312

BBA 51809

INHIBITION OF THE BLEACHING OF THE CAROTENOID CROCIN

A RAPID TEST FOR QUANTIFYING ANTIOXIDANT ACTIVITY

WOLF BORS, CHRISTA MICHEL and MANFRED SARAN

Abteilungftir Strahlenbiologie, GSF Forschungszenmm, 8042 Neuherberg (F.R.G.)

(Received July 12th, 1984)

Key words: Aikoxyl radical; Antioxidant; Carotenoid bleaching; Fatty acid hydroperoxide; Photolysis

The carotenoids crocin and canthaxanthin were bleached by photolytically produced alkoxyl radicals
from either tert-butyl hydroperoxide or fatty acid hydroperoxides. The inhibition of tbis bleaching by a range
of substances was quantified by competition kinetics to obtain relative rate constants for their reaction with
the alkoxyl radicals. We obtained these data for several classes of substances, such as phenolic antioxidants
and model phenols, polyhydric and heterocyclic radical scavengers and miscellaneous other substances. The
close correlation of relative rate constants in the described system with the known antioxidative capacities of
selected compounds recommends this assay system as a simple, rapid and quantitative test for the screening
of antioxidants. The relative rate constants did not correlate with the known rate constauts for reaction of
hydroxyl radicals with the substances mentioned above.

Introduction and their derivatives [2,3] and reaction -with the

stable radical 2,2-diphenyl-Z-picrylhydrazy!
The antioxidative capacity of both natural and (DPPH; Ref. 4) are more rapid assays. Quantita-
synthetic substances is an important aspect in food tive data, especially kinetic parameters, have been
chemistry that has led to the development of obtained by the latter method (51, by the “induc-
numerous assay procedures [l]. The determination tion time’ method [6], by the inhibition of the
of active oxygen, of peroxide or total carbonyl di-tert-butylperoxalate-induced chemiluminescence
value, of the amount of thiobarbituric acid-reac- [7], and by the HPLC-determination of the ratio of
tive material, of the bleaching of p-carotene and ks, tram vs. tram, tram diene hydroperoxides of
the Schaal oven test are all frequently used assays. linoleic acid [Sj.
These methods have the disadvantages that they It is generally assumed that antioxidants func-
allow only a qualitative comparison of antioxi- tion either by scavenging various types of radicals
dants and are very ‘time consuming. Hemin- or formed in autoxidation processes or by preventing
hemoprotein-induced peroxidation of fatty acids their formation through direct reduction of the
hydroperoxide precursors [%-II]. The radicals
which are formed during lipid autoxidation are
Abbreviations: DPPH, 2,2-diphenyi-l-picrylhydrazyl; BHA, alkyl (R’), peroxyl (ROO’) and alkoxyl (RO’)
Z(3)-tert-butyl-4-hydroxyanisole; BHT, 3,5-di-rert-butyl-4-hy-
radicals [ 12,131. Furthermore, singlet oxygen ( 10,;
droxytoluene; t-BuOOH, rut-butyl hydroperoxide; 13-LOOH,
13-hydroperoxylinoleic acid; JK-409, (4’-methoxybenzy!)-
Ref. 13) and the inorganic oxygen radicals hy-
2,4,5-trihydroxyphenylketone; DPPD, N, N’-diphenyl-p-phen- droxyl ( ‘OH) and superoxide anion (0;) {10,14!
ylenedizmine. have also been propcsed to participate in Iipid

0005-2760/84/$03.00 0 1984 Elsevier Science Publishers B.V

 313

peroxidation. The finding that antioxidants al- drocinnamic acid, 3,4-dihydroxytoluene, diphenyl-
leviate the toxicity of ozone implies that free radi- amine, ferulic acid (4-hydroxy-3-methoxycirmamic
cals are involved in this process too [15]. acid), 4-hydroxyacetophenone, 2-hydroxy-4-
Knowledge of the reactivity of antioxidants with methoxybenzaldehyde, nordihydroguaiaretic acid,
the radicals mentioned would not only result in propylgallate, sesamol (3,4-methylenedioxo-
quantitative data on the capability of a specific phenol), shikimic acid; Boehringer: 2,2’-
antioxidant, but would also pinpoint the contribu- azinobis(3-ethylbenzthiazoline-6-sulfonate)
tion of the individual radical species in the process (ABTS); Fluka: acetovanillon (4-hydroxy-3-
of lipid peroxidation. In the case of the inorganic methoxy-a~etophenone), 2-Bert-buty~ydroquinone,
oxygen radicals, only isolated and recent data exist chlorogenic acid, 3,4-dihy~oxybenzaldehyde, 2,3-,
on their reactivity with phenolic antioxidants 2,5- and 3,4_dihydroxybenzoic acid, 2,6- and 3,5-
116,171. However, we suggested earlier [18] that dimethoxyphenol, N, N’-diphenyl-p-phenylen-
organic oxygen radicals, in particular alkoxyl radi- ediamine, 3,5-di-tert-butyl-4-hydroxybenzoic acid,
cals, may participate in metabolic or pathological 2,4-di-fern-butylphenol, syringic acid (3,5-di-
processes and can, in many instances, simulate the methoxy-4-hydroxybenzoic acid), ellagic acid,
role which is usually attributed to the ‘free ‘OH guaiacol (2-methoxyphenol), histidine, hydro-
species’. It is known that alkoxyl radicals are far quinone, isoeugenol (2-methoxy-4-pro-
more reactive than peroxyl radicals in H-abstrac- penylphenol), 3- and 4-methox~henol, phenol,
tion reactions [19]. sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic
From our studies of the carotenoid crocin acid), toluhydroquinone, 2’,4’,6’-trihydroxy-
[20,21], which is efficiently bleached by radiolyti- acetophenone, 2’,4’,5’-trihydroxybutyrophenone,
tally produced RO radicals [20,22], we developed tryptophan, uric acid, vanillic acid (4-hydroxy-3-
a simple, rapid and reproducible method to obtain me~oxybenzoic acid); Roth: Phloretin; Serva:
the relative reaction rates of various antioxidants ascorbic acid, salicylaldehyde (2-hydroxybenz-
with RO’ in aqueous solution [22,23]. We de- aldehyde), salicylic acid (Zhydroxybenzoic acid),
liberately selected a water-soluble carotenoid, since 4-hydroxybenzoic acid ethyl and propyl ester,
comparisons with rate constants of the corre- vanillin (4-hydroxy-3-methoxybenz~dehyde), in-
sponding species ‘OH can only be obtained in osin, isoascorbic acid (erythorbic acid); Sigma:
aqueous solution [24]. It may be assumed that aspirin (acetylsalicylic acid), 2(3)-tert-butyl-4-hy-
abstraction of H.atoms from phenolic antioxidants droxyanisole (BHA), 3,5-di-tert-butyl-4-hydroxy-
occur with RO’, ROO’ as well as with the stable toluene (BHT), chlorpromazine, indomethacin,
DPPH radical [S]. Consequently, the initial chem- prometh~ne, sorbic acid, thiourea. Trolox c [26]
istry is the same for the DPPH method and for the was a gift from Hoffmann-LaRoche and (4’-
method described here. This H-abstraction mecha- methoxybenzyl)-2,4,5_trihydroxyphenylketone
nism also applies to the kinetic analysis of Porter (JK-409) was donated by J.F. Kurth (University of
and colleagues [S], and the ‘induction-period Bonn). 3’,4’-Dihydroxyacetophenone was
method used by Ingold and co-workers [6,11]. synthesized according to the method of Stephen
and Weizmann [27]. The substances were generally
Materials used without further purification. Scheme I depicts
the structures of those substances with more com-
Crocin was isolated from saffron 1251, which plicated systematic names.
was obtained as a powder from the local market. The hydroperoxides used as precursors of alk-
Canthaxanthin was a gift from Hoffmann- oxyl radicals were tert-butyl hydroperoxide (t-
LaRoche. Antioxidants and phenolic model com- BuOOH; as an 80% (v/v) solution in t-BuOH,
pounds were obtained at the highest purity availa- Peroxid Chemie, or as 70% (v/v) aqueous solution,
ble from the following companies: Aldrich: 2’,5’- Fluka) and 13-hydroperoxylinoleic acid (13-
and 2’,4’-dihydroxyacetophenone, caffeic acid LOOH, a gift from W. Grosch, Deutsche For-
(3,4-dihydroxycinnamic acid), 3,4-dihydroxydihy- schungsanstalt fur Lebensmittelchemie, Garching).
 CH,

(151 Trolox c 1371 chlorogenjc acid (381 NDGA (521 shikimic acid

OH 0

OH-OH
OH OH
C4-i

iL5) propylgallate (L61 :KLO9 IL71 phloretin (LB! e!icg;c ccid

004
CH,O
d
c--J‘G CH,

,k
G?Q
CH, b-l CH, di
162) ABTS (63) promethamlne (611 chlorpromazine (651 indomethocw

Scheme I. Structures of complex antioxidants.

Methods and after 5 min of photolysis. The decrease in

absorption after this time (the ‘bleaching rate’) is
The alkoxyl radicals from t-BuOOH or P3- termed ‘Q’. The experiments were then repeated
LOOH were generated by ultraviolet photolysis in the presence of various concentrations of anti-
(using a low-pressure mercury lamp, intensity 1.75 oxidants and the corresponding decrease in ah.-
J/m2) of aqueous solutions containing LO $‘vI sorption was termed ‘P”. To minimize the dilution
crocin and either 1 mM t-BuOOH or 0.2 mM error, care should be taken to add aliquots of
13-LOOH. Stoppered cuvettes (1 cm path-length, highly concentrated stock solutions of the individ-
3 ml volume) were placed next to the lamp, the ual substances, prepared in either water or pure
intensity of which was determined by actinometry t-BuOH. According to the equation for competi-
using a UVX-Radiometer (Ultra-Violet Products). tion reactions [30]:
Since photolysis of hydroperoxides results in RO’
and ‘OH as homolytic scission products [28], a V,/V=l+k,/k;[antioxidant]/[crocin],

sufficient amount of rert-butanol (t-BuOH; 0.1-l

M) was added to scavenge the ‘OH radicals. The a plot of V,/V vs. {antioxidant]/[crocin] gives a
(CL%,),-C(OH).CH, radical which is formed does straight line intersecting the ordinate at unity. The
not interfere in the assay 1211. Omitting t-BuOH slope of the line is the rate constant for the reac-
leads to a higher overall bleaching rate of crocin tion of the photolyticalljr produced radicals with
due to the attack of both RO’ and ‘OH (data not the antioxidant relative to the rate constant with
shown). However, this could not be evaluated for crocin (k, or k, are the respective absolute
competition kinetics owing to the different relative second-order rate constants).
rate constants of the substrates towards RO. and Some experiments were carried out in n-hexane
‘OH. solution containing PO PM canthaxanthin as a
Reaction of the alkoxyl radicals with crocin was reference compound instead of crocin. The bleach-
monitored by measuring its strong absorption at ing of the absorption at 450 nm (E = 1.1 j 10”
440 nm (E = 1.33 . lo5 ha-’ . cm-‘; Ref. 29) before M-’ . cm-‘; Ref. 31) was readily observed under
 315

TABLE I

RELATIVE RATE CONSTANTS WITH ALKOXYL AND HYDROXYL RADICALS FOR PHENOLIC ANTIOXIDANTS,
RADICAL SCAVENGERS AND RELATED SUBSTANCES IN AQUEOUS SOLUTION

ni. no inhibition.

Substance k rel k abs.. OH Ref.

(lo9 M-‘.s-‘)
t-BO’ 13-LO’ .OH

I. Antioxidants and model compounds

(a) Monophenols:
(1) phenol 0.001 0.006 0.424 14 .24
(2) 2-methoxyphenol (guaiacol) 0.004 0.006 0.455 15 33
(3) 3-methoxyphenol 0.002 0.002 0.667 22 33
(4) 4-methoxyphenol 0.010 0.022 0.545 18 33
(5) 3,Sdimethoxyphenol 0.008 0.001 0.455 15 33
(6) 2,6-dimethoxyphenol 0.030 0.030 0.545 18 33
(7) 3,4-methylenedioxophenol (sesamol) 0.025 0.008 a _ _ _
(8) 2-methoxy-4-propenylphenol (isoeugenol) 0.004 0.006 0.833 27.5 17
(9) 2,4-di-tert-butylphenol 0.031 0.032 b _ _
(10) 4-hydroxybenzoic acid ethylester 0.036 0.058 - _ _
(11) 4-hydroxybenzoic acid propylester 0.037 0.063 _ _ _
(12) 3,5-di-tert-butyl-4-hydroxytoluene (BHT) 0.053 0.017 b _
(13) 2(3)-terl-butyl-Chydroxyanisole (BHA) 0.056 0.029 a _
(14) 3,5-di-tert-butyl-4-hydroxybenzoic acid 0.042 0.055 _ _ _
(15) Trolox cd 0.230 0.320 1.045 34.5 17
(16) 2-hydroxybenzaldehyde (salicylaldehyde) 0.029 0.066 0.258 8.6 24
(17) 4-hydroxybenzaldehyde -=Z0.001 _b 0.030 1.0 24
(18) 2-hydroxy-4-methoxybenzaldehyde 0.017 0.006 _ _
(19) 4-hydroxy-3-methoxybenzaldehyde (vanillin) 0.012 0.009 b _ _
(20) 4-hydroxyacetophenone n.i. n.i. _ _ _
(21) 4-hydroxy-3-methoxyacetophenone
(acetovanillon) 0.028 0.030 _
(22) 2-hydroxybenzoic acid (salicylic acid) 0.001 a 0.001 a 0.360 12 24
(23) 4-hydroxy-3-methoxybenzoic acid (vanillic acid) 0.055 _ a.b 0.303 10 33
(24) 3,5-dimethoxy-4-hydroxybenzoic acid
(syringic a&id) 0.063 0.029 0.333 11 33
(25) 4-hydroxy-3-methoxycinnamic acid
(ferulic acid) 0.030 0.044 0.185 6.1 17
(26) 3,5-dimethoxy-4-hydroxycinnamic acid
(sinapinic acid) 0.050 0.056 0.458 15.1 17

(b) Di-, triphenols:

(27) hydroquinone 0.016 0.045 a 0.640 21 24
(28) toluhydroquinone (2,5-DHT) 0.043 0.130 _ _ _
(29) tert-butylhydroquinone 0.091 0.050 _ _ _
(30) 3,4-dihydroxytoluene (3,4-DHT) 0.018 0.055 0.485 16 34
(31) 3,4_dihydroxybenzaldehyde 0.025 0.060 0.145 4.8 35
(32) 3,4-dihydroxybenzoic acid 0.073 0.080 _ _
(33) 2,3_dihydroxybenzoic acid 0.020 0.060 _ - _
(34) 2,5-dihydroxybenzoic acid 0.005 0.012 = -
(35) 3,4dihydroxycinnamic acid (caffeic acid) 0.034 0.072 0.730 24.1 17
(36) 3,4-dihydroxydihydrocinnamic acid 0.036 0.027 a 0.697 23 36
(37) chlorogenic acid d 0.055 0.061 _
(38) nordihydroguaiaretic acid (NDGA) d 0.075 0.330 0.403 13.3 17
(39) 2’,5’-dihydroxyacetophenone 0.062 0.063 0.139 4.6 17
(40) 2’,4’-dihydroxyacetophenone 0.030 0.010 0.539 17.8 17
(41) 3’,4’-dihydroxyacetophenone 0.065 0.085 0.180 5.9 35
(42) 2’,4’,6’-trihydroxyacetophenone 0.009 _= _ _ _
316

TABLE I (continued)

Substance k abs. OH Ref

(109 IvI-:.s- ‘)
I3-LO’ -OH

(43) 2’,4’,5’-trihydroxybutyrophenone 0.80 0.155 _

(44) adrenalone 0.063 i? 0.205 0.303 10 37
(45) propylgallate d 0.067 0.167 0.364 12 24
(46) 4K 409 d 0.077 0.300
(47) phloretin d 0.013 0.008 a _
(48) ellagic acid ’ 0.600 a.b 0.490 _

(c) Others:
(49) acetylsalicyiic acid (Aspirin) -C 0.00: < 0.001 _ _
(50) ascorbic acid 0.72 b 1.60 0.333 11 24
(51) isoascorbic acid 1.25 1.35
(52) shikimic acid d < 0.001 0.002 _
(53) sorbic acid 0.041 0.170 _

II. Radical scavengers

(a) A mmes:
(54) thiourea 0.013 0.02 0.090 3 38
(55) diphenylamine 0.43 0.6 0.394 13 24
(56) N, N’-diphenyl-p-phenylenediamine (DPPD) - ’ 6.5 _
(57) picolinic acid 0.009 a _ _ _ _
(58) histidine _ 0.003 0.152 5 24
(59) tryptophan _ 0.008 0.364 12 24
(60) inosin 0.025 0.040 _ _
(61) uric acid 0.094 0.019 0.218 7.2 24
(b) Ideterocycles:
(62) 2,2’-azinobis(3-ethylbenzthiazoline-6-
sulfonate) (ABTS) d 0.174 1 .Ol 0.364 12 39
(63) promethazine d 0.550 0.64 0.294 9.7 40
(64) chlorpromazine d 0.62 1.45 0.267 8.8 40
(65) indomethacin d 0.059 0.068 _

III. Polyhydroxylated aliphates

(66) glycerol < o.ooi _ 0.058 1.9 24
(67) pentaerythritol < 0.001 _ 0.097 3.2 24
(68) D-sorbitoi < 0.001 _ _
(69) mannitol < 0.001 < 0.001 0.030 1.0 37
(70) inositol < 0.001 < 0.001 0.048 1.6 24
(71) o-( + )-galactose i 0.001 _ _
(72) ,L?-gentobiose < 0.001 < 0.001 _

IV. Compounds with low rate constants with hydroxyl radicals

(73) dimethylsulfone < O.OOi < 0.001 0.0002 0.006 24
(74) sodium oxalate _ _ a 0.0003 0.01 24
(75) urea _ i 0.001 0.00003 0.001 24

a Plotted lines do not intersect ordinate near unity.

b No linear competition plot (value taken from extrapolation to zero concentration).
’ Spectral overlap of DPPD/t-BuO’ adduct with crocin absorption, no competition possible.
d The structures of these substances are shown in Scheme I.

the irradiation conditions described above. spectra of the substances to be tested have to be
Obviously, substances which have strong ab- known beforehand. None of the substances as-
sorption maxima in the region of the carotenoids sayed in this paper absorbed at all in the region of
cannot be analyzed by this method. Therefore, the the visible band of crocin. Ft was a&o found that
 317

thiol compounds interacted with the carotenoid son [17,24,33-401. There is no obvious correlation
and could not be tested. except that those substances with low k.,, also
show low reactivity with RO’. The reference value
Results was that of crocin with ‘OH (3.3 .lO” M-’ . s-‘;
Ref. 21). The list also contains substances which
Generally, reactions involving initiation by have generally been used as radical scavengers,
radicals follow the so-called ‘square-root law’ [32]. such as several amines or heterocyclic compounds
In agreement with this hypothesis, plotting the of widely different structure. Some of the data
bleaching rate of crocin against the square root of have been published earlier [22,23]. For the com-
irradiation time representing the initiation process pounds indicated, the competition plots did not
did give straight lines for t-BuOOH and 13-LOOH. result in straight lines or did not intersect the
Table I lists the relative rate constants obtained ordinate at unity.
for a number of antioxidants, phenolic model The fact that such a competition method is also
compounds and plant phenolic substances with applicable in non-aqueous solvents allowed us to
the alkoxyl radicals t-BuO and 13-LO in aqueous determine the values listed in Table II, using the
solution. inhibition of the bleaching of canthaxanthin in
Where data are available, the rate constants for n-hexane.
reactions with ‘OH radicals are given for compari- This modification of the method is also used
preferentially for lipid-soluble antioxidants. Al-
though the number of substances is small, com-
TABLE II pared to the crocin assay in aqueous solution, the
generally higher reactivity with t-BuO radicals is
RELATIVE RATE CONSTANTS WITH ALKOXYL RADI-
CALS DETERMINED BY INHIBITION OF CANTHA- quite obvious.
XANTHIN BLEACHING IN n-HEXANE
Discussion
Substance k ret

t-BuO’ LO’ The applicability of our method for assaying

(1) phenol 0.320 antioxidative activity does not depend on the
(2) guaiacol 0.200 b knowledge of the mechanism of the bleaching of
(6) 2,6-dimethoxyphenol 0.260 crocin. Bleaching studies implicitly do not lend
(8) isoeugenol 0.029 b
themselves to mechanistic interpretations - except
(9) 2,4-di-t-butylphenol 0.300 c _
that the attack has to occur at the polyene chro-
(10) 4-hydroxybenzoic ethylester 0.052 0.056 d
(11) 4-hydroxybenzoic propylester 0.042 0.116 d mophore by disrupting the conjugated system
(13) BHA 0.315 c _ a [11,41]. As mentioned earlier, the photolysis of
(14) 3,5-di-t-butyl-4-hydroxybenzoic acid 0.350 0.098 hydroperoxides results in RO and ‘OH as homo-
(15) Trolox c 0.260 _
lytic scission products [28] and hydroxyl radicals
(16) salicylaldehyde 0.070 0.053
are scavenged by the alcohol solvents used for the
(18) 2-hydroxy-4-methoxybenzaldehyde 0.012 0.019
(19) vanillin 0.063 0.016 hydroperoxides. In the presence of unreactive sub-
(21) acetovanillon 0.098 0.001 strates and particularly in polar solvents [41], alk-
(23) vanillic acid 0.106 e oxyl radicals decay by intramolecular processes.
(24) syringic acid 0.315 _
Tertiary alkoxyl radicals form alkyl radicals and
(25) feruhc acid 0.305 0.013 c
(26) sinapinic acid 0.385
carbonyl compounds [42,43], whereas the
0.041
(42) 2’,4’,6’-trihydroxyacetophenone 0.009 0.013 cis,truns-octadeca-(9,11)-dienoic acid 13-0~~1 radi-
(43) 2’,4’,5’-trihydroxybutyrophenone 0.080 0.155 cal has been reported to rearrange to an epoxy
ally1 radical [44]. Methyl radicals (‘CH,), which
a Scavenger enhances bleaching of carotenoid.
are degradation products of t-BuO., have been
b Linear only at low concentrations of scavenger.
’ No linear competition plot. shown to be unreactive towards crocin [45].
d Data obtained with 9-LOOH as hydroperoxide precursor. The additional competition studies in n-hexane
e Plotted lines do not intersect ordinate near unity. were carried out because P-fragmentation or re-
318

arrangement of the alkoxyl radicals is less irn- tion rate constants of -OH, t-BuO or I?J-LCJ =C:h
portant in organic solvents [41]. The results in phenolic substances, we attempted to co:.relate the
Tabie PI mainly reflect the change in solvent polar- reactivity of the antioxidants with their structures
ity QIJ the stability of the akoxyl radicais: tertiary [53]. Until the identity of the radicals is estab-
alkoxyl radicals which are most prone to fi-frag- lished, however, mechanistic implications r”or the
mentation in polar soIvent [4S] are more stable function of antioxidants based on these structure-
and can be scavenged more effectively by the activity correlations may be premature. ‘We as-
phenolie compounds. This explains the increase in SUlTE that under our experimenta% conditions
k PB”O./kLO ratio for most of the substances. mainly alkoxyli radicals are present which are ca.-
One aspect which might be of particular impor- pable of both abstraction of W atoms from pheno-
tance is the hydro- or Bipophilicity of the sub- lit. antioxidants or other radica! scavengers and
stances under invest~ga~on. However, using the addition to olefinic structures such as the pofyene
method of Hansch and Leo [47j. No correiation of carotenoid crock. 1%w~nld also be interesting to
77 values with relative reaction rate constants was determine the stoichiometry of the RO reackm
found (V denotes the distribution coefficient of a with antioxidants which for the reaction of ROD
given substance in octanol/water mixtures). As with phenol&z a~t~oxidants, is approx. two j6].
Barclay et al. [48] have stressed recently, water- 133 conclusion, using a simple anti rapid assay
soluble antioxidants may be biologically important we have demonstrated in aqueous and in organic
both as synergists and as supplements to the solvents a correhtion of antioxidant potential and
lipid-soluble antioxidants by preferentially acting reactivity with radicals derived from photolysis of
at the Iipid/water interface. hyd~opero~des. Furthermore, for all the sub-
The data obtained by this method correspond stances tested, the assay yields definite kinetic
quite well with the antioxidative activity of the parameters which may be useful in aseertainiag
compounds tested. FQF exampIe, the highest rela- the mechanism of antioxidant action.
tive rate constants for reaction wiih 13-&O. were
obtained with: DPPD > ascorbic and isoascorbic Ac~~wle~geme~~
acid > diphenylamine > nordihydroguaiaretic acid
> Trolox c > JK 4G9 > propylgallate > 2’,3’,4’- We thank D. Tait and M. Erben-Russ for their
trihydroxybutyrophenone > toluhydroquinone. AU help in preparing the manuscript.
of these substances are known to be effective
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