Comparative evaluation of the antioxidant activity of some ortho-substituted mono- and dialkylphenols with the para-positioned hydroxymethyl group

Article abstract of DOI:10.1007/s11172-020-2937-x

The para-hydroxymethyl derivatives of 2,6-di-tert-butylphenol, 2,6-diisobornylphenol, 2-isobornyl-6-tert-butylphenol, 2-isobornyl-6-methylphenol, and 2-isobornylphenol were synthesized. A comparative evaluation of the antioxidant properties of the synthesized compounds was carried out in in vitro models. The activity of derivatives is predetermined by the number and bulkiness of the substituents at the ortho positions relative to the phenolic OH group and is consistent with the relationships previously identified by us for other phenolic antioxidants.

Full text of DOI:10.1007/s11172-020-2937-x

Russian Chemical Bulletin, International Edition, Vol. 69, No. 8, pp. 1573—1578, August, 2020
1573
Comparative evaluation of the antioxidant activity of some ortho-substituted
mono- and dialkylphenols with the para-positioned hydroxymethyl group*
E. V. Buravlev,a I. V. Fedorova,a O. G. Shevchenko,b and A. V. Kutchina
aInstitute of Chemistry, Komi Science Center, Ural Branch of the Russian Academy of Sciences,
48 ul. Pervomayskaya, 167000 Syktyvkar, Russian Federation.
Fax: +7 (821 2) 21 9916. E-mail: eugeneburavlev@gmail.com
bInstitute of Biology, Komi Science Center, Ural Branch of the Russian Academy of Sciences,
28 ul. Kommunisticheskaya, 167982 Syktyvkar, Russian Federation.
Fax: +7 (821 2) 24 0163
The para-hydroxymethyl derivatives of 2,6-di-tert-butylphenol, 2,6-diisobornylphenol,
2-isobornyl-6-tert-butylphenol, 2-isobornyl-6-methylphenol, and 2-isobornylphenol were
synthesized. A comparative evaluation of the antioxidant properties of the synthesized com-
pounds was carried out in in vitro models. The activity of derivatives is predetermined by the
number and bulkiness of the substituents at the ortho positions relative to the phenolic
OH group and is consistent with the relationships previously identified by us for other phenolic
antioxidants.
Keywords: phenols, alkylphenols, terpenephenols, hydroxymethyl derivatives, antioxidant
activity, membrane-protective activities, red blood cells, oxidative hemolysis.
It is known that the most common antioxidants are the
phenolic derivatives with the sterically hindered OH group
(sterically hindered phenols).1 Similarly to alkylphenols,
terpenephenols2 and their derivatives, viz. phenolic com-
pounds bearing the terpene substituents,3 are capable of
inhibiting oxidative processes.4—8 For example, it was
demonstrated that some para-alkoxymethyl terpene-
phenol derivatives have in vitro antioxidant activity,5
p-hydroxymethylphenol derivative bearing two isobor-
nyl substituents is efficient in vivo against benign pros-
tatic hyperplasia in male rats,9 and its parent com-
pound, 2,6-diisobornyl-4-methylphenol, is very active
in the model of experimental pathospermia in rats in-
creasing the antioxidant potential of spermatozoa and
reducing the percentage of degenerative forms of sper-
matozoa.10
Nevertheless, it is still challenging to study the ef-
fects of the alkyl and/or terpene substituents positioned
ortho to the phenolic OH group on biological activity of
the para-hydroxymethyl derivatives. Such research re-
quires increasing the range of the compounds by including
new analogs based on available alkyl- and terpenephenols
and an in vitro comparative evaluation of the new com-
pounds as inhibitors of the oxidative processes, which was
the aim of this work.
Results and Discussion
The known aldehydes 1—5 were subjected to the so-
dium borohydride reduction to give hydroxymethyl de-
rivatives 6—10 (Scheme 1). The 1H and 13C NMR spectral
data, IR spectral data, and elemental analysis data of
products 6, 8, and 10 confirm expectations on their struc-
ture. The spectral properties of compounds 7 and 9 are in
accord with those described earlier.11,12 The H and 13C
1
NMR spectra of derivatives 6—10 show signals of the
hydroxymethyl substituent at the para position to the
phenolic group along with the signals of 2-mono- or
2,6-disubstituted phenolic unit.
The radical scavenging activity (RSA) of compounds
6—10 was evaluated using DPPH assay, antioxidant activ-
ity (AOA) was examined using the substrates containing
natural lipids (mouse brain and testicular homogenates),
cytotoxicity, AOA, and membrane protective properties
were evaluated using mammalian red blood cells. A phe-
nolic antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT)
was used as a reference. Most of the above-mentioned test
systems were used previously for comparative evaluations
of AOA of different types of phenolic compounds.5—7,13—15
To obtain the wider data coverage on AOA of the synthe-
sized compounds we used not only the mouse brain homo-
genate but also the mouse testicular homogenate as
a substrate. The use of the latter is of the interest since the
spermatozoa and hormone-producing Leydig cells contain
high level of polyunsaturated fatty acid and, therefore, are
* Dedicated to Academician of the Russian Academy of Sciences
A. M. Muzafarov on the occasion of his 70th birthday.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 8, pp. 1573—1578, August, 2020.
1066-5285/20/6908-1573 © 2020 Springer Science+Business Media LLC

Buravlev et al.
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Russ. Chem. Bull., Int. Ed., Vol. 69, No. 8, August, 2020
Scheme 1*
active, while, para-hydroxymethyl derivative 7 bearing
two isobornyl units was the most active (Table 1). Note
that the activity of compound 7 assessed in the mouse brain
homogenate assay was much higher than that of BHT.
All synthesized compounds at a concentration of
10 mol L–1 exhibit high AOA in brain and testicular
homogenates of the laboratory animals and noticeably
decrease the content of the secondary products of the LPO
reacting with 2-thiobarbituric acid (TBA-reactive sub-
stances, TBA-RS) in the test samples relative to the con-
trol samples (see Table 1). In brain homogenates, we record
not only inhibition of the ascorbate-Fe2+-induced LPO
but also a statistically significant decrease in the concen-
tration of TBA-RS as compared with the intact samples,
in which no LPO was initiated. Despite that proliferative
activity in the brain and testis tissues differs significantly
and the LPO in them was initiated in different manner,
compound 10 with one ortho position terpene substituent
was the least active in both cases. When concentration of
the compounds was decreased to 1 mol L–1, low activity
was detected not only for compound 10 but also for
derivative 6 with two tert-butyl groups. Thus, AOA of
the para-hydroxymethyl derivatives in the above test sys-
tems is determined by the number and bulkiness of the
substituents at the ortho position relative to the phenolic
OH group.
R1 = But (1, 6),
(2—5, 7—10);
R2 = But (1, 3, 6, 8),
(2, 7), Me (4, 9), H (5, 10)
* Note. The numbering scheme for the carbon atoms is used for
the interpretation of the NMR spectra and may not corre-
spond to the IUPAC nomenclature. Compounds 2 and 7 are
meso stereoisomers, compounds 3—5 and 8—10 are racemates.
Scheme 1 shows the structures of one of the enantiomers.
very sensitive to reactive oxygen species.16—18 It was ex-
perimentally demonstrated that some antioxidants, for
instance, melatonin and -tocopherol, can suppress the
ascorbate-Fe2+-induced lipid peroxidation (LPO) in micro-
somes and mitochondria of the rat testicles and protect
Hemolytic activity measurements using red blood cells
isolated from mice show that neither of the synthesized
compounds has the pronounced activity at a concentration
of 10 mol L–1. Five hours after incubation with com-
pounds 6—10, the hemolysis level of the mouse red blood
cells does not exceed 6.9 0.2%.
the polyunsaturated fatty acids from oxidation.16,17
.
We found that RSA of the studied compounds is de-
termined by the number and bulkiness of the substituents
at the ortho positions to the phenolic OH group. Thus,
compound 10 with one terpene substituent was the least
The structure—activity relationships for derivatives
6—10 described above were corroborated by a comparative
Table 1. Comparative evaluation of RSA and AOA* of derivatives 6—10
Compound
RSA
TBA-RS/nmol mL–1
(inhibition (%))
mouse testicular
homogenate
mouse brain
homogenate
c = 10 mol L –1
c = 10 mol L–1
c = 1 mol L–1
c = 10 mol L–1
c = 1 mol L–1
Control
—
32.0 0.4
7.3 0.6
8.1 0.2
8.8 0.2
8.5 0.3
7.4 0.2
7.4 0.4
20.1 0.1
42.7 0.2
12.6 0.6
36.2 0.5
40.6 1.5
33.3 0.5
32.0 0.5
32.7 0.3
37.0 0.5
72.7 2.1
30.9 0.8
Intact sample
—
BHT
6
7
8
9
10
5.0 0.4
6.9 0.2
14.4 0.8
6.5 0.5
9.0 0.7
2.5 0.2
7.6 0.1
22.7 0.1
7.1 0.3
6.9 0.3
7.3 0.2
54.1 0.2
86.4 0.4
78.7 0.4
60.7 0.9
58.5 0.1
65.7 0.9
80.6 0.3
* The ability of compounds 6—10 to inhibit the accumulation of the products of the lipid peroxidation (LPO) reacting with 2-thio-
barbituric acid (TBA-reactive substances, TBA-RS) was estimated using the mouse testicular homogenate (3 h after the H2O2 initia-
tion of the LPO) and mouse brain homogenate (1 h after the ascorbate-Fe2+-initiation of the LPO). The control samples are the
samples containing no test compounds, the intact samples are the samples in which oxidation was not initiated.

Phenols with hydroxymethyl group
Russ. Chem. Bull., Int. Ed., Vol. 69, No. 8, August, 2020
1575
Table 2. Comparative evaluation of MPA of derivatives 6—10
at a concentration of 1 mol L–1 assessed using H2O2-induced
hemolysis of red blood cells
shows the higher antioxidant power in the animal model
assay than other phenolic antioxidants.9
In summary, in the present work we synthesized a se-
ries of mono- and dialkylphenols with the para position
hydroxymethyl group. The in vitro studies indicate that
the activity of the synthesized products is determined by
the number and bulkiness of the substituents at the ortho
position relative to the phenolic group, which is in agree-
ment with the relationships found by us earlier for other
phenolic antioxidants.19—23 In terms of the set of the
considered parameters, the lowest activity was observed
for para-hydroxymethyl derivative 10 with one terpene
substituent at the ortho position relative to the phenolic
OH group and the highest activity was found for com-
pound 7 with two isobornyl moieties.
Compound
MPA (hemolysis (%))
3 h
1 h
5 h
Control
16.4 1.5
6.7 1.2
11.5 1.2
1.8 0.0
2.0 0.2
5.4 1.0
16.4 1.7
40.0 0.9
37.0 1.7
37.7 0.7
3.3 0.0
25.6 1.2
37.7 0.7
34.2 0.7
56.8 1.6
49.2 1.0
52.0 0.4
5.4 0.3
30.4 1.0
48.8 1.0
49.4 0.3
BHT
6
7
8
9
10
evaluation of the membrane-protective activity (MPA)
(Table 2). Thus, compound 7 most efficiently prevented
H2O2-induced cell death and compounds 6 and 10 were
the least active. The high statistically significant AOA of
derivative 7 in the biological systems was confirmed by its
ability to efficiently inhibit accumulation of the secondary
LPO products and hemoglobin oxidation. For instance,
compound 7 induces a 1.4-times decrease in the concen-
tration of TBA-RS and 2.8-times and 1.8-times decrease
in methemoglobin/oxyhemoglobin (metHb/oxyHb) and
ferrylhemoglobin/oxyhemoglobin (ferrylHb/oxyHb) ra-
tios, respectively, as compared with the control. Finally,
this compound efficiently protected the erythrocyte heme
from the H2O2-induced damage, which was evidenced
from the decrease in the fluorescence of the products of
heme oxidative degradation (Table 3).
Experimental
1H and 13C NMR spectra of the synthesized compounds were
recorded with a Bruker Avance II 300 spectrometer at the work-
ing frequencies of 300.17 and 75.5 MHz, respectively. The
1H NMR signals were attributed using the NOESY technique,
the 13C NMR signals were assigned with the aid of the J-resolved
experiments and the HSQC and HMBC correlation techniques.
Diffuse reflectance IR spectra were recorded with a Shimadzu
IR Prestige 21 FT-IR spectrophotometer in KBr pellets. Elemental
analysis was performed on a vario Micro cube analyzer operating
in CHNS mode. The spectrophotometric measurements were
carried out on a Thermo Spectronic Genesys 20 spectrophoto-
meter. The reaction course was monitored by TLC on the
precoated plates Sorbfil (LLC Imid). The spots of the compounds
were visualized by treatment of the TLC plates with a KMnO4
solution (KMnO4 (15 g), H2O (300 mL), concentrated H2SO4
(0.5 mL)). Melting points were measured with a Sanyo
Gallenkamp MPD350 apparatus and are given uncorrected.
Aldehyde 1 was synthesized by the known procedure,24 deriva-
tives 2—5 were described by us earlier.11,15,23,25. Sodium boro-
hydride NaBH4 (Daejung Co.), BHT, and DPPH (both Alfa
It should be emphasized that in the cell test system
(red blood cells) derivatives 7 and 8 bearing the para posi-
tion hydroxymethyl group were significantly more active
than BHT; while, their advantages were not such pro-
nounced in non-cellular bioassays (mouse lipid homo-
genates) (see Table 1). It is also notable that compound 7
Table 3. Comparative evaluation of AOA of derivatives 6—10 at a concentration of 1 mol L–1 assessed
using H2O2-induced hemolysis of red blood cells
Compound
TBA-RS/nmol L–1
metHb/oxyHb
ferrylHb/oxyHb
Ifl/arb. units
Control
2.17 0.04
1.87 0.04
1.93 0.05
1.51 0.04
1.67 0.05
2.03 0.03
2.02 0.05
2.20 0.18
1.88 0.10
2.73 0.19
0.78 0.04
1.20 0.14
2.69 0.09
1.96 0.05
1.09 0.06
1.14 0.03
0.54 0.01
0.61 0.01
0.93 0.03
0.75 0.04
0.75 0.02
9.20 0.12
7.87 0.10
9.14 0.18
6.22 0.04
7.57 0.21
8.73 0.47
8.24 0.17
BHT
6
7
8
9
10
* The forms of hemoglobin, in which the iron atom (a complexing agent) has different formal charges, are
as follows: oxyhemoglobin (oxyHb, Fe2+), methemoglobin (metHb, Fe3+), ferrylhemoglobin (ferrylHb,
Fe4+). Methemoglobin and ferrylhemoglobin, in which the formal charges of heme iron are higher than in
oxyhemoglobin, cannot function as oxygen carriers.

Buravlev et al.
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Russ. Chem. Bull., Int. Ed., Vol. 69, No. 8, August, 2020
Aesar) were used as purchased. Alfa Aesar silica gel (0.06—0.2 mm)
was used for the column chromatography.
: 12.22 (C(10´)); 20.22 (C(9´)); 21.38 (C(8´)); 27.59 (C(5´));
29.79 (C(CH3)3); 34.29 (C(3´)); 34.50 (C(CH3)3); 40.33 (C(6´));
45.39 (C(4´)); 46.31 (C(2´)); 48.39 (C(7´)); 49.53 (C(1´)); 66.13
(CH2OH); 123.89, 124.96 (C(3), C(5)); 128.83, 131.38, 135.73
(C(2), C(4), C(6)); 153.18 (C(1)).
4-Hydroxymethyl-2-methyl-6-(1,7,7-trimethylbicyclo[2.2.1]-
hept-exo-2-yl)phenol (9). Beige powder. Yield 0.207 g (75%).
The spectral properties are in agreement with those described
earlier.12
4-Hydroxymethyl-2-(1,7,7-trimethylbicyclo[2.2.1]hept-exo-
2-yl)phenol (10). Beige powder, m.p. 109—111 C. Yield 0.200 g
(77%). Found (%): C, 78.54; H, 9.33. C17H24O2. Calculated (%):
C, 78.42; H, 9.29. IR (KBr), /cm–1: 3345, 3130, 3096, 3021
(OH); 2949, 2976, 2747, 1440 (CH3, CH2); 1610 (C=C); 1252,
1117 (C—O); 822 (=C—H). 1H NMR (CDCl3), : 0.79, 0.84,
0.89 (all s, 3 H each, C(8´)H3, C(9´)H3, C(10´)H3); 1.28—1.53
(m, 2 H, 1 H(5´), 1 H(6´)); 1.55—1.74 (m, 2 H, 1 H(3´),
1 H(6´)); 1.78—1.95 (m, 2 H, 1 H(4´), 1 H(5´)); 2.17—2.32 (m,
1 H, 1 H(3´)); 3.13 (t, 1 H, 1 H(2´), J = 8.8 Hz); 4.60 (s, 2 H,
CH2OH); 5.07 (s, 1 H, ArOH); 6.73, 7.04 (both d, 1 H each,
1 H(5), 1 H(6)), J = 8.0 Hz, J = 8.0 Hz); 7.31 (s, 1 H, 1 H(3)).
13C NMR (CDCl3), : 12.31 (C(10´)); 20.31 (C(9´)); 21.39
(C(8´)); 27.49 (C(5´)); 33.84 (C(3´)); 39.88 (C(6´)); 45.42, 45.55
(C(4´), C(2´)); 48.08 (C(7´)); 49.85 (C(1´)); 65.63 (CH2OH);
115.04, 125.70 (C(5), C(6)); 127.66 (C(3)); 129.92, 132.27 (C(2),
C(4)); 154.55 (C(1)).
Some physicochemical studies of the synthesized compounds
were performed on the equipment of the Center for Collective
Use "Chemistry" of the Institute of Chemistry of Komi Science
Center, the Ural Branch of the Russian Academy of Sciences
(RAS). Biological activity of the synthesized compounds was
studied using the equipment of the Center for Collective Use
"Molecular Biology" of the Institute of Biology of Komi Science
Center, the Ural Branch of the RAS. For the experiments, the
laboratory mice from the scientific collection of experimental
animals of the Institute of Biology of the Komi Scientific Center,
the Ural Branch of the RAS were used (http://www.ckp-rf.ru/
usu/471933/). The animals were kept and all experiments with
them were carried out in accordance with the "Regulations on
the vivarium of experimental animals" of the Institute of Biology
of the Komi Scientific Center, the Ural Branch of the RAS
(protocol No. 1 dated 01.24.2017), taking into account the
sanitary-hygienic and bioethical aspects (http://www.ckp-rf.ru/
usu/471933/).
Synthesis of alcohols 6—10 (general procedure). To a solution
or a suspension of aldehyde 1—5 (1.0 mmol) in anhydrous MeOH
(15 mL), NaBH4 (0.189 g, 5.0 mmol) was added at ~5 C (bath
temperature) and the mixture was stirred for 1.5 h. After the
reaction completion, the mixture was warmed to room tem-
perature, treated with 2 M NaOH (12 mL), stirred 5 min, di-
luted with diethyl ether (30 mL), and stirring was continued for
15 min. The organic layer was separated, washed with 2 M NaCl
to pH ~7.0, dried with anhydrous K2CO3, and co-evaporated
with small amount of pentane. The product was collected by
filtration and washed with petroleum ether. Compound 6 was
additionally purified by silica gel column chromatography (elu-
tion with CHCl3).
4-Hydroxymethyl-2,6-di-tert-butylphenol (6). Colorless pow-
der, m.p. 141—143 C (cf. Ref. 26: 140—145 C (MeOH)). Yield
0.106 g (45%). Found (%): C, 76.49; H, 10.44. C15H24O2.
Calculated (%): C, 76.23; H, 10.24. IR (KBr), /cm–1: 3568,
3522 (OH); 2945, 2953, 2914, 2872, 1481, 1435 (CH3, CH2);
1584 (C=C); 1204, 1113, 1013 (C—O); 881 (=C—H). 1H NMR
spectral data are in agreement with those described previously.26
13C NMR (CDCl3), : 30.26 (2 C(CH3)3); 34.34 (2 C(CH3)3);
66.10 (CH2OH); 124.44 (C(3), C(5)); 131.62, 136.13 (C(2),
C(4), C(6)); 153.52 (C(1)).
4-Hydroxymethyl-2-{(1S,2R,4R)-1,7,7-trimethylbicyclo-
[2.2.1]heptan-2-yl}-6-{(1R,2S,4S)-1,7,7-trimethylbicyclo[2.2.1]-
heptan-2-yl}phenol (7). Colorless powder. Yield 0.317 g (80%).
The spectral properties are in agreement with those described
earlier.11
Radical scavenging activity of compounds 6—10 was esti-
mated using the DPPH assay as earlier described.27 The test
compounds at a concentration of 10 mol L–1 were added to
a solution of DPPH in MeOH, stirred for 30 min and then the
optical density of the solution was measured at  = 517 nm.
Antioxidant activity of the compounds was estimated by their
ability to inhibit the LPO in the mouse brain and testicular homo-
genates.28—30 Removed mouse brain and testicles were homo-
genized in 10% saline (pH 7.4), centrifuged for 10 min to obtain
supernatant (S1). The test compounds were added to the super-
natant as the solutions in acetone at the final concentrations of
1 and 10 mol L–1. After 30 min, the LPO was initiated by add-
ing either freshly prepared FeCl2 and ascorbic acids31,32 (mouse
brain homogenate) or H2O2 (testicular homogenate). The
test samples were incubated at 37 C for 1—3 h under gentle
agitation using a Biosan ES-20 orbital shaker-incubator.
The optical density of the samples was measured at  = 532 nm;
the TBA-RS content was calculated using its extinction coeffi-
cient of 1.56•105 L mol–1 cm–1 30,33
.
Hemolytic activity (cytotoxicity), membrane-protective and
antioxidant properties were evaluated using mouse red blood cell
suspension in phosphate buffered saline (pH 7.4).
4-Hydroxymethyl-2-tert-butyl-6-(1,7,7-trimethylbicyclo-
[2.2.1]hept-exo-2-yl)phenol (8). Beige powder, m.p. 125—127 C.
Yield 0.263 g (83%). Found (%): C, 79.86; H, 10.01. C21H32O2.
Calculated (%): C, 79.70; H, 10.19. IR (KBr), /cm–1: 3638,
3597, 3416 (OH); 2953, 2874, 2729, 1458, 1437 (CH3, CH2);
1593 (C=C); 1204, 1169, 1130 (C—O); 877 (=C—H). 1H NMR
(CDCl3), : 0.79, 0.85, 0.87 (all s, 3 H each, C(8´)H3, C(9´)H3,
C(10´)H3); 1.32—1.47 (m, 11 H, 1 H(5´), 1 H(6´), C(CH3)3));
1.56—1.79 (m, 2 H, 1 H(3´), 1 H(6´)); 1.84—2.02 (m, 2 H,
1 H(4´), 1 H(5´)); 2.24—2.38 (m, 1 H, 1 H(3´)); 2.89 (t, 1 H,
1 H(2´), J = 8.5 Hz); 4.60 (s, 2 H, CH2OH); 4.80 (s, 1 H, ArOH);
7.15, 7.19 (both s, 1 H each, 1 H(3), 1 H(5)). 13C NMR (CDCl3),
Cytotoxicity of compounds 6—10 was measured in vitro by
their ability to induce erythrocyte hemolysis. The solutions of
the compounds in acetone were added to the erythrocyte suspen-
sion to a final concentration of 10 mol L–1 and the samples
were incubated at 37 C for 5 h using a Biosan ES-20 orbital
shaker-incubator.
Membrane-protective and antioxidant activities of com-
pounds 6—10 were assessed by the degree of inhibition of the
induced hemolysis, inhibition of the accumulation of the second-
ary LPO products, and oxidation of oxyHb in erythrocytes. For
this, the test compounds were added to the erythrocyte suspen-
sion, to a final concentration of 1 mol L–1, after 30 min the

Phenols with hydroxymethyl group
Russ. Chem. Bull., Int. Ed., Vol. 69, No. 8, August, 2020
1577
hemolysis was initiated by adding a H2O2 solution (0.006%). The
reaction mixtures were incubated at 37 C for 5 h under gentle
agitation on an orbital shaker-incubator. Every 60 min, an aliquot
was taken, centrifuged for 5 min (1600 g), and the amount of
hemoglobin released was determined by reading optical density
of the supernatant at  = 524 nm.34 The degree of hemolysis was
calculated as the ratio of the hemoglobin content in supernatant
over the hemoglobin content in the completely hemolyzed
sample. The TBA-RS content was determined spectrophoto-
metrically as described above. To estimate the accumulation of
the products of the hemoglobin oxidation, the absorption spec-
tra were analyzed at  = 540—640 nm range. The content of
oxyHb, metHb, and ferrylHb was calculated using the corre-
sponding extinction coefficients.35 The concentration of the heme
degradation products formed upon oxidation of the membrane-
bound hemoglobin with the reactive oxygen species was de-
termined from the Ifl value at a maximum of  = 464—468 nm
(a Fluorat-2-Panorama spectrofluorometer, excitation at
 = 321 nm, emission at  = 400—600 nm, 2-nm steps).36—38
Each experiment was performed four-six times. The experi-
mental results in Tables 1—3 are expressed as an arithmetic
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mean and a standard error (M
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This work was financially supported by the Russian
Foundation for Basic Research (Project No. 18-03-
00950_a) and the Ministry of Science and Higher Edu-
cation of the Russian Federation (State Assignment
No. AAAA-A18-118011120004-5).
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Received April 15, 2020;
in revised form May 7, 2020;
accepted June 19, 2020