Redox intermediates of flavonoids and caffeic acid esters from propolis: An EPR spectroscopy and cyclic voltammetry study

Free Radical Biology & Medicine, Vol. 18, No. 5, pp. 901-908, 1995 1995 Elsevier Science Ltd Printed in the USA. All rights reserved 0891-5849/95 $9.50 + .00

Pergamon

0891-5849(94)00232-0

Original Contribution REDOX ESTERS

INTERMEDIATES OF FLAVONOIDS AND CAFFEIC ACID FROM PROPOLIS: AN EPR SPECTROSCOPY AND CYCLIC VOLTAMMETRY STUDY

PETER RAPTA,* VLAD1M[R MI~[K, *$ ANDREJ STA~KO,* and IMR1CH VRABEL* *Faculty of Chemistry, Slovak Technical University, Bratislava, Slovak Republic; qnstitute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava, Slovak Republic; and *National Cancer Institute, NIH, Bethesda, Maryland, USA

(Received 18 April 1994; Revised 11 October 1994; Accepted 2 December 1994) A b s t r a c t - - T h e redox properties of flavonoids: chrysin (1), tectochrysin (2), galangin (3), isalpinin (4), pinostrobin (5), pinobanksin (6), pinobanksin-3-acetate (7), and of caffeic acid ester (8) and diacetylcaffeic acid ester (9), all isolated from propolis, were investigated by cyclic voltammetry in acetonitrile. The choice of aprotic solvent lowered the reactivity of the radical intermediates and made possible to identify redox steps and intermediates not detected so far. The oxidation potentials (vs. saturated calomel electrode) of the investigated compounds were in the region of 1.5 V for 3 and 4; 1.9 V for 1, 2, and 5; 2.0 V for 6 and 7; 1.29 V for 8; and 2.3 V for 9. These oxidation potentials were mainly influenced by the presence of a double bond in 2,3-position and substituent R ~ in position 3. Comparison with our earlier data revealed that flavonoids, 1-4, and caffeic acid ester 8 with lower oxidation potentials showed the maximal lipid antioxidant activity, whereas those with higher potentials (5, 6, 7, and 9) are less active. On reduction of 1 - 9 several one-electron-steps were typically observed in the potential regions: - 1.5 V, - 1.8 V, and - 2 V, where in simultaneous EPR experiments anion radicals of 1 and 3 were observed with the center of unpaired spin density on ring A. Upon oxidation of flavonoids 1 - 4 carbonyl carbon-centered radicals, .C(O)R, were identified as consecutive products using the EPR spin trapping technique. Keywords---Flavonoids, Caffeic acid esters, Radical intermediates, Redox reactions, EPR, Spin trapping, Cyclic voltammetry, Antioxidants, Free radicals

ide, 2s which dismutates to give hydrogen peroxide) 3 The prooxidant action of flavonoids may also be linked to their direct reduction of Fe(III) to Fe(II), resulting in the production of -OH radicals through Fenton chemistry. 28 During autooxidation of several water-soluble flavonoids in aqueous buffer, o-semiquinone radicals, the one-electron oxidation products, have been detected using EPR-spin stabilization technique with Mg 2÷ as the stabilizing metal ion. 2s Propolis, a honeybee hive product, and its phenolic constituents and their derivatives were shown to exhibit several positive pharmacological properties, among them antiproliferative activity in human tumor cells, 14 antiinflammatory, Is antibacterial, 16 antiviral, ~7 immunomodulatory, Is and antioxidant. ~9 In this work a complex redox behavior of series of flavonoids and caffeic acid esters isolated from propolis was investigated using cyclic voltammetry and EPR spectroscopy coupled with electrochemical apparatus. The obtained electrochemical and EPR data were compared with the previously reported antioxidant potential of these substances. 2° In addition, the structure and reactivity

INTRODUCTION

Flavonoids, phenolic plant constituents, showed a number of positive pharmacological properties such as vasoprotective, t antiinflammatory, 2'3 antiviral, 4 antifungal, 5 and antiproliferative activity on tumor cells. 5 Some of these effects (i.e., antiproliferative activity) may be attributed to the topoisomerase II dependent DNA cleavage induced by flavonoids; 6 others (such as vasoprotective and antiinflammatory) may be linked to their antioxidant properties. The latter property of flavonoids was investigated in great detail, and a number of structural requirements for antioxidant activity were elucidated (for review see ref. 7). The antioxidant properties of flavonoids were associated with their ability to form stable "antioxidant" radicals. 9'1° Prooxidant activity of flavonoids has also been reported 11.12,28 and seems to be linked to their ability to autoxidize in the presence of dissolved oxygen to produce superoxAddress correspondence to: Vladimlr M i ~ , National Cancer Institute, NIH, Bldg. 10, Rm. B1B50, Bethesda, MD 20892, USA; Email: [email protected] 901

902

P. RAPTA et al.

Table 1. Electrochemical Data Obtained From Cyclic Voltammograms of the Investigated Flavonoids and Caffeic Acid Esters

OH Compound 1 2 3 4

R~

Chrysin Tectochrysin Galangin Isalpinin

O

R2

Ep,

E~

E~

H H OH OH

OH OCH3 OH OCH3

1.86i 1.88i 1.54i 1.42i

-0.26 ~ -0.18 r -0.1 r ~0 r

-

H OH OCOCH3

OCH3 OH OH

1.95 i 2i 2.03 i

-0.T -0.T -0.23 r

-2.2 i - 1.35i - 1.45i

E2

1.5i 1.45i 1.45i 1.64q

- 1.72q - 1.68i - ! .76q

E~ -2.02 r

E~

E2;

-0.45 -0.62

0.47 0.45 0.4

-0.45 -0.4 -0.17

0.46 0.85 0.92

2,3-dihydro

5 Pinostrobin 6 Pinobanksin 7 Pinobanksin-3 -acetate

- 1.7i - 1.82i

~ R~

-2 i ~-2.2 i

//CH2

CH=CH--C--O~CH2--CH2--C x

\ CH3

R2 / CH3 C H 2 - - C H ~ C \ CH3 8 Caffeic and 9 Diacetylcaffeic acid esters

OH

OH

1.29~

OCOCH3

OCOCH3

2.3 ~

0.13 i

--1.52 i

-0.12 r

-1.72 i

--2.15 i

0.28 -0

Ep,--anodic peak potential. F~--cathodic peak potential on reverse scan. --cathodic peak potential of the first reduction wave. --cathodic peak potential of the second reduction wave. --cathodic peak potential of the third reduction wave. 'a--cathodic peak potential on reverse scan. 'a--anodic peak potential on reverse scan. i--irreversible; q--quasireversible; r--reversible.

~

~

of radicals generated during electrochemical redox p r o c e s s e s in a c e t o n i t r i l e ( A C N ) is d e s c r i b e d . T h e c h o i c e o f aprotic s o l v e n t l o w e r e d the r e a c t i v i t y o f radical p r o d u c t s a n d m a d e it p o s s i b l e to i d e n t i f y r e d o x steps and i n t e r m e d i a t e s n o t o b s e r v a b l e so far in a q u e ous solutions.

MATERIALS AND METHODS

T h e i n v e s t i g a t e d c o m p o u n d s ( T a b l e 1) w e r e i s o l a t e d f r o m p r o p o l i s as d e s c r i b e d e a r l i e r ) '2° A c e t o n i t r i l e w a s o b t a i n e d f r o m F l u k a ( R o n k o n k o m a , N Y ) . S p i n trap 2methyl-2-nitroso propane (MNP) was acquired from A l d r i c h . 5 , 5 - d i m e t h y l p y r r o l i n e N - o x i d e ( D M P O ) , purc h a s e d f r o m S i g m a , w a s f r e s h l y d i s t i l l e d at 75°(2 and 0.5 torr and s t o r e d at - 2 5 ° C b e f o r e use. Cyclic voltammograms were obtained under argon a t m o s p h e r e u s i n g the e l e c t r o c h e m i c a l s y s t e m P A R 2 7 0 e m p l o y i n g three e l e c t r o d e s w i t h p l a t i n u m w o r k and

a u x i l i a r y e l e c t r o d e s and r e f e r e n c e saturated c a l o m e l e l e c t r o d e ( S C E ) e q u i p p e d w i t h L u g g i n capillary. In situ e l e c t r o c h e m i c a l E P R e x p e r i m e n t s w e r e card e d o u t in a V a r i a n fiat cell, and E P R s p e c t r a w e r e recorded using a Bruker 200D spectrometer. I N D O c a l c u l a t i o n s w e r e c a r d e d out to d e t e r m i n e the u n p a i r e d spin d e n s i t y d i s t r i b u t i o n a n d c o r r e s p o n d i n g splitting c o n s t a n t s in a n i o n radical o f c h r y s i n e ; G E O M O p r o g r a m w a s u s e d f o r c a l c u l a t i o n s , 29 and the g e o m e t r y w a s o p t i m a l i z e d b y A M I - m e t h o d 3° w i t h MOPAC-program.3

RESULTS AND DISCUSSION

T h e c o m m o n structural f o r m u l a e o f the f l a v o n o i d s and c a f f e i c a c i d esters a l o n g w i t h the e l e c t r o c h e m i c a l d a t a o b t a i n e d f r o m c y c l i c v o l t a m m o g r a m s are s u m m a r i z e d in T a b l e 1. R e p r e s e n t a t i v e c y c l i c v o l t a m m o g r a m s are s h o w n in F i g u r e 1.

Redox intermediates of flavonoids

a)

903

c)

/

I

xo

(1) 0t4

/~J / /

o

/

J

/ t

!

I

,,"

I

I

/

/

/

I

/

|

/

l

Volts I

'

I

'

I

'

I

3

2

1

0

-1

-2

-3

/

OH

0

Volts

'

I

~

I

~

2

1

0

-1

I

-2

-3

d)

b)

HO

CH = CH -- C-- O-- CH2-- C~-- C'~CH~

0.

/I

(8)

Ii

~

,

I/I

,

!

/ / /

I I

x \

\/

I I

-

-

-'-"---o~'/

,,~co---~ ?-c~ =c~-c-o-%- %-c ~CH,

~/~a,

(9)

s

Volts

Volts

'

I

'

I

i

I

I

'

I

i

2

1

0

-1

-2

-3

3

2

1

0

I

'

-1

-2

l

-3

Fig. 1. Cyclic voltammogramsof (a) 1 mM chrysin,(b) pinobanksin-3-acetate,(c) caffeicacid ester, and (d) diacetylcaffeicacid ester in 0.1 M tert-butylammoniumperchlorate ACN solutions.Anodicoxidation--full line; cathodicoxidation--dashedline.

Anodioxi c dation One totally irreversible anodic peak (full lines in Fig. 1 a-d) in the region from 1V to 3V versus SCE was observed by oxidation of all investigated compounds. The investigated substrates can he divided into two groups based on the values of their oxidation potentials. The compounds in the first group (1, 2, 3, 4, and 8) show an anodic peak in the region from 1.2 to 1.9 V, as shown in Figure 1 a,b for chrysin and caffeic acid ester, respectively. The compounds in the second group (5, 6, 7, and 9), with characteristic cyclic voltammograms shown in

Figure 1 c,d, have higher oxidation potentials (2-2.3 V). The oxidation potentials of substances 1-9 correlate well with their lipid antioxidant activity found previouslyEo: the lipid antioxidant activities of the compounds with the lower oxidation potentials (1, 2, 3, 4, and 8) were about 30% higher compared to those with higher oxidation potentials (5, 6, 7, and 9). Thus, the highest antioxidant activity was found for caffeic acid ester (8) possessing the lowest oxidation peak potential Ep~ = 1.29 V (Fig. 1 b). In contrast, diacetylcaffeic acid ester (9) with the highest anodic peak potential Ep~ = 2.3 V (Fig. 1 d) exhibited the lowest antioxidant activity.E°

904

P. RAPTA et al.

O I Bu-N-COR o

a)

b)

~ 1 COR H O.

t

experiment

N simulation

aNtNo)l.42

a,, 2.14

)0.75

in mT

Fig. 2. Experimental and simulated EPR spectra of (a) DMPO and (b) MNP spin adducts observed in the anodic oxidation of galangin. The splitting constants used in the simulations are listed under the simulated spectra.

From the investigations of the heights of the oxidation peaks in cyclic voltammograms and their logarithmic analysis (Cyclic Voltammetry Software Condecon TM 710), a two-electron oxidation step is evident for all substrates. In all cyclic voltammograms one or two consecutive products were observed in the reverse scans (full lines in Fig. 1 a-d). One of them can be again oxidized in a reversible step, with the exception of caffeic acid ester. These reduction peaks originate from the newly formed oxidation products. To confirm this, the potential scan was started at -0.5 V and was increased to

the beginning of the oxidation peak. Then, in a scan reversal, no reduction peaks were observed in this potential region. This demonstrates that the consecutive products originate from oxidation steps at higher potentials. The reduction of these products at very low potentials (0-0.2 V vs. SCE) indicates the ease of their autooxidation. Autooxidation most likely explains why isalpinin, which has the lowest oxidation potential among the investigated flavonoids, does not have the highest lipid antioxidant activity. 2° It should be noted at this point that some deviations in the relationship between electrochemical oxidation potentials and lipid

9O5

Redox intermediates of flavonoids 2

0-. H

R2

0

0 2 -2e

C

y-.1 O...H" 0

A

D

O\ H. .0+.

B

solvent

0..H 0 C'

l) ~

Scheme 1.

antioxidant activity are to be expected due to the differences in the reaction mechanisms of the electrochemical oxidation and of the lipid antioxidant processes. Whereas the electrochemical oxidation potentials of secondary oxidation products can clearly be separated from those of the parent compound, when measuring lipid antioxidant effect, an overall activity of the parent compound and of its secondary oxidation products is measured. When the electrochemical anodic oxidation of the test compounds was carried out in the cavity of the EPR spectrometer no free radical products could be detected by the direct EPR. Therefore, spin traps DMPO and MNP were used. The EPR spectra shown in Figure 2 a were detected when electrochemical oxidations of 1, 2, and 3 were performed in the presence of DMPO in ACN solutions. The splitting constants aN = 1.42 mT, a . = 2.14 mT were confirmed by simulation and are characteristic of the carbon-centered adducts. 21 Further insight into the nature of these carboncentered radicals was obtained when the experiments were performed in the presence of the nitroso spin trap MNP; a single triplet simulated using aN = 0.75 mT (Fig. 2 b) was obtained, which is characteristic of MNP/.C(O)R adducts. 22 The -C(O)R radical is certainly a product of a multistep reaction. The experimental results are in agreement with the reaction mechanism shown in Scheme I. The oxidation of A (Scheme I) is a two-electron step according to the cyclic voltammetry. The abstraction of two electrons (possibly in two rapidly coupled oneelectron steps) is probably taking place at the pyranone ring because its structural properties (substituent R I and a double bond in 2,3 position) are decisive for the oxidation potentials of these compounds. Formally,

intermediates B can be formulated after two electron abstraction. The ionized pyranone ring then undergoes fragmentation, resulting in the formation of .C(O)R radical, which was identified by spin trapping experiments. The most probable fragmentation route leading to the observed .C(O)R adducts is the splitting of bonds adjacent to the carbonyl group of the pyranone ring accompanied by the formation of radicals C' and D' (Scheme I). Based on the mass spectrometric data the formation of species, C' is the preferred pathway because a corresponding fragment was found in mass spectrum of A . 23'24 Its formation can be understood as a retro Diels-Alder cleavage. Radical products oC(O)R were observed only with substances 1-4, which have double bond in 2,3-position, which favors the retro Diels-Alder cleavage. The presence of intermediates C or C' (Scheme I) is evident from cyclic voltammograms, where easily oxidizable consecutive products are found in the potential region chracteristic of semichinoidal structures. 25'16

Cathodic reduction The cyclic voltammograms observed in reduction of flavonoids 1, 3 and caffeic acid esters 8, 9 are shown in Figure 1 a - d (dashed lines). The investigated compounds exhibited double or triple peak voltammograms. Their number is probably related to the degree of separation of two or three individual one electron steps. 27 One of these steps involves formation of anion radicals which was demonstrated for chrysin using a direct EPR (Fig. 3, experimental and simulated spectra are shown). Less stable radicals were obtained by reduction of galangin (very poorly resolved EPR spec-

906

P. RAPTA e t

al. In

experiment

I

!

simulation

!

hydrogen splittings in mT 0.019; 0.047; 0.102; 0.102; 0.107; 0.355; 0.369; 0.522 Fig. 3. Experimental and simulated EPR spectra of anion radical generated by cathodic reduction of chrysin. The hydrogen splitting constants used in the simulation are listed under the simulated spectrum.

trum, not shown). Other flavonoids and caffeic acid esters gave no detectable EPR signals. The assigment of the splitting constants obtained from the simulation of the chrysin anion radical (Fig. 3) was based on INDO calculations of the unpaired

spin density distribution. Figure 4 shows the calculated hyperfine coupling constants (c'aCaH)and assignment of the experimental values (exPaH) to the individual hydrogen nuclei in the anion radical of chrysin. The three highest experimentally found hyperfine splittings:

Redox intermediates of flavonoids

907

(0.102) -0.247 1 m

(0.019)

IlO

l

,

tiP

( experimental ) calculated

0.006

-0.024

(0.o4,

0

0

\

(0a -

tt-5 Fig. 4. Structure of the chrysin anion radical. The INDO-calculated splitting constants are given (all values are in mT); the assignment of the experimentally measured splitting constants (Fig. 3) is also shown (data in brackets). Note that the calculated values also show the sign of the splitting constants, which can be predicted from the spin polarization model. The sign of the splitting constant is not needed to predict the EPR splitting pattern to first order. 33

0.335, 0.369, and 0.522 mT, were attributed to the two ortho and one para-hydrogens in the ring B, based on the calculated values for these positions: C~caH°= 0.435 mT, ~Can° = 0.442 mT, and calcaHP = 0.497 mT. The splitting of 0.107 mT was assigned to the hydrogen in the position 3 (C~caH3 = 0.153 mT), and the two equivalent splittings of 0.102 mT to the two meta-hydrogens on ring B. The calculated values overestimate the size of the meta-hydrogen splittings (C~Canm'm = 0.247, 0.250 mT), which is frequently a problem of semiempirical methods. Common EPR experience shows that the meta-hydrogen splittings are usually about one third (1/5 in some cases) of the ortho-hydrogens, which is in agreement with our assignment of the experimental s p l i t t i n g s (an °'° = 0.335, 0.369 mT, and aHm'm ----0.102, 0.102 mT). Finally, the smallest experimentally detected hydrogen splittings, 0.047 mT and 0.019 mT, were assigned to the meta-ketyl protons on the C ring in the positions 6 and 8, respectively, based on the calculated values: calcaH6 = 0.024 mT, ~acaHs = 0.015 mT, calcan( o _ 8)5 = 0.015 mT, ~ a , ( o - n)7 = 0.0057 mT, and also on the fact that the spin densities on hydroxyl hydrogens are typically very low and do not resolve in EPR spectra. Splitting constants of a similar magnitude as the experimental splittings assigned to the C-ring hydrogens were also found for meta-hydrogens in phenyl ketyl radicals. 32 To confirm the assignment of the experimentally found splitting constants an attempt was made to deuterate 5 and 7 hydroxyl groups of chrysin by adding a small amount of D20 to acetonitrile solution. Unfortunately, no EPR-observable radicals could be detected when D20 was present, probably due to the protolysis

of the anion radical. Because of the limited availabilty of the purified plant material, no further attempts to deuterate hydroxyl groups of chrysin could be made. An interesting feature of the investigated flavonoids is that in the oxidation as well as in the reduction processes consecutive products are formed with moderate redox potentials (around 0 V) that may contribute to the antioxidant activities of the test compounds. The activation of the hydroxylated phenyl ring is probably responsible for this phenomenon and becomes active only after redox processes and consecutive reactions. This is in line with the redox behavior of caffeic acid ester (8), which already possesses the ortho-hydroxylated phenyl ring in its basic structure, has the lowest oxidation potential (1.29 V), and shows the maximal antioxidant activity. 2° By acetylation of the hydroxyl groups of the caffeic acid ester (9), the oxidation potential considerably increases (to 2.3 V), and antioxidant activity dramatically decreases. 2° Using cyclic voltammetry and EPR spectroscopy, this study demonstrates a negative correlation between lipid antioxidant properties of flavonoids and caffeic acid esters isolated from propolis and their oxidation potential. This reflects probably the ease of these compounds to react with the oxidizing chain-carrying radicals. Oxidation of 1, 2, and 3 gave secondary carbonyl carbon-centered radicals, which were demonstrated by spin trapping. In the reduction step, EPR-observable anion radicals were demonstrated in case of chrysin and galangin. - - This work was supported by the Slovak Grant Agency grants GA-SAV-280/91 and GAV-914.

Acknowledgements

908

P. RAerA et al. REFERENCES 16.

1. Marcollet, M.; Bastide, P.; Tronche, P. Angioprotective effects of anthocyanisides of Vaccinum myrtillus shown by the release of lactate dehydrogenase (LDH) and their cardiac isoenzymes in rats subjected to swimming test. C. R. Soc. BioL 163:17861789; 1969. 2. Brasseur, T. Anti-inflammatory properties of flavonoids, J. Pharm. Belg. 44:235-241; 1989. 3. Tubaro, A.; Del Negro, P.; Bianchi, P.; Romussi, G.; Della Loggia, R. Topical anti-inflammatory activity of a new acylated flavonoid. Agents. Act. 26:229-230; 1989. 4. Selway, J. W. T. Antiviral activity of flavones and flavans. In: Cody, V.; Middleton, E., Jr.; Harborne, J. B., eds. Plantflavonoids in biology and medicine. New York: Alan Liss; 1986:521 536. 5. Smith, D. A.; Banks, W. Biosynthesis, elicitation and biological activity of isoflavonoid phytnalexyns. Phytochem. 25:979-995; 1986. 6. Yamashida, Y.; Kawada, S.; Nakano, H. Induction of mammalian topoisomerase II dependent DNA cleavage by nonintercalative flavonoids, genistein and orobol. Biochem. Pharmacol. 39:737-744; 1990. 7. Bors, W.; Heller, W.; Michel, C.; Saran, M. Flavonoids as antioxidants: Determination of radicals scavenging efficiencies. In: Packer, L.; Glazer, A. N., eds. Methods in enzymology. Vol. 186. London: Academic Press; 1990:343-355. 8. Suchy, V.; Tekelovfi, D.; Gran~ai, D.; Nagy, M.; DolejL L. Isolation of flavonoids from ethanolic extract of propolis. J. Ceskoslov. Farm. 34:405-408; 1985. 9. Bors, W.; Saran, M. Radical scavenging by flavonoid antioxidants. Free Radical Res. Comms. 2:289-294; 1987. 10. Darmon, N.; Fernandes, Y.; Cambon-Gros, C.; Mutjavila, S. Quantification of the scavenger capacity of different flavonoids with regard to the superoxide ion. Food Addit. Contain. 7:6063; 1990. 11. Canada, A. T.; Giannella, E.; Nguien, T. D.; Mason, R. P. The production of reactive oxygen species by dietary flavonols. Free Radic. Biol. Med. 9:441-449; 1990. 12. Hodnick, W. F.; Milosavljevic, E. B.; Nelson, J. H.; Pardini, R. S. Electrochemistry of flavonoids: Relationship between redox potentials, inhibition of mitochondrial respiration, and production of oxygen radicals by flavonoids. Biochem. Pharmacol. 37:2607-2611; 1988. 13. Hodnick, W. F.; Kung, F. S.; Ruettger, W. J.; Bohmont, C. W.; Pardini, R. S. Inhibition of mitochondrial respiration and production of toxic oxygen radicals by flavonoids. Biochem. Pharmacol. 35:2345-2357; 1986. 14. Guarini, L.; Su, Z. Z.; Zucker, S.; Lin, J.; Grunberger, D.; Fisher, P. B. Growth inhibition and modulation of antigenic phenotype in human melanoma and glioblastoma multiforme cells by caffeic acid methyl ester (CAPE). Cell MoL Biol. 38:513-527; 1992. 15. Dobrowolski, J. W.; Vohora, S. B.; Sharma, K.; Shah, S. A.; Naqvi, S. A.; Dandiya, P. C. Antibacterial, antifungal, antiamoe-

17. 18.

19.

20. 21. 22.

23. 24. 25.

26. 27.

28.

29. 30. 31. 32. 33.

bic, antiinflammatory, and antipyretic studies on propolis bee product. J. Ethnopharmacol. 35:77-82; 1991. Grange, J. M.; Davey, R. W. Antibacterial properties of propolis [bee glue]. J. Royal Soc. Med. 83:159-160; 1990. Serkedjieva, J.; Manolova, N.; Bankova, V. Anti-influenza virus effect of some propolis constituents and their analogues (esters of substituted cinnamic acid). Z Nat. Prod. 55:294-302; 1992. Dimov, V.; lvanovska, N.; Bankova, V.; Popov, S. Immunomodulatory action of propolis: IV. Prophylactic activity against gram-negative infections and adjuvant effect of the water-soluble derivative. Vaccine 10:817-823; 1992. Krol, W.; Czuba, Z.; Scheller, S.; Gabrys, J.; Grabiec, S.; Shani, J. Anti-oxidant property of ethanolic extract of propolis [EEP] as evaluated by inhibiting the chemoluminiscence of luminol. Biochem. Int. 21:593-597; 1990. M i ~ , V.; OndriaL K.; Gergel, D.; Bullov~, D.; Suchy, V.; Nagy, M. Lipid peroxidation of lecithin liposomes depressed by some contituents of propolis. Fytoterapia 62:215- 220; 1991. Buettner, G. R. Spin trapping: ESR parameters of spin adducts. Free Radic. Biol. Med. 3:259-303; 1987. Janzen, E. G. The application of ESR spin trapping in the detection of gas phase free radicals produced from photolysis of gas phase organic molecules. Creat. Detect. Excit. State 4:83-138; 1976. Takayama, M.; Fukai, T.; Hano, Y.; Nomura, T. Mass spectrometry of prenylated flavonoids. Heterocycles 33:405-434; 1992. Kingston, D. G. I. Mass spectrometry of organic compounds: VI. Electron-impact spectra of flavonoid compounds. Tetrahedron 27:2691-2700; 1971. Ryba, O.; Peminek, J.; Pospi~il, J. Antioxidant agents and stabilizers: IV. Influence of structure on polarographic half-wave potentials of alkylated hydroquinones. Coll. Czechoslov. Chem. Comm. 30:843-850; 1965. Kissinger, P. T.; Heineman, W. R. Cyclic voltammetry. J. Chem. Educ. 60:702-706; 1983. Geissmann, T. A.; Friess, S. L. Flavanones and related compounds: VI. The polarographic reduction of some substituted chalcones, flavones and flavanones. J. Am. Chem. Soc. 71:38933902; 1949. Hodnick, W. F.; Kalyanaraman, B.; Pristos, C. A.; Pardini, R. S. The production of hydroxyl and semiquinone free radicals during the autoxidation of flavonoids. In: Simic, M. G.; Taylor, K. A.; Ward, J. F.; von Sonntag, C., eds. Oxygen radicals in biology and medicine. New York: Plenum Press; 1988:149152. Rinaldi, D. Quantum chemistry program exchange (QCPE) #217. Dewar, M. J. S.; Zoebish, E. G.; Healy, E. F.; Stewart, J. J. P. A general purpose quantum mechanical molecular model. J. Am. Chem. Soc. 107:3902-3913; 1985. Stewart, J. J. P. Quantum chemistry program exchange (QCPE) #217. Sta~ko, A. Intermediate radical products formed in the catalytic systems of nickel with organometallic compounds. Chemicke Zvesti 37:95-137; 1983. Leffler, J. E. An introduction to free radicals. New York: John Wiley & Sons; 1993.