Lipoylcarnosine: Synthesis, Study of Physico-Chemical and Antioxidant Properties, Biological Activity

Article abstract of DOI:10.1134/S199075081804008X

Abstract—: Synthesis of lipoylcarnosine (LipС), a conjugated molecule based on two natural antioxidants, carnosine and α-lipoic acid, is described and its physico-chemical, antioxidant properties and biological activity are characterized. According to reversed-phase HPLC with a UV detector, purity of the final product was 89.3%. The individuality of the obtained sodium salt of LipС was confirmed by tandem HPLC-mass spectrometry. LipC demonstrated high resistance to hydrolysis with serum carnosinase. The antioxidant activity of LipC evaluated by the reaction with the formation of thiobarbituric acid reacting substances and kinetic parameters of iron-induced chemiluminescence was higher than that of carnosine and lipoic acid. LipC did not affect viability of SH-SY5Y human neuroblastoma cells, differentiated to the dopaminergic phenotype, at concentrations not exceeding 5 mM. In the concentration range of 0.1–0.25 mM LipC protected neuronal cells against 1-methyl-4-phenylpyridinium (MPP⁺)-induced toxicity.

Full text of DOI:10.1134/S199075081804008X

ISSN 1990-7508, Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry, 2018, Vol. 12, No. 4, pp. 308–315. © Pleiades Publishing, Ltd., 2018.
Original Russian Text © S.L. Stvolinsky, N.A. Antonova, O.I. Kulikova, A.V. Lopachev, D.A. Abaimov, I. Al-Baidani, O.M. Lopacheva, T.N. Fedorova, A.P. Kaplun,
G.M. Sorokoumova, 2018, published in Biomeditsinskaya Khimiya.
Lipoylcarnosine: Synthesis, Study of Physico-Chemical
and Antioxidant Properties, Biological Activity
S. L. Stvolinskya, *, N. A. Antonovab, O. I. Kulikovaa, c, A. V. Lopacheva, D. A. Abaimova, I. Al-Baidanib,
O. M. Lopachevaa, d, T. N. Fedorovaa, A. P. Kaplunb, and G. M. Sorokoumovab, †
aResearch Center of Neurology, Volokolamskoe shosse 80, Moscow, 125367 Russia
bMoscow Technological University, Moscow, 119454 Russia
cPeoples’ Friendship University of Russia, Moscow, 117198 Russia
dMoscow State University, International Biotechnological Center, Moscow, Russia
*e-mail: slstvolinsky@mail.ru
Received March 12, 2018
Abstract—Synthesis of lipoylcarnosine (LipС), a conjugated molecule based on two natural antioxidants,
carnosine and α-lipoic acid, is described and its physico-chemical, antioxidant properties and biological
activity are characterized. According to reversed-phase HPLC with a UV detector, purity of the final product
was 89.3%. The individuality of the obtained sodium salt of LipС was confirmed by tandem HPLC-mass
spectrometry. LipC demonstrated high resistance to hydrolysis with serum carnosinase. The antioxidant
activity of LipC evaluated by the reaction with the formation of thiobarbituric acid reacting substances and
kinetic parameters of iron-induced chemiluminescence was higher than that of carnosine and lipoic acid.
LipC did not affect viability of SH-SY5Y human neuroblastoma cells, differentiated to the dopaminergic
phenotype, at concentrations not exceeding 5 mM. In the concentration range of 0.1–0.25 mM LipC pro-
tected neuronal cells against 1-methyl-4-phenylpyridinium (MPP+)-induced toxicity.
Keywords: carnosine, α-lipoic acid, lipoylcarnosine, antioxidant, carnosinase, parkinsonism
DOI: 10.1134/S199075081804008X
INTRODUCTION
philicity [11]. These features of carnosine require its
modification in order to increase stability and improve
bioavailability [12].
At present, the search for and development of neu-
roprotective drugs still represents an important task,
and many biomedical studies are directed to its solu-
tion [1, 2] because of the high social significance and
wide spread of neurodegenerative and vascular dis-
eases of the brain. The main molecular mechanism
underlying the death of nerve tissue is oxidative stress
(OS) [3–5] and antioxidants protect cells and tissues
against OS [6]. One of the promising antioxidants is
the natural neuropeptide carnosine. It exhibits not
only the properties of a direct antioxidant, a radical
Another promising antioxidant is α-lipoic acid
(LA), a small molecule containing two oxidized or
reduced (in the case of dihydrolipoic acid (DHLA))
thiol groups. Although DHLA is the preferred form, in
which the compound exists in the body tissues, both
DHLA and LA can scavenge reactive oxygen species
(ROS) and reduce endogenous antioxidants (glutathi-
one, vitamins E and C). This combination of proper-
ties explains high interest in this compound as a prom-
scavenger, but also an antiglycating agent, a metal ion ising therapeutic agent [13]. Despite the fact that LA
chelator, a molecular chaperone, and an inducer of
antioxidant systems under OS conditions [7]. The
antioxidant and neuroprotective effects of carnosine
have been demonstrated in various models in both in
vitro and in vivo experiments [7, 8]. In pilot studies
carnosine increased the effectiveness of basic therapy
in patients with discirculatory encephalopathy [9] and
Parkinson’s disease [10]. However, the action of car-
nosine in the body is limited by its hydrolysis, cata-
lyzed by a specific enzyme, carnosinase, and low lipo-
effectiveness has been demonstrated in diabetes, car-
diovascular, neurodegenerative, and autoimmune dis-
eases, its further application requires additional stud-
ies [14, 15].
It should be noted that manufacturers of food addi-
tives have proposed compositions containing a mix-
ture of carnosine and LA, designed to increase insulin
sensitivity and slow down the aging process [16, 17]. A
pharmacological preparation based on a mixture of
LA and carnosine, which effectively cures the syn-
drome of phantom pains, has also been described [18].
†
Deceased.
308

LIPOYLCARNOSINE: SYNTHESIS, PROPERTIES, BIOLOGICAL ACTIVITY
309
Recently a new substance named by authors as equipped with a Finnigan Surveyor LC Pump Plus
ADM_09 (sodium salt of lipoylcarnosine, LipC) was constant pressure pump (Thermo Fisher Scientific,
synthesized from carnosine and LA [19]. LipC was USA). Direct injection of a LipC sample into the mass
able to neutralize the superoxide radical in vitro in the detector was performed using a Hamilton chromatog-
xanthine-xanthine oxidase system and in the oxalipla- raphy syringe (with volume of 500 μL), which pro-
tin-induced oxidative stress model, as well as to reduce vided liquid flow of 10 μL/min by means of a con-
neuropathic pain in in vivo experiments [19, 20]. At trolled pump.
The 1H-NMR spectra were recorded on a Brucker-
BioSpin 300 GmbH spectrometer (Germany) at a fre-
quency of 300 Hz (number of scans: 11, number of
dots: 128000).
the same time, there is a clear need to assess the effec-
tiveness and mechanisms of the antioxidant effect of
this compound. In this context it seems promising to
study the neuroprotective effect of LipC under condi-
tions of modeling various pathological CNS pro-
cesses.
The aim of this work was to obtain LipC, to study its
physico-chemical characteristics, resistance to hydrolysis
by carnosinase, and antioxidant properties, and to evalu-
ate its effect on the viability of human SH-SY5Y neuro-
blastoma cells, differentiated to the dopaminergic phe-
notype under the conditions of 1-methyl-4-phenylpyri-
dinium (MPP+)-induced toxicity.
Preparation of LipC
LipC was synthesized according to the procedure
described by Nativi et al. [19, 20]. The scheme of its
synthesis is shown in Fig. 1.
The synthesized LipC was purified by adsorption
column chromatography on silica gel.
Adsorption column chromatography. The stationary
phase powder (40 g of silica gel) was suspended in a
mixture of isopropanol with an ammonia solution
(25%, 3 : 1) used as a mobile phase and transferred to
a 3 cm diameter column. LipC (400 mg) was dissolved
in 5 mL of the same mixture and applied onto the col-
umn. During the chromatography, the collected frac-
tions (1 mL each) were analyzed for the presence of
LipC by thin layer chromatography (TLC).
Thin layer chromatography. TLC was carried out
using silica gel plates (TLC Silicagel 60 F254 Merck),
20 × 20 cm, 0.2 mm thick, on an aluminum substrate
in the solvent system: isopropanol−aqueous ammonia
(25%, 3 : 1). Substances on chromatograms were
detected using 0.1 M KMnO4 in 0.1 M CH3COOH.
Nuclear magnetic resonance (NMR). The spectra
were recorded using an NMR instrument with a fre-
quency of 300 Hz. The proton NMR spectrum was
performed using the following parameters: number of
scans: 11; number of points: 128000.
HPLC-tandem mass spectrometry. The purity of
the resultant LipC was confirmed by tandem HPLC-
mass spectrometry; analytical phase A: 0.1% formic
acid in deionized water with 5% acetonitrile; mobile
phase B: 0.1% formic acid in acetonitrile supple-
mented with 5% deionized water. The chromato-
graphic gradient elution was performed as follows: 1%
B (0–2 min), 1% to 50% B (2–12 min), 50% B (12–
17 min), 1% B (17–20 min); flow: 0.5 mL/min [21].
MATERIALS AND METHODS
Reagents
The following substances were used in the study:
carnosine (Yonezawa Hamari Chemicals, Ltd.,
Japan), 100 U/mL penicillin-streptomycin solution,
Hanks’ solution, Minimum Essential Medium
(MEM) with Earle’s salts with glutamine, MEM/F-12
medium without glutamine (PanEco, Russia), 10%
fetal calf serum (PAA Laboratories, USA), α-lipoic
acid, retinoic acid, 2-O-tetradecanoyl-morpholine-
13-acetate, 1-methyl-4-phenylpyridinium (MFP+)
(Sigma, USA), soy phosphatidylcholine (PCh) (SPC-
100, Lipoid, Germany); cyclohexylcarbodiimide
(DCC), N-hydroxysuccinimide, sodium hydrogen-
carbonate, sulfuric acid, potassium permanganate,
acetic acid, chloroform, ethyl alcohol, sodium chlo-
ride, dimethylsulfoxide (DMSO), ethyl acetate, hex-
ane, tetrahydrofuran (THF), dimethylformamide
(DMF), acetone of chemically pure grade (Khimmed,
Russia).
Column chromatography was performed using Sil-
ica Gel MN Kieselgel 60 (0.04−0.063 mm, Merck,
Germany).
Human neuroblastoma SH-SY5Y cell culture
(ATCC, USA) was used in the experiments.
Equipment
Determination of LipC Resistance to Hydrolysis
by Serum Carnosinase
Resistance of LipC and free carnosine to serum
carnosinase was evaluated by the method proposed by
Pegova et al. [22]. The study was performed using
pooled serum from several donors. Serum carnosinase
was activated by incubation with 5 mM CdCl2 at 37°C
The human neuroblastoma cells were cultured in
96 well plates (Nunc, Denmark) using a cell incubator
(ShelLab, USA). The optical density in the MTT test
was measured using a Synergy H4 hybrid multi-mode
microplate reader (BioTek Instruments, Inc., USA).
Mass-spectrometric analysis was performed using
an LCQ Fleet MS quadrupole ion trap mass-spec-
trometer with electrospray ionization, that was for 30 min. Then, a solution of carnosine or LipC was
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STVOLINSKY et al.
O
O
OH
+
N
S S
HO
O
1
2
NH
N
O
O
O
DCC
THF
N
OH
+
H2N
NH
O
O
O
S S
3
4
NH
N
O
O
Na2CO3
O−
Na+
DMF/H2O
NH
5
NH
S
S
O
Fig. 1. The scheme of LipC synthesis 1—LA, 2—N-hydroxysuccinimide, 3—N-hydroxysuccinimide ester of lipoic acid, 4—car-
nosine, 5—LipC; DCC—dicyclohexylcarbodiimide, THF—tetrahydrofuran, DMF—dimethylformamide.
added to the incubation mixture at a final concentra- final concentration of 75 nM 3 days after retinoic acid
administration [24].
tion of 2 mM. The samples were incubated for 0–15–
30–45–60–90–120–150–180 min and then mixed
with cold 20% TCA. Denatured proteins were precip-
itated by centrifugation at 16000 g for 30 min at 5°C
and the supernatant was used for analysis. Samples
incubated without the addition of carnosinase (serum)
were used as controls. The content of LipC and car-
nosine in the samples was determined by tandem
HPLC-mass spectrometry, as described above. The
response of the instrument in the conventional inte-
gration units from the starting samples (0 min) con-
taining 2 mM carnosine and LipC was defined as
100%, the remaining samples were normalized to the
peak area of the starting sample. The data were pro-
cessed using Xcalibur 2.1 Foundation 1.0.1.
Evaluation of cell viability. Cell viability was evalu-
ated using the MTT test. The method is based on
reduction of yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) to blue for-
mazan in living cells [25].
To assess LipC effect on cell viability, the test com-
pound was added to the wells at final concentrations of
0.1–0.25–0.50–0.75–1.0–5.0 mM, and the plates
were incubated for 24 h. The medium was then
replaced with the MTT solution (0.5 mg/mL), pre-
pared using the culture medium. After 3 h, the
medium with MTT was carefully removed without
affecting the formed formazan crystals. The crystals
were dissolved by adding 100 μL of DMSO to each
well, and absorbance was measured at 570 nm and
660 nm wavelengths using a Synergy H4 microplate
reader (BioTek Instruments, Inc., USA). The readings
at 660 nm and negative control were subtracted from
Human Neuroblastoma SH-SY5Y Cell Culture
Human neuroblastoma SH-SY5Y cells were cul- the optical density at 570 nm. Wells containing all
components except cells were used as a negative con-
trol. Data represent a percentage of the signal level in
wells with intact cells. The results were processed using
Microsoft Excel and GraphPad Prism 6.
tured using a mixture of media containing MEM
Eagle with Earle’s salts with glutamine and F-12 with-
out glutamine in a ratio 1 : 1 with the addition of
100 U/mL penicillin-streptomycin and 10% fetal
bovine serum. The culture was maintained in a cell
incubator at 37°C, 90% humidity, 5% CO2. Every
3 days the medium was refreshed [23]. Cell differenti-
ation to the dopaminergic phenotype was induced by
a decrease in serum concentrations to 1% and subse-
quent culturing for 7 days in the presence of retinoic
acid at a final concentration of 10 μM, followed by
To determine the cytoprotective effect of LipC, a
solution of 2 mM MPP+ (final concentration) was
added to wells with the cells, and after addition of
LipC (the concentration range 0.1–0.25–0.50 mM)
cells were incubated for 24 h. LipC and MPP+ were
not added into control wells. After that MTT test was
performed according to the procedure described
addition of 2-O-tetradecanoylphorbole 13-acetate at a above.
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LIPOYLCARNOSINE: SYNTHESIS, PROPERTIES, BIOLOGICAL ACTIVITY
311
RT: 14.86
AA: 84695
was performed daily, then three times on day 14,
day 21, and day 28. The stability was evaluated by the
method of targeted mass spectrometry analysis of
LipC. For this purpose, the area of the chromato-
graphic peak was determined from the total ion cur-
rent of the fragment ions formed during the fragmen-
tation of the parent LipC ion with m/z = 415.3 (Fig. 2).
NL: 4.76E3
TIC: ITMS + c ESI Full
ms2 415.10@cid24.00
[110.00-600.00] MS
100
80
60
40
20
0
2 12
14
16
18
20
22
RESULTS AND DISCUSSION
Time, min
Physico-Chemical Characteristics of LipC
Fig. 2. A mass chromatogram based on the total ion cur-
rent of the fragment ions formed upon fragmentation of
the parent LipC ion with m/z 415.1 at a normalized colli-
sion energy of 34 eV.
Results of adsorption column chromatography in
the isopropanol−ammonia (3 : 1) system and TLC
analysis of fractions have shown that the synthesized
compound is eluted in fractions 40–82. In the samples
obtained from each fraction, the distribution of LipC,
LA, and carnosine was investigated by TLC. Accord-
ing to TLC, in the solvent system used, the retention
factor (Rf) was 0.52 for LipC, 0.61 for LA, and 0.17 for
carnosine.
Determination of the Antioxidant Activity of LipC,
Carnosine and LA
Formation of products reacting with thiobarbituric
acid (TBA-RP). The antioxidant activity of carnosine,
LA, and LipC was evaluated by the ability to prevent
the increase in TBA-RP under conditions of induced
ROS formation, using soy phosphatidylcholine (PCh)
as the oxidation substrate [26]. The formation of ROS
in the modeled lipid peroxidation system, was initiated
using the Fenton reaction:
For further investigation, fractions 40 to 82 were
pooled and portion dried on a rotary evaporator.
According to reversed-phase HPLC using a UV detec-
tor, the purity of the resulting final product (LipC) was
89.3% (Fig. 3, Table 1).
The structure of the obtained compound was con-
firmed by 1H-NMR and mass spectrometry.
Fe2+ + H2O2 = Fe3+ + OH− + iOH.
The final concentration of the investigated antiox-
idants—carnosine, LA, and LipC in the samples was
50 μM, 250 μM and 1000 μM.
The method of iron-induced chemiluminescence.
The antioxidant activity of the test compounds was
evaluated using the model of Fe2 +-induced oxidation
of the total fraction of low and very low density lipo-
proteins (LP) isolated from sera from healthy individ-
uals. Samples containing a suspension of LP without
addition of test compounds were used as a control.
The method is based on the measurement of che-
miluminescence (CL) parameters under conditions of
oxidation of the LP suspension by divalent iron ions.
The following kinetic parameters of CL were
recorded: a rapid CL burst (h, mV) reflecting the sta-
tionary level of lipid hydroperoxides; the duration of
the CL latent period (lag-period, τ, s) characterizing
the LP resistance to oxidation, which depends on their
endogenous antioxidant activity. The measurements
were carried out on the Luminometer LKB1251 che-
miluminometer (Sweden) [27].
1
Nuclear magnetic resonance. In the H-NMR
(D2O) spectrum, signals with chemical shifts (ppm)
were detected: H-NMR (D2O δ, ppm): 7.98 (1H, s,
1
NH–CH=N); 7.05 (1H, s, N–CH=C); 4.27 (1H, m,
CH–S); 3.43 (2H, m, CH2–NH); 3.06–2.91 (2H, m,
CH2–C=CH); 2.80 (4H, m, CH2–CH2S); 2.28
(2H, t, CH2–C=O (carnosine)); 1.99 (2H, t,
CH2C=O (LA)); 1.74; 1.43; 1.18 (6H, m, CH2 (LA
except CH2C=O)). The results obtained correspond
to the signals for the substance ADM_09 described
in [19].
Mass spectrometry analysis. Figure 4 shows the
mass spectrum of the synthesized LipC. The molecu-
lar mass of LipC is 414 g/mol, which corresponds to
the molecular peak of this compound with a mass-to-
charge ratio m/z 415 = [M + H]+ (protonated LipC).
Also the mass spectrum is characterized by the pres-
ence of the peak of the LipC sodium adduct with
m/z 437 = [M + Na]+ and a set of LipC fragments,
among which there are fragments with m/z values of
156, 209, 226, corresponding to histidine, DHLA, and
carnosine.
Determination of LipC Stability
LipC stability was assessed using HPLC chroma-
Thus, according to the physico-chemical charac-
tography-mass spectrometry (HPLC-MS) as follows: teristics studied, the compound LipC synthesized in
an aqueous solution of the substance (10 μg/mL) was this study completely corresponds to the substance
stored at 4°C; during the first week of storage the study ADM_09 [19].
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STVOLINSKY et al.
AU
2.62
1
3
5
4
6
2
210 nm
230 nm
250 nm
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 min
Fig. 3. Determination of the purity of the obtained LipC by the HPLC method.
415.07
100
90
80
70
60
50
40
30
20
10
437.07
156.00
259.93
226.93
188.87
109.87
254.00
459.07
160.80 209.87
0
100
150
200
250
300
350
400
450
500
m/z
Fig. 4. Mass spectrum of LipC (obtained by direct syringe injection and using electrospray ionization).
Study of the Antioxidant Activity of LipС
Formation of TBA-RP. Table 2 shows that LipC has
the strongest antioxidant effect; it decreased the TBA-
RP content in all studied concentrations versus con-
trol values: 50 μM by 72.1%, 250 μM by 74.4%, 1000
μM by 76.8%. At the same time, the increase of LipC
concentrations from 50 μM to 1000 μM insignifi-
cantly changed the TBA-RP content. The effective-
ness of the antioxidant effect of carnosine increased
with the increase in its concentration, while the anti-
A comparative evaluation of the effectiveness of the
antioxidant effect of LipC, LA, and carnosine was
performed using two complementary methods: the
formation of TBA-RP and informative kinetic param-
eters of iron-induced CL. The tested compounds were
used in the following range of concentrations: 50–
250–1000 μM.
Table 1. Evaluation of purity of the synthesized compound, LipC by means of HPLC: calculation results (the method of
calculation: normalization of response at 210 nm to the total peak areas)
Peak no.
Retention time, min
Peak area, AU s
Area, %
1
2
3
4
5
6
9.41
9.79
10.94
11.26
55.06
0.21
4.90
0.097
1.17
89.31
0.34
7.96
0.16
1.90
11.70
11.92
Total duration of chromatography
16.58
0.205
Total peak area
61.642
0.33
Normalization
100%
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LIPOYLCARNOSINE: SYNTHESIS, PROPERTIES, BIOLOGICAL ACTIVITY
313
Table 2. The effect of the compounds tested on TBA-RP products under conditions of induction of ROS formation
(nmol/mL)
Concentration
of compounds tested,
µM
TBA-RP concentration
carnosine, µM
LA, µM
LipC, µM
PCh (control)
20.42 1.02
50
250
1000
14.90 0.44*
9.29 0.48*
4.34 1.14*
8.84 0.44*
9.64 0.48*
22.72 1.13
5.69 0.28*
5.23 0.26*
4.74 0.24*
* p < 0.01 versus PCh.
Table 3. The effect of the compounds tested on the CL parameters: the level of rapid burst (h) and latent period duration (τ)
h, % of control
LA
τ, % of control
Concentration,
µM
carnosine
98
LipC
carnosine
LA
LipC
78 4.5*#
151 14*#+
189 24**#+
233 20**#++
50
250
2
86
3
105.5 5.5
131.5 1.5*
164.7 2.3**+
100
100
70 4*
68 1*
59 1*
64.5 2*
60.8 5*#
1000
44.5 1.5*
125 3*
#
+
Results are expressed as percent of control defined as 100%. * p < 0.05, ** p < 0.01 versus control; p < 0.05 versus carnosine; p <
++
0.05,
p < 0.01 versus LA.
oxidant effectiveness of LA decreased and even a small
prooxidant effect was observed at 1000 μM concentra-
tion: the TBA-RP content increased by 11.3%.
Iron-induced CL. Data on the effect of the tested
compounds on the level of the rapid burst of CL (h)
and on the latent period of CL (τ) are presented in
Table 3.
Table 3 shows that at a low concentration (50 μM)
LipC prevented the growth of lipid hydroperoxides
more effectively compared to carnosine (by 20.4%)
and LA (by 9.3%). At 250 μM their effectiveness was
comparable. At a higher concentration (1000 μM), the
LipC efficacy was comparable to that of LA, and was
lower by 26.8% compared to carnosine.
Carnosine increased the duration of the latent
period of CL versus control; starting at 250 μM, it
exhibited a concentration dependent effect (250 μM,
31.5%, 1000 μM, 64.7%). LA promoted an increase in
the duration of the latent period of CL at 1000 μM (by
25% compared to control), while its lower concentra-
tions were ineffective. LipC demonstrated the most
pronounced effect on the increase in this CL parame-
ter at all tested concentrations both versus carnosine
(50 μM, 44%, 250 μM, 44%, 1000 μM, 86%) and ver-
sus LA (50 μM, 51%, 250 μM, 89%, 1000 μM, 86%).
Thus, LipC, the new derivative of two natural anti-
oxidants, α-lipoic acid and L-carnosine, is character-
ized by high antioxidant activity, due to the ability to
prevent the growth of primary and secondary lipid
peroxidation products demonstrated using different
model approaches. The study of direct antioxidant
activity supplements the literature data on its superox-
ide-scavenging activity in the xanthine oxidase reac-
tion. It should be noted that Nativi et al. did not com-
pare the effectiveness of the antioxidant action of
LipC with carnosine, while compared to LA the effec-
tiveness of the antioxidant action was lower [20].
Determination of LipC Resistance to Serum Carnosinase
and Stability in Aqueous Solution
It is known that carnosine in the body undergoes
hydrolysis to β-alanine and L-histidine by blood
serum carnosinase. The recognition of the peptide by
the enzyme occurs via a non-proteinogenic amino
acid, β-alanine [28]. Figure 5 shows that due to β-ala-
nine modification, LipC demonstrates absolute resis-
tance to hydrolysis by carnosinase for 3 h, while car-
nosine during this time is almost completely hydro-
lyzed by the enzyme.
These results indicate that LipC exhibits higher
antioxidant activity at all studied concentrations, it
most effectively prevented the growth of TBA-RP in a
concentration-independent manner (its concentra-
tion in the sample), while its effect on the growth of
lipid hydroperoxides was already observed at a low
concentration (50 μM) compared to carnosine
and LA.
The obtained results indicate a high stability of
LipC to hydrolysis by serum carnosinase.
Investigation of the stability of LipC in aqueous
solution during prolonged storage showed that during
a month LipC in aqueous solution was not destroyed
at 4–6°C.
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150
150
100
50
1
100
50
0
2
3
0
0
50
100
150
200
Time, min
0
0.10 0.25 0.50 1.00 5.00
LipC concentration, mM
Fig. 5. Kinetics of LipC hydrolysis by carnosinase. 1—
LipC + carnosinase, 2—LipC without carnosinase, 3—
carnosine + carnosinase.
Fig. 6. The effect of LipC used in the concentration range
of 0.1–5 mM in the culture medium on the survival of SH-
SY5Y human neuroblastoma cells differentiated to the
dopaminergic phenotype, as determined by the MTT test.
The mean value in the wells with intact cells was defined as
100%.
The Effect of LipC on the Viability of Human
Neuroblastoma SH-SY5Y Cells, Differentiated
to the Dopaminergic Phenotype
The LipC toxicity assay was performed on human
inase. Results of the evaluation of the antioxidant
neuroblastoma SH-SY5Y cells, differentiated to the
dopaminergic phenotype, which were used to create a effect by the reaction with thiobarbituric acid and
cellular model of Parkinsonism. In the concentration
range from 0.1 mM to 5 mM, the cell viability assessed
by the MTT test was comparable to the control values
thus indicating the lack of LipC toxicity (Fig. 6). Ear-
lier it was also shown that LipC had no toxic effect on
the astrocyte culture [20].
informative parameters of iron-induced chemilumi-
nescence, have shown that LipC has a more pro-
nounced antioxidant effect, as compared to carnosine
and LA. Experiments with human SH-SY5Y neuro-
blastoma cells, differentiated to the dopaminergic
phenotype revealed the absence of cytotoxicity at con-
centrations up to 5 mM and demonstrated a neuropro-
The absence of toxicity made it possible to evaluate
the neuroprotective effect of LipC on the cell model of
Parkinsonism. In in vivo experiments Parkinsonism is
modeled using the neurotoxin 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP), which selectively
affects the neurons of the nigrostriatal pathway. Treat-
ment of cells with the oxidized form of MPTP,
1-methyl-4-phenylpyridinium (MPP+), is the cellular
analogue of this model. We evaluated the neuropro-
tective effect of LipC on the human neuroblastoma
culture differentiated to the dopaminergic phenotype
after MPP+ administration. Using the MTT test, it
was shown that incubation of cells with 2 mM MPP+
for 24 h resulted in a 22% decrease in the viability
compared to intact cells. LipC at concentrations of
0.1 mM and 0.25 mM exhibited a protective effect,
increasing the viability up to 100%; this effect can be
attributed to the ability of LipC to bind free radicals
formed under conditions of OS induced by MPP+
(Fig. 7).
150
*
*
100
50
0
CONCLUSIONS
The synthesized compound lipoylcarnosine
(LipC) is a conjugate of two natural antioxidants,
L-carnosine and α-lipoic acid (LA). It is character-
ized by high resistance to hydrolysis by serum carnos-
Fig. 7. The cytoprotective effect of LipC used at concen-
trations of 0.1 and 0.25 mM on the human neuroblastoma
cell culture under conditions of oxidative stress induced by
+
+
MPP ; * p < 0.05, versus cells treated with MPP .
BIOCHEMISTRY (MOSCOW), SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY Vol. 12 No. 4 2018

LIPOYLCARNOSINE: SYNTHESIS, PROPERTIES, BIOLOGICAL ACTIVITY
315
tective effect in the model of MPP+-induced cell
death modeling the damage of dopaminergic neurons
in Parkinsonism. Based on the results obtained in this
study it can be concluded that the demonstrated
advantages of LipC in comparison to carnosine and
LA open the prospect of further studies of its protec-
tive action on various experimental models of pathol-
ogies accompanied by the development of oxidative
stress.
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Ray & Terry’s Longevity Products © Copyright Ray and
Terry’s Health Products Inc. All Rights Reserved, 2011.
info@rayandterry.com.
COMPLIANCE WITH ETHICAL STANDARDS
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The authors declare that they have no conflict of
interest. This article does not contain any studies that
involve animals or human participants.
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Translated by A. Medvedev
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