow phase. The rapid absorbance increase had a lag period. To characterize more exactly the above absorbance changes, time-courses of the appearance of ascorbate- and sodium hydrosulfite-reducible components were investigated (Fig. 4). The ascorbate-reducible component reached a maxim u m value 1 min after the reaction was started and decreased to a constant value at 480 nm when the ratio of H202 to rutin was 0.5. The fraction of the slow absorbance decrease became smaller as the ratio increased. The increase of an ascorbatereducible component at 620 nm was much slower than that at 480 nm. The appearance of a sodium hydrosulfite-reducible component at 480 nm was slower than that of the ascorbate-reducible component at 480 nm but faster than that of a sodium hydrosulfite-reducible component at 620 nm. The kinetics of the appearance of the sodium hydrosulfite-reducible component at 620 nm were similar to that of the ascorbate-reducible component at 620 nm. The data suggest that the ascorbatereducible component with an absorption maxi-

448

A

AA

CAq002

0.16

I

I

I

I

I

t

I

~

I

AA

I

0.2

0.04

0.1

0.02

0.1 C

;

/f--

-

0

q00, -0.01

-0.01

-

0.08

/ /

:2

0.04

/X=4BOnm

I

I I I I i I 300 400

~ 50O

i i i I i 6O0

I 4

i

700

Wclvelength (nm]

Fig. 5. Peroxidase-dependent oxidation of rutin at pH 4. The reaction mixture (2.5 ml) contained 10 ~M rutin, 10 ~M H202, 0.8 ktg/ml horseradish peroxidase and 50 mM NaH2PO4-acetic acid (pH 4). Trace 1, rutin; trace 2, oxidized product; trace 3, 2 plus l mM sodium ascorbate. The inset shows time-courses of absorbance change at the wavelengths indicated. An arrow indicates the addition of H202.

m u m at a b o u t 480 n m was different from the c o m p o n e n t with an a b s o r p t i o n m a x i m u m at a b o u t 620 nm, a n d that the s o d i u m hydrosulfite-reducible c o m p o n e n t at 480 n m m a y be f o r m e d from the a s c o r b a t e - r e d u c i b l e c o m p o n e n t at 480 nm. The a b s o r p t i o n b a n d in the red region seems to consit of at least two c o m p o n e n t s . The effects of p H on the f o r m a t i o n of the p r o d u c t s were e a m i n e d (Fig. 5 a n d 6). A t p H 4, the a b s o r p t i o n m a x i m u m of rutin was o b s e r v e d at a b o u t 260 a n d 350 nm. W h e n rutin was oxidized at this p H , the c o m p o u n d o b t a i n e d h a d an abs o r p t i o n m a x i m u m at a b o u t 300 n m a n d a s h o u l d e r at a b o u t 440 n m (Fig. 5). T h e r e was n o a b s o r p t i o n b a n d at a r o u n d 600 nm, in c o n t r a s t to the p r o d ucts o b t a i n e d at p H 7.8 (Fig. 3). A s c o r b a t e red u c e d almost all of the oxidized p r o d u c t to rutin at p H 4. A difference s p e c t r u m of oxidized m i n u s a s c o r b a t e - r e d u c e d h a d a negative a n d a positive p e a k at 420 nm a n d 350 nm, respectively. A p e a k of the difference s p e c t r u m of a s c o r b a t e - r e d u c i b l e c o m p o n e n t c h a n g e d between 420 a n d 480 n m d e p e n d i n g on p H , substrate c o n c e n t r a t i o n , etc. Time-courses of the a b s o r b a n c e changes at 290, 350 a n d 440 n m at p H 4 were very similar to each other, with a half-time of a b o u t 12 s (Fig. 5, inset).

"o---

o- - -c~ -0.02 ]

/

-0.04~- # 0

N

~,=480nrn

I_0.03

/i / / i 5

I 6 pH

I 7

1 8

I 9

I

I

P

I

11-0.04

5

6

7

8

9

pH

Fig. 6. Effects of pH on the formation of ascorbate- and sodium hydrosulfite-reducible products. The reaction mixture (2.5 ml) contained 40 /~M rutin, 20 p.M H202, 0.4 k~g/ml horseradish peroxidase and sodium phosphate (pH 5-9). A buffer solution of pH 4 was obtained as described in Fig. 5. Reactions were started by adding H202. 3 min after the addition, absorption spectra were recorded and reductants were added subsequently as in Fig. 3. Left figure: absorbance increase (~ = 350-390 nm) (e e) and decrease at 480 nm (O. . . . . . O) and at 620 nm (a) by the addition of 1 mM sodium ascorbate. Right figure: O, a sodium hydrosulfite-reducible component; A, a sodium hydrosulfite-nonreducible component.

A s the p H b e c a m e higher, an a s c o r b a t e - r e d u c ible c o m p o n e n t which a b s o r b e d blue light a n d red light decreased a n d increased, respectively (Fig. 6). F o r m a t i o n of rutin from an a s c o r b a t e - r e d u c i b l e c o m p o n e n t b e c a m e smaller as the p H was increased from 4 a n d was not observed when the p H was higher than 6. A s o d i u m hydrosulfite-reducible c o m p o n e n t increased as the p H was increased from 4 to 7 a n d then reached a c o n s t a n t value. A s o d i u m h y d r o s u l f i t e - n o n r e d u c i b l e c o m p o n e n t was f o r m e d m a x i m a l l y at p H 6 T h e s t o i c h i o m e t r y for the o x i d a t i o n of rutin b y H202 was e x a m i n e d at p H 4, since only one p r o d u c t was f o r m e d at this p H (Fig. 5). H 2 0 2 in c o n c e n t r a t i o n n e a r l y e q u i m o l a r with rutin was required for c o m p l e t e o x i d a t i o n (A = 380 nm) and the f o r m a t i o n of a p r o d u c t ( k = 290 a n d 420 nm) (Fig. 7). Two isosbestic p o i n t s were observed during the titration, suggesting that the p r o d u c t f o r m e d was not t r a n s f o r m e d further. This result indicates that the a s c o r b a t e - r e d u c i b l e c o m p o n e n t which a b s o r b s blue light is a two-electron oxidized

449 A 0.05 L

I

I

I

I

I

0.1

1.0

L ~

0

--

0.5

-0.05

~

.

,

o 300

400

500 Wgve[engt h

-0.1C

I 0

I

I

5 H202

°05

'~\ \ \ p H 6

I 10

I

(pM)

Fig. 7. Stoichiometry for oxidation of rutin by H202. The reaction mixture (2.5 ml) contained 10 btM rutin, 0.8 ~ M horseradish peroxidase and 50 m M NaHzPO4-acetic acid (pH 4). Extent of absorbance changes were plotted as a function of H202 concentration. (3 (3, oxidation of rutin followed at 350 nm; (3 . . . . . . (3, formation of a product at 420 nm; e, formation of a product at 290 nm.

product of rutin, rather than dimers or polymers. At pH 7.8, it was difficult to determine the stoichiometry precisely because of transformation of the ascorbate-reducible component to ascorbate-nonreducible component (Figs. 4 and 8). However, when it was determined in the presence of a high concentration of horseradish peroxidase (4 /~g/ml), postulating that the HzO2-dependent oxidation was completed at the peak of absorbance increase at 480 nm (see Fig. 4), the stoichiometry for rutin oxidation by H202 was nearly I (not shown). Hydrogen ion concentrations were changed after the HzO2-dependent oxidation was completed (Fig. 8). By increasing the pH from 4 to 6, an absorption band in the red region appeared and the absorption intensity in the blue region became small. An absorption band in the ultraviolet region shifted from 300 to 340 nm as a result of the above pH change. The absorption characteristics of the products obtained by changing pH from 4 to 6 were similar to those of the products obtained by oxidizing rutin at pH 6. The products obtained by changing pH from 4 to 6

600

700

800

{nm)

Fig. 8. Effects of pH on absorption spectra. Reaction mixtures contained 40 /~M rutin, 40 v M H z O 2, 0 . 8 / ~ g / m l horseradish peroxidase and 50 m M sodium phosphate (pH 6) or NaH2PO4-acetic acid (pH 4). After reactions were completed, the pH was shifted. - - , an oxidized product of rutin at p H 4; . . . . . 7, pH was shifted from 4 to 6; . . . . . , oxidized products of rutin at pH 6: . . . . . . , pH was shifted from 6 to 4.

consisted of at least three components; i.e., an ascorbate-reducible component and hydrosulfitereducible and -nonreducible components as in Fig. 3. The pH effects were irreversible. By decreasing the pH from 6 to 4, after reactions were completed at pH 6, the absorption band in both the blue and red region became small. The absorbance decrease in the red region was greater than in the blue region. The absorption intensity in the ultraviolet region increased and a shoulder was found at 300 nm when a difference spectrum before and after the pH change wa splotted (not shown). The products formed by changing pH from 6 to 4 consisted of the three components, an ascorbate-reducible component and sodium hydrosulfite-reducible and -nonreducible components. Discussion

Rutin is a potent antioxidant [3,5,13] as well as an electron donor to peroxidase [8]. The experiments in this paper were designed to elucidate the mechanism of the antioxidative function of rutin. Since the antioxidative function of rutin may be attributed to its ability to scavenge radicals [14,15], the oxidation products formed by tert-butyl hy-

450

droperoxide were compared to those formed by a horseradish peroxidase-H202 system. The products obtained by the above two systems had absorption bands in both the blue and red regions at pH 7.8. An ascorbate-reducible product formed by tert-butyl hydroperoxide had broad absorption maxima at about 480 and 600 nm (Fig. 2) as did that formed by peroxidase-dependent oxidation (Fig. 3). The data suggest that the ascorbatereducible products formed by the above two systems are very similar to each other. As the pH of the reaction mixture was decreased, an ascorbate-reducible component which absorbed blue light increased and one that absorbed red light decreased (Fig. 6). The absorption band of the ascorbate-reducible component in the blue region was shifted to a shorter wavelength by the pH change. These data suggest that the absorption characteristics of the ascorbate-reducible component can be affected by acid-dissociation of hydroxyl groups. A hydroxyl group at C-7 of rutin seems to contribute to the acid-dissociation because an acidic hydroxyl group, 4'-OH, is probably oxidized (see later). A hydroxyl group at C-7 of rutin is known to dissociate at mild alkaline pH [16]. On increasing the pH from 4 to 6 after the H2OR-dependent oxidation was completed, the products obtained were partially reduced by ascorbate (not shown), while the product formed at pH 4 was nearly completely reduced by ascorbate (Fig. 5). This result indicates that the product formed at pH 4 can be converted to other substances by nonenzymatic reactions only by increasing pH. However, it is also possible to consider that the partial reduction by ascorbate at pH 6 may be due to the decrease of redox potential by increasing pH. This possibility is excluded due to the fact that the complete reverse reaction was not possible. As a reaction sequence of rutin oxidation by the peroxidase-H202 system, the following reactions are considered: A ~ B ~ C, where A is rutin, B is an ascorbate-reducible component of an absorption maximum at 480 nm, and C is a sodium hydrosulfite-reducible component with an absorption maximum at 480 nm. The formation of B from A is deduced from the data that only an ascorbate-reducible component was formed at pH

4 by two-electron oxidation (Fig. 7). The transformation of B to C is deduced from the difference in kinetics of the appearance of B and C in Fig. 4 and from the formation of C from B by shifting the pH from 4 to 6 (Fig. 8). The compounds which had an absorption band in the red region seem to be formed from B (Fig. 8). However, further experiments are required to firmly establish the reaction sequence. At pH 4, ascorbate reduced the oxidized product to rutin (Fig. 5), but at pH higher than 4 ascorbate could reduce some of the oxidized components, and rutin was not always formed by the reduction (Fig. 6). This indicates that ascorbate can transform the oxidized components to compounds other than rutin by reduction at pH higher than 4. To estimate the oxidized products of rutin, apigenin, luteolin, kaempferol and quercetin oxidation by horseradish peroxidase were examined. Rates of the oxidation were in order to luteolin > quercetin > kaempferol > rutin >> apigenin. During luteolin and quercetin oxidation, two absorption bands were observed in the visible region at pH 7.8 and the absorption band in the longer wavelength disappeared when these compounds were oxidized at pH 4 (unpublished data); these are similar to the pH effects shown in Figs. 3 and 5. The oxidized products were completely reduced by ascorbate at pH 4. The oxidation of apigenin and kaempferol did not accompany the absorption increase in the visible region. These data suggest an important function of 3'- and 4'-OH groups in the formation of ascorbatereducible products which had absorption bands in the visible region. Participation of 3'- and 4'-OH groups in the oxidation was supported by the data that aluminium chloride, which can chelate o-OH groups, inhibited quercetin and luteolin oxidation as well as rutin oxidation, but did not inhibit kaempferol oxidation. The important function of o-OH groups in ring B for the oxidation of flavonoids has been pointed out by Younes and Siegers [3]. Oxidation of 5- and 7-OH groups, which are common to the flavonoid compounds examined in this paper, seems not be easy, since apigenin is not a potent electron donor to horseradish peroxidase (see above) and carotenoid or lipid radicals formed in illuminated chloroplasts [17].

451 Slower oxidation of r u t i n t h a n quercetin a n d luteolin might be a t t r i b u t e d to d i s t u r b a n c e of the a p p r o a c h of the o-OH groups of r u t i n to the catalytic site of peroxidase by 3-rutinoside. This c o n s i d e r a t i o n is supported by the data that quercitrin, quercetin 3-rhamnoside, is a better electron d o n o r to peroxidase t h a n r u t i n [8].

Acknowledgements I t h a n k Professor T. Oniki for reading the revised version. T h a n k s are also due to Professor T. Egashira for his e n c o u r a g e m e n t d u r i n g this work.

References 1 Letan, A. (1966) J. Food Sci. 31,518-523 2 Takahama, U. (1985) Phytochemistry 24, 1443-1446 3 Younes, M. and Siegers, C.P. (1981) Planta Med. 43, 240-244 4 Takahama, U. (1983) Plant Physiol. 71,598-601

5 Takahama, U., Youngmarm, R.J. and Elstner, E.F. (1984) Photobiochem. Photobiophys. 7, 175-181 6 Takahama, U. (1983) Photochem. Photobiol. 38, 363-367 7 McClure, J.W. (1974) in The Flavonoids (Harborne, J.B. et al., eds.), pp. 970-1055, Chapman and Hall, London 8 Takahama, U. (1984) Plant Physiol. 74, 852-855 9 Kameta, M. and Sugiyama, N. (1971) Bull. Chem. Soc. Japan 44, 3211 10 Takahama, U. (1985) Photochem. Photobiol. 42, 89-91 11 Barz, W. and Hoesel, W. (1979) in Recent Advance in Phytochemistry (Swain, T. et al., eds.), Vol. 12, pp. 339-369, Plenum Press, New York 12 Wagner, H. (1979) in Recent Advance in Phytochemistry (Swain, T. et al., eds.), Vol. 12, pp. 589-616, Plenum Press, New York 13 Whittern, C.C., Miller, E.E. and Pratt, D.E. (1984) J. Am. Oil Chem. Soc. 61, 1075-1078 14 Cavallini, L., Bindoli, A. and Siliprandi, N. (1978) Pharmacol. Res. Commun. 10, 13-136 15 Thompson, M., Williams, C.R. and Elliot, G.E.P. (1976) Anal. Chem. Acta 85, 375-381 16 Harborne, J.B. (1973) Phytochemical Methods, Chapman and Hall, London 17 Takahama, U. (1982) Plant Cell Physiol. 23, 859-864