Synthesis and Antioxidant Activity of Myrtanylthiotriazoles

Article abstract of DOI:10.1007/s10600-017-2150-9

Thio-derivatives of triazole-substituted myrtanols were synthesized in 78–95% yields and were demonstrated to have membrane-protective and antioxidant properties based on their ability to inhibit H₂O₂-induced hemolysis of erythrocyte

Full text of DOI:10.1007/s10600-017-2150-9

DOI 10.1007/s10600-017-2150-9
Chemistry of Natural Compounds, Vol. 53, No. 5, September, 2017
SYNTHESIS AND ANTIOXIDANT ACTIVITY
OF MYRTANYLTHIOTRIAZOLES
1
1
Yu. V. Gyrdymova, M. Ya. Demakova,
2
1*
O. G. Shevchenko, D. V. Sudarikov,
1
1
L. L. Frolova, S. A. Rubtsova,
1
and A. V. Kuchin
Thio-derivatives of triazole-substituted myrtanols were synthesized in 78–95% yields and were demonstrated
to have membrane-protective and antioxidant properties based on their ability to inhibit H O -induced
2
2
hemolysis of erythrocytes and LPO processes in brain lipids (in vitro).
Keywords: cis- and trans-myrtanols, triazoles, antioxidants, membrane-protective properties, erythrocytes, oxidative
hemolysis, lipid peroxidation (LPO).
Cis- and trans-myrtanols occur in essential oils of plants in the family Lamiaceae and possess antibacterial properties
[1]. Compounds containing triazoles are known to exhibit broad spectra of biological activity, e.g., anti-inflammatory, analgesic
[2], antitumor [3], antihelminthic [4], antifungal [5], antibacterial [6, 7], antiviral [8, 9], anticonvulsant [10], and antioxidant
[11–16]. Individual monoterpenoid derivatives also exhibit high antioxidant activity [17–19]. Therefore, the synthesis and
characterization of the antioxidant properties of triazole-containing myrtanol derivatives are highly crucial.
Herein, syntheses of monoterpenoids containing triazoles from cis- and trans-myrtanols (1, 2) are reported.
Myrtanylthiotriazoles 5a,b and 6a,b were prepared from monoterpenol p-toluenesulfonates (3 and 4) and heterocyclic
thiols. Tosylates 3 and 4 were synthesized from cis-(1) and trans-myrtanols (2) by the published procedure (Scheme 1) [20].
a
S
b
Terp-OH
Terp
Terp-OTs
R
3, 4
1, 2
5a,b; 6a,b
10
10
1
2
9
6
N
N
1
2
9
6
N
N
Terp =
; R =
1'
4
7
7
HN
N
4'
4
Ph
Ph
5
a
6
b
a. TsCl, Py; b. RSH, Cs CO , TBAI, EtOH (DMF), reflux, 8 h
2
3
Scheme 1
The formation of sulfides 5a,b and 6a,b was consistent with IR absorption bands for C=N at 1516–1560, 1470, and
–1
1360 cm ; N–H, 3110; and N–N, 1270 (5a and 6a) of the heterocyclic moiety and C–H absorption bands for the terpene
–1
moieties (2870–2980 cm ).
13
PMR and C NMR spectra of 5a,b and 6a,b contained resonances for the terpene and triazole fragments.
13
The triazole-ring C–S resonances were observed in C NMR spectra at 154–157 ppm. Resonances of myrtanyl C-10 appeared
at 39–41 ppm and were characteristic of C–S bonds.
1) Institute of Chemistry, Komi Scientific Center, Ural Branch, Russian Academy of Sciences, 48 Pervomaiskaya,
Syktyvkar, 167982, e-mail: sudarikov-dv@chemi.komisc.ru; 2) Institute of Biology, Komi Scientific Center,
28 Kommunisticheskaya, Syktyvkar, 167982. Translated from Khimiya Prirodnykh Soedinenii, No. 5, September–October,
2017, pp. 762–766. Original article submitted December 2, 2016.
©
0009-3130/17/5305-0895 2017 Springer Science+Business Media New York
895

60
50
40
30
20
10
0
C
5a
6a
5c
6c
C
5a
6a
5b
6b
1 mM
5c
6c
0.1 mM
- A
- B
Fig. 1. Effects of myrtanylthiotriazoles at concentrations of 1 and 0.1 mM on TBA-AP content in brain homogenate 1 h after LPO
2+
initiation (initiators: A, ascorbate/Fe ; B, H O ). C is the control sample without the studied compounds.
2
2
3-Mercapto-1,2,4-triazole is known to exist in two tautomeric forms, i.e., thiol (A) and thione (B) (Scheme 2) with
the latter dominating [21]. Thus, reaction of tosylates 3 and 4 with thione form B formed thioamides 5c and 6c in yields of 89
and 95%, respectively (Scheme 2).
N
N
HS
S
N
S
N
A
HN N
B
S
6'
N
a
N
3, 4
Terp N
+
4'
HN N
N
5c, 6c
Terp =
3, 5
4, 6
a. TsCl, Py; b. RSH, Cs CO , TBAI, EtOH (DMF), reflux, 8 h
2
3
Scheme 2
Formation of the thioamides was confirmed by IR and NMR spectral data. Thus, IR spectra of conjugates 5c and 6c
–1 13
showed absorption bands characteristic of C=S vibrations at 1090 cm . C NMR spectra of these same compounds exhibited
resonances for the heterocyclic-ring C=S at 167 ppm and for myrtanyl C-10 at 53–55 ppm that were characteristic of C–N
bonds.
Antioxidant activity (AOA) of synthesized myrtanylthiotriazoles 5a–c and 6a–c was studied in extracellular and
cellular model systems (in vitro).
Mammal blood erythrocytes are convenient subjects for testing the AOA of various compounds and plant extracts
[22]. Brain lipids of laboratory animals are widely used as an extracellular model for assessing AOA [23–25].
Studies of the AOA of the synthesized thiotriazoles in the extracellular model system showed that all compounds
without phenyls (5a, 6a, 5c, 6c) were highly active at a concentration of 1 mM (Fig. 1).
Thioamide 6c exhibited the greatest inhibitory activity if the concentrations were reduced by an order of magnitude
(0.1 mM).
The antioxidant and membrane-protective activities of the synthesized compounds in the cellular model system were
studied for all compounds except 6b and 6c, which were highly cytotoxic even at a concentration of 0.1 mM according to
preliminary investigations.
Figure 2 shows results for the membrane-protective activity of myrtanylthiotriazoles 5a–c and 6a in an erythrocyte
oxidative hemolysis model.
896

50
40
30
C
5b
5c
20
10
5a
6a
0
1
2
3
4
5
Incubation time, h
Fig. 2. Effect of myrtanylthiotriazoles (5a–c and 6a) at a concentration
of 0.1 mM on degree of H O -induced hemolysis of erythrocytes after
2
2
incubation for 1–5 h.
All studied compounds exhibited membrane-protective activity. However, sulfanyltriazoles 5a and 6a with
unsubstituted triazoles in their structures possessed the greatest ability to inhibit H O -induced hemolysis during the whole
2
2
incubation period.
The ability of the myrtanylthiotriazoles to prevent oxidation of hemoglobin and lipids in the cellular model confirmed
that they had antioxidant activity. Thus, compounds 5a, 6a, and 5c slowed the conversion rate of oxyhemoglobin to
methemoglobin by 1.5–1.6 times whereas 6a and 5c decreased the accumulation rate of LPO secondary products by
1.3–1.4 times.
Thus, myrtanylthiotriazoles of various structures were synthesized. Myrtanylsulfanyl-1H-triazoles 5a and 6a possessed
the greatest antioxidant activity for H O -induced oxidative stress according to a mammal blood erythrocyte test system,
2
2
possibly because the triazole contained a free H that was susceptible to hemolytic cleavage to form a resonance-stabilized
electron-saturated triazole radical that probably trapped free radicals in the system. Thiotriazoles 6b and 6c were cytotoxic
because of the trans-myrtanyl moiety in their structures. Studies of the antioxidant activity in a non-cellular model system
with substrate obtained from brain tissue showed that all thiotriazoles without phenyl substituents were active regardless of the
myrtanyl isomer.
Myrtanylsulfanyltriazoles 5a and 6a were most promising for further research according to the comprehensive study
of antioxidant activity in the two model test systems.
EXPERIMENTAL
IR spectra were recorded from thin films or KBr pellets on a Shimadzu IR Prestige 21 FTIR spectrometer. PMR and
13
C NMR spectra were recorded in CDCl using solvent resonances as internal standards on a BrukerAvance-300 spectrometer
3
1
13
13
1
(300.17 MHz for H and 75.48 MHz for C). C NMR spectra were recorded in J-modulation mode. Resonances of H and
C were fully assigned using two-dimensional homo- ( H– H COSY, H– H NOESY) and heteronuclear experiments
( H– C HSQC, H– C HMBC). Mass spectra were recorded on a Shimadzu GCMS-QP 2010 Plus instrument at ion-source
13
1
1
1
1
1
13
1
13
temperature 200°C in mass scan range m/z 2–400 with electron-impact ionization at 70 eV. Optical rotation angles were
measured on an Optical Activity PolAAr 3001 automated digital polarimeter (England). TLC used Sorbfil plates, CHCl
3
solvent, and phosphomolybdic acid developer. Column chromatography used AlfaAesar silica gel (0.06–0.2 mm) and eluents
petroleum ether–EtOAc (5:1, 1:1), CHCl –Et O (10:1), CHCl –i-PrOH (5:1), and petroleum ether–Et O (5:1, 50:1).
3
2
3
2
20
Cis- and trans-myrtanols were synthesized from commercially available (–)-ꢀ-pinene (Alfa Aesar), [ꢁ] –27.9°
D
(neat). Sulfides were synthesized using 3-mercapto-1,2,4-triazole, 3-mercapto-4-methyl-1,2,4-triazole, and 3-mercapto-2,4-
diphenyl-1,2,4-triazole (Alfa Aesar).
(–)-cis-Myrtanol, (1S,2R,5S)-6,6-dimethylbicyclo[3.1.1]heptane-2-methanol (2) was prepared by the literature
20
20
method [26], [ꢁ] –20.5° (c 1.9, CHCl ), lit. [ꢁ] –20.9° (c 4.0, CHCl ) [26]. The spectral characteristics agreed with those
D
3
D
3
published [27].
897

(–)-trans-Myrtanol, (1S,2R,5S)-6,6-dimethylbicyclo[3.1.1]heptane-2-methanol (3) was prepared by the literature
20
20
method [27], [ꢁ] –27.9° (c 1.9, CHCl ), lit. [ꢁ] –28.5° (c 4.0, CHCl ) [27]. The spectral characteristics agreed with those
D
3
D
3
in the literature [27].
Tosylates of (–)-cis- (3) and (–)-trans-myrtanols (4) were synthesized as before [28].
General Method for Synthesizing Thio-derivatives. Thiol (1.5 mmol) was dissolved in DMF (5 mL), treated with
Cs CO (1 mmol) and TBAI (1 mmol), stirred for 5 min, and treated with a solution of tosylate 3 or 4 (1 mmol) in DMF.
2
3
The syntheses were carried out with constant stirring and refluxing for 8 h. The reaction was monitored by TLC. When
the reaction was finished, the solvent was distilled off. The residue was extracted with EtOAc. The extract was washed with
saturated KHCO solution. The combined organic fractions were combined and dried over Na SO . The target sulfides were
3
2
4
isolated by column chromatography over silica gel.
2-[(1R,2S,5R)-(1,2,4-Triazolylsulfanyl)methyl]-6,6-dimethylbicyclo[3.1.1]heptane (5a). Yield 91%, yellow oily
20
–1
liquid, R 0.11 (CHCl –Et O, 10:1), [ꢁ] –23.4ꢂ (c 1.18, CÍCl ). IR spectrum (KBr, ꢃ, cm ): 3110 (N–H), 2980–2870
f
3
2
D
3
+
1
(Ñ–Í), 1516–1560, 1470, 1360 (C=N), 1270 (N–N). Mass spectrum (EI, 70 eV), m/z (I , %): 237.20 (70) [M ]. H NMR
rel
spectrum (300 MHz, CDCl , ꢄ, ppm, J/Hz): 0.88 (1Í, d, J = 9.7, H-7ꢁ), 1.02 (3Í, s, Í-8), 1.18 (3Í, s, H-9), 1.49–1.65 (1Í,
3
m, Í-3ꢁ), 1.73–2.14 (5H, m, Í-1, 3ꢀ, 4ꢁ, 4ꢀ, 5), 2.28–2.42 (2Í, m, Í-2, 7ꢀ), 3.28 (2Í, dd, J = 7.7, 6.7, Í-10), 8.13 (1Í, s,
13
Í-4ꢅ), 11.96 (1Í, br.s, NH). C NMR spectrum (75 MHz, CDCl , ꢄ, ppm): 21.77 (Ñ-3), 23.16 (Ñ-8), 26.02 (Ñ-4), 27.88
3
(Ñ-9), 33.23 (Ñ-7), 39.63 (Ñ-10), 41.02 (Ñ-2), 41.15 (Ñ-5), 44.64 (Ñ-6), 45.31 (Ñ-1), 148.03 (Ñ-4ꢅ), 156.96 (Ñ-1ꢅ).
2-{[(1R,2S,5R)-(1,5-Diphenyl-1H-1,2,4-triazolyl)sulfanyl]methyl}-6,6-dimethylbicyclo[3.1.1]heptane (5b).
26
Yield 78%. White powder, mp 246–247ꢂÑ, R 0.10 (CHCl –i-PrOH, 5:1), [ꢁ] +35.2ꢂ (c 0.4, CÍCl ). IR spectrum (KBr, ꢃ,
f
3
D
3
–1
cm ): 2980–2860 (Ñ–Í), 1560–1516 (Ñ=N), 1622, 1494 (Ñ Í ), 1470, 1360 (C=N), 1270 (N–N). Mass spectrum
(EI, 70 eV), m/z (I , %): 391.20 (73) [M] , 392.20 (24) [Ì + 1] . H NMR spectrum (300 MHz, CDCl , ꢄ, ppm): 0.80 (3Í,
6
5
+
+ 1
rel
3
s, H-8), 1.19 (3Í, s, Í-9), 1.30–1.46 (2Í, m, Í-3ꢁ, 7ꢁ), 1.72–1.98 (5H, m, Í-1, 3ꢀ, 4ꢁ, 4ꢀ, 5), 2.01–2.14 (1Í, m, Í-7ꢀ),
13
2.26–2.40 (1Í, m, Í-2), 7.19–7.61 (10Í, m, 2 Ñ Í ). C NMR spectrum (75 MHz, CDCl , ꢄ, ppm): 20.09 (Ñ-8), 21.87
6
5
3
(Ñ-3), 23.72 (Ñ-7), 24.16 (Ñ-4), 26.61 (Ñ-9), 34.84 (Ñ-2), 38.66 (Ñ-10), 39.50 (Ñ-6), 40.73 (Ñ-5), 44.89 (Ñ-1), 126.71 (Ñ-6ꢅ),
127.39, 128.13, 128.42, 129.56, 129.75, 128.86 (Ñ-7ꢅ–11ꢅ, 13ꢅ–17ꢅ), 134.40 (Ñ-12ꢅ), 153.16 (Ñ-4ꢅ), 154.67 (Ñ-1ꢅ).
2-(1R,2S,5R)-(4-Methyl-2,4-dihydro-3-thione-1,2,4-triazolyl)-6,6-dimethylbicyclo[3.1.1]heptane (5c). Yield 89%,
27
–1
white powder, mp 112–113ꢂC, R 0.27 (CHCl –Et O, 10:1), [ꢁ] –8.1ꢂ (c 0.3, CÍCl ). IR spectrum (KBr, ꢃ, cm ):
f
3
2
D
3
+
2980–2870 (Ñ–Í), 1560–1516, 1470, 1360 (C=N), 1270 (N–N). Mass spectrum (EI, 70 eV), m/z (I , %): 251.05 (21) [M ].
H NMR spectrum (300 MHz, CDCl , ꢄ, ppm, J/Hz): 0.90 (1Í, d, J = 9.9, H-7ꢁ), 1.19 (6Í, d, Í-8, 9), 1.59–1.73 (1Í, m,
rel
1
3
Í-3ꢁ), 1.76–2.06 (5Í, m, Í-1, 3ꢀ, 4ꢁ, 4ꢀ, 5), 2.28–2.41 (1H, m, Í-7), 2.68–2.83 (1Í, m, Í-2), 3.56 (3Í, s, Í-6ꢅ), 4.20 (2Í,
13
t, J = 8.07, Í-10), 7.74 (1Í, s, Í-4ꢅ). C NMR spectrum (75 MHz, CDCl , ꢄ, ppm): 18.93 (Ñ-3), 23.08 (Ñ-8), 25.84 (Ñ-4),
3
27.20 (Ñ-9), 32.69 (Ñ-6ꢅ), 32.91 (Ñ-7), 38.65 (Ñ-6), 39.82 (Ñ-2), 41.20 (Ñ-5), 43.26 (Ñ-1), 54.41 (Ñ-10), 138.70 (Ñ-1ꢅ),
162.01 (Ñ-4ꢅ).
2-[(1R,2S,5R)-(1,2,4-Triazolylsulfanyl)methyl]-6,6-dimethylbicyclo[3.1.1]heptane (6a). Yield 90%. oily liquid,
–1
R 0.18 (CHCl –Et O, 1:1). IR spectrum (KBr, ꢃ, cm ): 3109 (NH), 2978–2871 (Ñ–Í), 1560–1516, 1470, 1360 (C=N), 1270
f
3
2
+
1
(N–N). Mass spectrum (EI, 70 eV), m/z (I , %): 237.15 (80) [M ]. H NMR spectrum (300 MHz, CDCl , ꢄ, ppm, J/Hz): 0.77
rel
3
(3Í, s, Í-8), 1.18 (3Í, s, H-9), 1.32 (2Í, d, J = 10.2, H-3ꢁ, 7ꢁ), 1.70–1.82 (3Í, m, Í-3ꢀ, 4ꢁ, 4ꢀ), 1.85–1.93 (2H, m, Í-1, 5),
2.00–2.10 (1Í, m, Í-7ꢀ), 2.19–2.32 (1Í, m, Í-2), 3.01–3.17 (2Í, m, Í-10), 8.15 (1Í, s, Í-4ꢅ), 13.13 (1Í, br.s, NH).
13
C NMR spectrum (75 MHz, CDCl , ꢄ, ppm): 20.00 (Ñ-8), 21.83 (Ñ-3), 23.27 (Ñ-7), 24.10 (Ñ-4), 26.56 (Ñ-9), 34.99 (Ñ-2),
3
38.91 (Ñ-10), 39.44 (Ñ-6), 40.68 (Ñ-5), 44.83 (Ñ-1), 147.90 (Ñ-1ꢅ), 156.47 (Ñ-4ꢅ).
2-{[(1R,2S,5R)-(1,5-Diphenyl-1H-1,2,4-triazolyl)sulfanyl]methyl}-6,6-dimethylbicyclo[3.1.1]heptane (6b). Yield
27
89%. White powder, mp 155–156ꢂÑ. R 0.20 (petroleum ether–Et O, 5:1), [ꢁ] –7.4ꢂ (c 0.5, CÍCl ). IR spectrum (KBr, ꢃ,
f
2
D
3
–1
cm ): 3026 (NH), 2980–2870 (Ñ–Í), 1595 (Ñ Í ), 1560–1515 (C=N), 1270 (N–N), 644 (Ñ–S). Mass spectrum (EI, 70 eV),
m/z (I , %): 389.20 (20) [M ], 390.20 (24) [Ì + 1] . H NMR spectrum (300 MHz, CDCl , ꢄ, ppm, J/Hz): 0.87 (3Í, s, H-8),
6
5
+
+ 1
rel
3
1.20 (3Í, s, H-9), 1.30–1.40 (2Í, m, Í-3ꢁ, 7ꢁ), 1.72–1.83 (3Í, m, Í-3ꢀ, 4ꢁ, 4ꢀ), 1.88 (2H, d, J = 5.8, H-1, 5), 5.00–5.12 (1Í,
13
m, Í-7ꢀ), 2.26–2.40 (1Í, m, Í-2), 3.14–3.33 (2Í, m, Í-10), 7.19–7.56 (10Í, m, 2Ñ Í ). C NMR spectrum (75 MHz,
6
5
CDCl , ꢄ, ppm): 20.07 (Ñ-8), 21.84 (Ñ-3), 23.30 (Ñ-7), 24.13 (Ñ-4), 26.58 (Ñ-9), 34.79 (Ñ-2), 38.62 (Ñ-10), 39.47 (Ñ-6),
3
40.68 (Ñ-5), 44.84 (Ñ-1), 126.63 (Ñ-6ꢅ), 127.35, 128.11, 128.40, 129.56, 129.74, 128.84 (Ñ-7ꢅ–11ꢅ, 13ꢅ–17ꢅ), 134.34 (Ñ-12ꢅ),
153.53 (Ñ-4ꢅ), 154.70 (Ñ-1ꢅ).
2-(1R,2R,5R)-(4-Methyl-2,4-dihydro-3-thione-1,2,4-triazolyl)-6,6-dimethylbicyclo[3.1.1]heptane (6c). Yield 95%,
26
–1
yellowish powder, mp 88–89ꢂÑ, R 0.43 (CHCl –Et O, 10:1), [ꢁ] –24.5ꢂ (c 0.8, CÍCl ). IR spectrum (KBr, ꢆꢃ, cm ):
f
3
2
D
3
898

+
2980–2870 (Ñ–Í), 1560–1516, 1470, 1360 (C=N), 1270 (N–N). Mass spectrum (EI, 70 eV), m/z (I , %): 251.15 (35) [M ],
rel
+ 1
252.15 (23) [Ì + 1] . H NMR spectrum (300 MHz, CDCl , ꢄ, ppm): 0.17 (3Í, s, H-8), 0.57 (3Í, s, Í-9), 0.71–0.86 (2Í, m,
3
Í-3ꢁ, 7ꢁ), 0.86–1.04 (1H, m, Í-3ꢀ), 1.06–1.19 (3Í, m, Í-1, 4ꢁ, 5), 1.19–1.30 (1Í, m, Í-4ꢀ), 1.42–1.53 (1Í, m, 7ꢀ), 3.37
13
(2Í, m, Í-10), 7.36 (1Í, s, Í-4ꢅ). C NMR spectrum (75 MHz, CDCl , ꢄ, ppm): 18.72 (Ñ-3), 19.74 (Ñ-8), 23.18 (Ñ-7), 23.62
3
(Ñ-4), 26.33 (Ñ-9), 32.33 (Ñ-6ꢅ), 33.96 (Ñ-2), 38.88 (Ñ-6), 40.43 (Ñ-5), 42.34 (Ñ-1), 53.30 (Ñ-10), 162.22 (Ñ-4ꢅ), 166.50 (Ñ-1ꢅ).
Antioxidant activity of the myrtanylthiotriazoles was assessed from the ability to inhibit lipid peroxidation (LPO)
processes in brain lipids of laboratory mice [23–25]. Excised brain was homogenized (10%) in normal saline (pH 7.4) and
centrifuged for 10 min. Then, the supernatant (S1) containing H O, proteins, DNA, RNA, and lipids (cholesterol, galactolipids,
2
individual phospholipids, and gangliosides) was collected. The studied compounds were added to S1 as Me CO solutions
2
(final concentration 0.1 and 1 mM). After 30 min, LPO was initiated by adding freshly prepared FeSO (3 M) and ascorbic
4
acid (300 M). Then, the test samples were incubated for 1 h at 37°C with gentle stirring. The contents of LPO secondary
products that reacted with 2-thiobarbituric acid (TBA-AP) were determined by spectrophotometry at ꢇ = 532 nm using
a Thermo Genesys Spectronic 20 spectrophotometer (USA) [23–25, 29].
Toxicity and antioxidant and membrane-protective activities of the compounds were studied using a suspension
(0.5%, v/v) of laboratory-mouse erythrocytes in phosphate-buffered saline (PBS, pH 7.4). Toxicity of the compounds was
assessed (in vitro) from their ability to induce hemolysis. Solutions of the compounds were added to erythrocyte suspension
and incubated at 37°C for 5 h in a Bioscan ES-20 thermostatted shaker (Latvia). Cytotoxicity was assessed from the degree of
erythrocyte hemolysis after incubation for 1, 3, and 5 h. Membrane-protective and antioxidant activities were determined
from the degrees of inhibition of H O -induced hemolysis, suppression of accumulation of LPO secondary products, and
2
2
oxidation of oxyhemoglobin in erythrocytes. For this, hemolysis was initiated by adding H O solution (0.006%) to Me CO
2
2
2
suspensions of erythrocytes 30 min after the studied compounds were added. Then, the reaction mixtures were incubated in a
thermostatted shaker at 37°C with gentle mixing for 5 h. Aliquots of the incubation mixture were taken every hour and
centrifuged for 5 min (1,600 g). Hemolysis was determined at ꢇ = 524 nm from the hemoglobin content in the supernatant on
a Thermo Spectronic Genesys 20 spectrophotometer (USA). The percent hemolysis was calculated from the ratio to total
hemolysis of the sample. The content of LPO secondary products that reacted with TBA-AP was determined
spectrophotometrically [29]. Accumulation of hemoglobin oxidation products after the incubation was assessed by total
hemolysis of an aliquot of erythrocyte suspension, centrifugation to precipitate erythrocyte particulates, and analysis of
the absorption spectrum in the range 540–630 nm using a Fluorat-02-Panorama spectrofluorimeter (Lumex, St. Petersburg).
The contents of various hemoglobin species (oxyHb and ferrylHb) were calculated considering the corresponding extinction
coefficients [30].
Each experiment was performed in 4–5 repetitions. Results were processed statistically using Microsoft Office Excel
2007 software.
Antioxidant and membrane-protective activity were studied at the Molecular Biology Center for Collective Use, Inst.
Biol., Komi SC, UB, RAS. Animals from the scientific collection of experimental animals of the Inst. Biol., Komi SC, UB,
RAS, were used in the work (http://www.ckp-rf.ru/usu/471933/).
ACKNOWLEDGMENT
The work was supported financially in part by the RFBR (Project 16-53-00171 Bel_a) using equipment at the Khimiya
Center for Collective Use, Inst. Chem., Komi SC, UB, RAS.
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