с 01.01.2018 по 01.01.2020
Кемерово, Кемеровская область, Россия
Кемерово, Кемеровская область, Россия
Кемерово, Кемеровская область, Россия
Кемерово, Кемеровская область, Россия
Кемерово, Кемеровская область, Россия
Москва, г. Москва и Московская область, Россия
Транс-коричная кислота является фенольным соединением с широким спектром биологической активности, включая антиоксидантную и антибактериальную способность. Обладает высоким потенциалом для применения в пищевой и косметической промышленности. Целью работы являлось выделение транс-коричной кислоты из шлемника байкальского (Scutellaria baicalensis) и исследование ее геропротекторной направленности на модели нематоды Caenorhabditis elegans. Объектами исследования являлись корневая культура S. baicalensis и ее экстракт, транс-коричная кислота, полученная из экстракта корневой культуры S. baicalensis, и штамм C. elegans. Выделение транс-коричной кислоты проводили методом высокоэффективной жидкостной хроматографии. Исследование геропротекторной активности транс-коричной кислоты осуществляли путем оценки влияния на продолжительность жизни, стрессоустойчивость и репродуктивные способности нематод C. elegans при концентрациях 10, 50, 100 и 200 мкмоль/л. Продолжительность жизни нематоды изучали в процессе культивирования при 20 °С в течение 61 дня. Для оценки стрессоустойчивости при окислительном стрессе в каждую ячейку планшета добавляли 15 мкл 1М параквата, при температурном стрессе – повышали температуру до 33 °С. Репродуктивные способности оценивали с использованием нематод, которые культивировали в S-среде с добавлением Escherichia coli OP50 и транс-коричной кислоты в необходимой концентрации в течение 72 ч. Максимальная продолжительность жизни наблюдалась у нематод, обработанных 50 мкмоль/л транс-коричной кислоты (9,8 %). При окислительном стрессе транс-коричная кислота приводила к увеличению выживаемости во всем диапазоне испытуемых концентраций, а под действием температуры снижала процент выживших нематод. При концентрации транс-коричной кислоты 100 мкмоль/л омечено увеличение репродуктивности в 1,48 раз. Основываясь на полученных данных, транс-коричную кислоту из S. baicalensis можно рекомендовать как биологически активное соединение с геропротекторной активностью. Однако необходимы дополнительные исследования на других модельных организмах с подробными исследованиями токсичности.
Транс-коричная кислота, корневая культура, Scutellaria baicalensis, Caenorhabditis elegans, геропротектор, продолжительность жизни, стрессоустойчивость, репродуктивность
Introduction
Life expectancy in the first-world countries has
significantly increased due to proper hygiene and
nutrition. According to the World Health Organization,
there are 125 million people aged 80+ in the world [1].
This explains a growing need for developing drugs to
prevent age-related diseases.
Old age is a major risk factor for common chronic,
neurodegenerative, and oncological diseases such as
cancer, cardiovascular diseases, multiple sclerosis,
Parkinson’s disease, etc. [2]. Aging is often accompanied
by genomic damage, mitochondrial dysfunction, telomere
shortening, epigenetic changes, proteostasis dysregulation,
disruption of intercellular communication, and other
processes. Cellular aging is defined as a response to stress
that gives cells an irreversible proliferative capacity,
thus causing the body to age [3].
Creating anti-aging drugs is a long process with
many variables and it is difficult to assess their effect
in clinical trials [4, 5]. Metformin is one of the drugs
with an anti-aging effect that is used to treat diabetes
mellitus [6]. However, we should distinguish between
drugs that are aimed at reversing the aging process and
geroprotective drugs that can prevent premature aging
and increase life expectancy [2, 7, 8].
Polyphenols, whose molecules contain one or
more phenolic hydroxyl groups, exhibit antioxidant,
antitumorous, cardioprotective, anticancerous, and
antimicrobial properties [9]. There is growing evidence
that phenolic acids, especially hydroxycinnamic acids,
have an effect on the regulation of lipid metabolism. For
example, caffeic, ferulic, and coumaric acids significantly
reduce hepatic lipids in rats with high cholesterol [10].
Trans-cinnamic acid (3-phenylpropenoic acid) is
the main phenolic compound in plants [11]. Many
studies have reported its geroprotective activity due to
antibacterial, antidiabetic, anticancerous, and antiaging
properties [12, 13].
Many medicinal plants growing in the Siberian
Federal Okrug are sources of geroprotective compounds
[14]. For example, the Baikal skullcap (Scutellaria
baicalensis) contains flavonoids (quercetin, rutin,
catechin, luteonin, etc.), phenolic acids (caffeic, ferulic,
p-coumaric, p-hydroxybenzoic, and cinnamic), vitamins,
carotenoids, and terpenes [15–19]. In order to preserve
the diversity of its species, trans-cinnamic acid should
be isolated from this plant’s cell cultures in vitro [20].
Previous studies have found a significant amount of
trans-cinnamic acid in the in vitro root culture extract
of S. baicalensis [21].
A number of model organisms are used to study the
effect of bioactive substances of plant origin on the
aging process. They include nematodes (Caenorhabditis
elegans), fruit flies (Drosophila melanogaster),
yeasts (Saccharomyces cerevisiae), short-lived fish
(Nothobranchius furzeri), and rodents (mice and rats) [1].
In this study, we used nematodes C. elegans as a
preclinical experimental model mainly due to their short
lifespan under normal growth conditions [22]. This
feature of nematodes allows scientists to study processes
that affect aging and life expectancy. In addition, using
C. elegans is cost-effective since they feed on inexpensive
microorganisms such as Escherichia coli bacteria.
Also, growing worms can be fully automated using a
flow cytometry apparatus, where they are distributed
in analytical plates, as well as robots that place the
experimental samples in the wells [23]. Nematodes
have a transparent body and do not need to be stained
at all growth stages, so their internal organs are easily
visible under a microscope. At the subcellular and
tissue levels, fluorescent label reporters are used to
study the distribution of expressing genes and their
protein products. Powerful phase-contrast microscopes
enable scientists to observe the division and death of
individual worm cells [24, 25].
C. elegans has a lifespan of only three weeks, which
makes it a convenient model to use. With sufficient
food, optimal temperatures, and population control,
nematodes can reach the adult growth stage in three
days. Their embryogenesis is faster compared to other
model organisms. At 20–25°C, the development of each
cell can be traced in just 10–12 h. After embryonic
development, nematode larvae go through several stages
(L1–L4) before becoming adults [26]. The studies into
the lifespan of nematodes are usually carried out in
Petri dishes using liquid and solid nutrient media [27].
Thus, C. elegans can be used as model organisms to
study the geroprotective properties of various bioactive
substances.
In this study, we aimed to isolate trans-cinnamic
acid from the Baikal skullcap (S. baicalensis) and study
its geroprotective activity in the C. elegans nematode.
Study objects and methods
Our study objects were:
– in vitro root culture of the Baikal skullcap (Scutellaria
baicalensis);
– in vitro root culture extract of the Baikal skullcap
(S. baicalensis);
– trans-cinnamic acid obtained from the in vitro root
culture extract of the Baikal skullcap (S. baicalensis); and
– soil nematodes Caenorhabditis elegans (strain N2
Bristol).
Germinated sterile seeds of S. baicalensis were used
to obtain the root culture (Botanical Garden of the
Immanuel Kant Baltic Federal University, Kaliningrad).
The seeds were sterilized in several stages: they were
washed with detergent, placed in 95% ethanol for 30 s,
and transferred to a 6% NaOCl solution for 30 min.
After sterilization, the seeds were rinsed with sterile
distilled water and then washed three times with it
for 20 min. The seedlings grew for 14–28 days on a
nutrient medium containing 50.00 mg of B5 macrosalts,
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Федорова А. М. [и др.] Техника и технология пищевых производств. 2022. Т. 52. № 3. С. 582–591
10.00 mg of B5 microsalts, 5.00 mL of Fe-EDTA,
10.00 mg of thiamine, 1.00 mg of pyridoxine, 1.00 mg
of nicotinic acid, 30.00 g of sucrose, 100.00 mg of
inositol, 0.05 mg of 6-benzylaminopurine, 1.00 mg of
indoleacetic acid, and 20.00 g of agar. The seedlings were
transformed with the soil agrobacteria Agrobacterium
rhizogenes 15834 Swiss (Moscow, Russia). The in
vitro root cultures of S. baicalensis were cultivated for
5 weeks. They were first grown in 100 mL flasks (40
mL of medium) and then transplanted into 300 mL
flasks (100 mL of medium). The initial weight of the
root culture ranged from 0.5 to 1.0 g [21].
The extract of the S. baicalensis root culture was
obtained by water-alcohol extraction. For this, the dried
and crushed plant roots were treated with 30.0 ± 0.2%
ethyl alcohol (1:86) at 70.0 ± 0.1°C for 6.0 ± 0.1 h. The
extraction was performed in an EKROS PE-4310 water
bath (Ekroskhim, Russia) with a reflux condenser [28].
Then, trans-cinnamic acid was isolated from the
obtained water-alcohol extract of the S. baicalensis
root culture by high-performance liquid chromatography
(HPLC) on a liquid chromatograph (Shimadzu
LC-20 Prominence, Japan). The process of isolation
and purification consisted of several stages, namely:
1. The extract of the S. baicalensis root culture was
evaporated under vacuum at 50°C max;
2. Diethyl ether was added to the evaporated residue
in three repetitions;
3. The ether fraction obtained was chromatographed
on PF silica gel in an n-hexane-acetone gradient
(1:0 → 0:1) to isolate hydroxycinnamic acids; and
4. Trans-cinnamic acid was isolated by subsequent
rechromatography on PF silica gel in n-hexanechloroform
(1:0 → 0:1).
The trans-cinnamic acid isolated from the
S. baicalensis root culture extract was at least 95% pure.
Infra-red (IR) spectroscopy was performed to analyze
the chemical composition of trans-cinnamic acid on an
FSM-1202 apparatus (Infraspek, St. Petersburg, Russia).
IR spectra were recorded in potassium bromide disks
(Fluka, Germany) in the range of 4000–400 cm–1 with
a resolution of 4 cm–1 and the number of scans being 30.
Air was used as a reference sample and it was recorded
before the analysis of each sample. The Fspec (4.0.0.2)
and Aspec (1.1) software was used to control the apparatus
and process spectral data.
Next, we assessed the effect of trans-cinnamic
acid on the lifespan, stress (oxidative and thermal)
resistance, and reproductive abilities of C. elegans
nematodes. The N2 Bristol strain was provided by the
Laboratory for the Development of Innovative Medicines
and Agrobiotechnologies (Moscow Institute of Physics
and Technology, Dolgoprudny, Russia). The nematodes
were fed on Escherichia coli OP50 provided by the
V.A. Engelhardt Institute of Molecular Biology (Moscow,
Russia). We used a total of 100 nematodes for all the
stages of the study. The control nematodes were not
treated with solutions of trans-cinnamic acid. However,
they were used in the lifespan and reproductivity tests
and were subjected to oxidative and thermal stress.
To assess the effect of trans-cinnamic acid on
nematodes, we used a stock solution of this acid in
10 mmol/L of dimethyl sulfoxide. Then, the acid was
titrated by diluting stock solutions in sterile distilled
water to concentrations of 2000, 1000, 500, and 100 μM.
Each well with nematodes was filled with 15 μL of
freshly prepared stock solutions, thus obtaining working
concentrations of trans-cinnamic acid of 2000, 1000,
500, and 100 μmol/L, respectively. The stocks were
stored at 4°C.
At the first stage, nematodes were cultivated on
solid agar. For this, a daily culture of E. coli OP50 was
obtained by inoculating one bacterial colony, which was
previously grown on L-broth (15 g bacteriological agar,
10 g tryptone, 5 g yeast extract, 5 g NaCl, 1000 mL
distilled water), in 5–10 mL of L-broth (10 g tryptone,
5 g yeast extract, 10 g NaCl, 800 mL distilled water).
The bacteria were cultivated at 37°C for 24 h with
intensive stirring. After the incubation of E. coli OP50,
50 μL of the overnight culture was inoculated into
Petri dishes with an NGM medium (3 g NaCl, 17 g
bacteriological agar, 2.5 g peptone, 975 mL distilled
water) and incubated at 37°C for 24 h. Then, an NGM
medium was prepared for cultivating nematodes. For
this, the autoclaved NGM agar medium was cooled to
55°C in an EKROS PE-4310 water bath (Ekroskhim,
Russia) for 15 min. Then, 1 mL of 1 M CaCl2, 1 mL of
5 mg/mL cholesterol in alcohol, 1 mL of 1 M MgSO4,
and 25 mL of 1 M KPO4 buffer were added to the cooled
agar. The nematodes were transferred to new NGM agar
dishes in two ways: by a loop and by a piece of agar.
The first method involved hooking a nematode with a
calcined and cooled bacteriological loop and planting
it on a bacterial lawn in the center of a new Petri dish
with NGM agar. In the second method, a 5×0.5 cm
piece of agar containing a nematode was cut out with
a sterile scalpel from an NGM dish and transferred to
the center of the new dish surface down. The dishes
were incubated at 20°C.
At the second stage, the nematodes were synchronized.
For this, 5–10 mL of sterile water was added to the
Petri dish with a nematode and pipetted until its eggs
were completely attached to the agar. The liquid from
the dish was placed in a 50 mL centrifuge tube and
centrifuged for 2 min (1200 rpm). Then, the supernatant
was removed and the precipitate was washed with 10
mL of distilled water and centrifuged as described
above. After repeated centrifugation, the supernatant
was removed, and 5 mL of a freshly prepared mixture
of 1 mL of 10 N NaOH, 2.5 mL of household bleach,
and 6.5 mL of H2O was added to the precipitate. The
mixture was thoroughly mixed on a vortex (Biosan,
Latvia) for 10 min with a break every 2 min to observe
the hydrolysis of nematodes under an Axio Observer Z1
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Fedorova A.M. et al. Food Processing: Techniques and Technology. 2022;52(3):582–591
microscope (Karl Zeiss, Germany). After that, 5 mL of
M9 medium was added to neutralize the reaction. The
resulting mixture was centrifuged for 2 min (2500 rpm).
The supernatant was removed, and 10 mL of sterile
water was added to the precipitate, with washing and
centrifugation repeated 3 times. In the fourth repetition,
the precipitate was washed with 10 mL of S-medium
and the test tube with nematode eggs was placed on a
slow shaker for a day at room temperature so that the
nematodes could enter the L1 stage.
When the nematodes reached the L1 stage, an
overnight bacterial culture of E. coli OP50 was added
to the S-medium. The culture had been previously washed
from the L-broth and resuspended in the S-medium to
a concentration of 0.5 mg/mL. Then, 120 μL of the
suspension containing bacteria and nematodes was added
to each well of a 96-well plate (TPP, Switzerland). The
plate was sealed with a film and left for 48 h at 20°C.
After that, 15 μL of 1.2 mM 5-fluoro-2-deoxyuredin
(FUDR) was poured into each well and left for a day at
20°C to prevent the nematodes from reproduction. At
the end of incubation, the worms entered the L4 stage.
Then, 15 μL of a solution with trans-cinnamic acid in
different concentrations was added to the wells and the
plates were cultivated at 20°C on day 5.
Next, we analyzed the effects of trans-cinnamic acid
on the lifespan of C. elegans nematodes, their resistance
to oxidative and thermal stress, as well as reproductive
ability.
To assess the effect of trans-cinnamic acid on the
lifespan of C. elegans, we used the acid at concentrations
of 0 (control), 10, 50, 100, 200 μmol/L. The experiment
was carried out in 96-well plates in the liquid S-medium
for the cultivation of nematodes in 6 repetitions. The
numbers of live and dead nematodes were counted
every 4–7 days during 61 days of the experiment. The
experiment was considered completed when all the
control nematodes died.
To determine the resistance of C. elegans to oxidative
stress, we added 15 μL of 1 M paraquat to each well
and continued incubation in the thermostat at 20°C.
The numbers of live and dead nematodes were counted
twice: after 24 and 48 h of incubation.
The resistance of C. elegans to thermal stress was
studied by increasing the temperature to 33°C. Live
and dead nematodes were counted after 24 and 48 h
of incubation.
The effect of trans-cinnamic acid on the reproductive
ability of C. elegans was analyzed as follows. The
synchronized nematodes at stage L1 in the S-medium
with E. coli OP50 were placed in 48-well 270 μL
plates and 30 μL of trans-cinnamic acid at the required
concentration was immediately added to them. Thus,
L1 larvae developed to the sexually mature stage of
L4 in the presence of trans-cinnamic acid throughout
the experiment for 72 h. Trans-cinnamic acid at each
concentration was added in triplicate. When the
nematodes reached stage L4, each well was filled
with 300 μL of a lysis solution prepared in a 2-fold
concentration and containing 2 mL of 10 M NaOH
and 5.0 mL of bleach in 3.0 mL ddH2O. The plate was
covered with an adhesive film, placed in a MaxMate
plate shaker (USA), and vortexed for 5 min at 1800 rpm.
Then, the wells were filled with repeat nematodes for
each test condition of each sample. The lysed nematodes
were transferred into a 2 mL Eppendorf-type tube and
centrifuged for 2 min at 1100 g at room temperature in
an Eppendorf 5424 centrifuge (Eppendorf, USA). The
supernatants were collected and transferred into a new
Eppendorf tube, with 1 mL of M9 buffer added to the
egg precipitate. The mixture was vigorously stirred and
centrifuged for 2 min at 1100 g at room temperature.
Then the procedure was repeated. Namely, the M9
washing medium was transferred into a new Eppendorf
tube, and 1 mL of distilled water was added to the egg
precipitate. The mixture was intensively stirred and
used later to count the number of eggs formed in the
L4 stage nematodes under the action of trans-cinnamic
acid. For the count, 100 μL was taken from 1 mL of the
Figure 1. Trans-cinnamic acid obtained from the water-alcohol extract of the Scutellaria baicalensis root culture in vitro:
a) structure; b) IR-spectrum
Рисунок 1. Транс-коричная кислота, полученная из водно-спиртово го экстракта корневой культуры in vitro Scutellaria baicalensis:
a) структура; b) ИК-спектр
a b
3500 3000 2500 2000 1500 1000 500
20
40
60
80
100
0
Wavelength, cm-1
Transmission, %
O
OH
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Федорова А. М. [и др.] Техника и технология пищевых производств. 2022. Т. 52. № 3. С. 582–591
aqueous suspension of eggs previously obtained after
the nematode lysis stage and transferred to a 96-well
plate. The samples were placed in duplicate for each
concentration of trans-cinnamic acid. The count
was performed on an Axio Observer Z1 microscope
(Karl Zeiss, Germany). If the well contained more
than 100 eggs, they were additionally diluted and
recounted.
Results and discussion
The results of IR spectroscopy of trans-cinnamic
acid obtained from the in vitro root culture extract of
Scutellaria baicalensis are shown in Fig. 1 and Table 1.
As we can see in the IR spectrum of trans-cinnamic
acid (phenylpropenoic acid) isolated from S. baicalensis,
the 3064 cm–1 band is due to the stretching vibrations
of the acid’s diene fragment =C-H, while the 3026 cm–1
band is determined by the C-H stretching vibrations
in the benzene ring. These bands can be considered as
characteristic for trans-cinnamic acid.
In this case, the 1680 cm–1 band can also be considered
as the C=C of the diene fragment. The 1631 cm–1 band is
due to the stretching vibrations of the carboxyl fragment.
The 1576, 1451, 1420, and 1176 cm–1 bands correlate
with the stretching vibrations of the aromatic fragment’s
С-Н bonds. The absorption band at 1451 cm–1 is due to
the deformation vibrations of the carboxyl fragment’s
C-O-H. The bands at 1332, 1313, and 1221 cm–1 result
from the О-Н deformation vibrations and С-О stretching
vibrations, including those of the carboxyl fragment.
The 1285 cm–1 band is associated with the stretching
vibrations of the C-O bond.
The band at 979 cm–1 correlates with the diene
fragment in the trans-form. The monosubstituted ring
Table 1. Vibrational frequencies and their correlation with the structural fragments of trans-cinnamic acid obtained from the wateralcohol
extract of the Scutellaria baicalensis root culture in vitro
Таблица 1. Характеристичные колебательные частоты и их соотнесение с основными структурными фрагментами образца транс-коричной
кислоты, полученной из водно-спиртового экстракта корневой культуры in vitro Scutellaria baicalensis
Reference Wavelength, cm–1 Type of vibrations/bonds of structural fragments
O
OH
O
OH
O
OH
O
OH
O
OH
O
OH
O
OH
3064 Stretching vibrations of the diene fragment = С-Н
O
OH
O
OH
O
OH
O
OH
O
OH
O
OH
O
OH
3026 Stretching vibrations of the С-Н bond
O
OH
O
OH
O
OH
O
OH
O
OH
O
OH
O
OH
1680 С=С of the diene fragment
O
OH
O
OH
O
OH
O
OH
1631 Stretching vibrations C=O of the carboxyl group
O
OH
O
OH
O
OH
O
OH
O
OH
O
OH
O
OH
1576, 1451, 1420, 1176 Planar stretching vibrations of the С-С bonds of aromatic
fragments
O
OH
O
OH
O
OH
O
OH
O
OH
O
OH
O
OH
1332, 1313, 1221 Planar deformation vibrations of the О-Н bond
O
OH
O
OH
O
OH
O
OH
O
OH
O
OH
O
OH
1285 Stretching vibrations of the C-O bond
O
OH
O
OH
O
OH
O
OH
O
OH
979 Planar deformation vibrations of the ring hydroxyl groups
O
OH
O
OH
O
OH
O
OH
766 Out-of-plane deformation vibration of the C-C bond
588
Fedorova A.M. et al. Food Processing: Techniques and Technology. 2022;52(3):582–591
is characterized by an out-of-planedeformation vibration
of the C-C bond at 766 and 711 cm–1.
Thus, the spectral activity corresponded to the
structural features of trans-cinnamic acid.
Figure 2 shows the effect of trans-cinnamic acid
(0 (control),10, 50, 100, and 200 μmol/L) isolated from
the water-alcohol extract of the S. baicalensis in vitro
root culture on the lifespan of Caenorhabditis
elegans nematodes.
As we can see in Fig. 2, on day 8 of the experiment,
trans-cinnamic acid at all the concentrations under study
(10, 50, 100, and 200 μmol/L) increased the lifespan of
worms (by 18.1, 26.3, 24.1, and 36.6%, respectively).
From day 13 to 34, 200 μmol/L of trans-cinnamic
acid did not have a positive effect on the lifespan of
nematodes, unlike the other concentrations. From day
34 to 61, all the concentrations increased the percentage
of surviving nematodes. The highest increase in lifespan
(9.8%) was observed in the nematodes treated with
50 μmol/L of trans-cinnamic acid.
Figures 3 and 4 show the effect of trans-cinnamic
acid isolated from the water-alcohol extract of the
S. baicalensis in vitro root culture on stress resistance.
As can be seen in Fig. 3, all the concentrations of
trans-cinnamic acid (10–200 μmol/L) had a positive
effect on the resistance of nematodes to oxidative stress
during 24 and 48 h, i.e. increased their survival, compared
to the control. We also found a gradual decrease in
the percentage of surviving nematodes during 24-h
oxidative stress with increased concentrations of transcinnamic
acid.
After 5 days of nematode incubation in the presence
of trans-cinnamic acid, the experimental plate was
transferred to a 33°C incubator. Dead worms were
counted after 24 h of nematode incubation at elevated
temperature and after 48 h of incubation under prolonged
thermal stress.
According to Fig. 4, trans-cinnamic acid significantly
reduced the percentage of surviving nematodes under
Figure 2. The effect of trans-cinnamic acid isolated from
the water-alcohol extract of the Scutellaria baicalensis
in vitro root culture on the lifespan
of Caenorhabditis elegans nematodes
Рисунок 2. Влияние транс-коричной кислоты, выделенной
из водно-спиртового экстракта корневой культуры in vitro
Scutellaria baicalensis, на продолжительность жизни нематод
Caenorhabditis elegans
0
20
40
60
80
100
120
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
Control 10 μmol/L 50 μmol/L
100 μmol/L 200 μmol/L
85.9
90.7
86.8
94.3
92.9
70.7
69.7
73.4
81.8
80.7
Survival, %
Control 1 0 μ m o l / L 5 0 μ m ol/L 100 μmol/L 200 μmol/L
Cultivation time, h
24 h 48 h
85.9
90.7
86.8
94.3
92.9
70.7
69.7
73.4
81.8
80.7
Survival, %
Control 1 0 μ m o l / L 5 0 μ m ol/L 100 μmol/L 200 μmol/L
Cultivation time, h
24 h 48 h
Figure 3. The effect of trans-cinnamic acid isolated from
the water-alcohol extract of the Scutellaria baicalensis
in vitro root culture on the resistance of nematodes
to oxidative stress
Figure 3. Рисунок 3. Влияние транс-коричной кислоты,
выделенной из водно-спиртового экстракта корневой
культуры in vitro Scutellaria baicalensis, на устойчивость
нематод при окислительном стрессе
96.3
95.1
96.0
94.9
93.9
80.6
74.6
78.9
71.8
65.2
Control 10 μmol/L 50 μmol/L 100μmol/L 200μmol/L
Survival, %
Cultivation time, h
24 ч 48 ч
85.9
90.7
86.8
94.3
92.9
70.7
69.7
73.4
81.8
80.7
Survival, %
Control 1 0 μ m o l / L 5 0 μ m ol/L 100 μmol/L 200 μmol/L
Cultivation time, h
24 h 48 h
Figure 4. The effect of trans-cinnamic acid isolated from
the water-alcohol extract of the Scutellaria baicalensis
in vitro root culture on the resistance of nematodes
to thermal stress
Рисунок 4. Влияние транс-коричной кислоты, выделенной
из водно-спиртового экстракта корневой культуры in
vitro Scutellaria baicalensis, на устойчивость нематод к
температурному стрессу
589
Федорова А. М. [и др.] Техника и технология пищевых производств. 2022. Т. 52. № 3. С. 582–591
thermal stress with increased concentrations from
10 to 200 μmol/L.
Figure 5 shows the effect of trans-cinnamic
acid isolated from the water-alcohol extract of the
S. baicalensis in vitro root culture on the reproductive
abilities of C. elegans nematodes.
As we can see in Fig. 5, trans-cinnamic acid at
the concentrations of 10, 50, and 200 μmol/L did not
significantly affect the reproductive performance of
nematodes, compared to the control. The concentration
of 100 μmol/L was the most effective since it produced
1.48 times more eggs, compared to the control.
Conclusion
Modern medical gerontology is looking for ways
to increase life expectancy with a focus on geroprotectors
– special compounds that reduce the rate of
aging. Plants are the main source of geroprotectors,
including Scutellaria baicalensis. Since this is a rare
plant included in the Russian Red Data Book, we used
its root culture as a source of trans-cinnamic acid.
In particular, we isolated trans-cinnamic acid from
the water-alcohol extract of the S. baicalensis in vitro
root culture b HPLC. The isolated bioactive compound
was at least 95% pure. According to IR spectroscopy, the
spectral activity corresponded to the structural features of
trans-cinnamic acid.
To study the geroprotective activity of trans-cinnamic
acid, we evaluated its effect in various concentrations on
the lifespan, oxidative and thermal stress resistance, as
well as reproductivity of Caenorhabditis elegans used
as a model organism. As a result, we drew the following
conclusions:
– all the studied concentrations of trans-cinnamic acid
increased the lifespan of C. elegans worms, with the
highest increase achieved by 50 μmol/L;
– all the concentrations of trans-cinnamic acid (10–
200 μmol/L) had a positive effect on the resistance of
nematodes to oxidative stress increasing their survival,
compared to the control;
– under thermal stress, increased concentrations of transcinnamic
acid significantly reduced the percentage of
surviving nematodes; and
– trans-cinnamic acid at a concentration of 100 μmol/L
increased the reproductive capacity of nematodes,
producing 1.48 times more eggs, compared to the
control. The remaining concentrations did not have
such an effect.
Based on our data, trans-cinnamic acid can be used
as a bioactive substance with geroprotective properties.
However, further research is needed on other model
organisms with detailed toxicity studies to determine
the full potential of trans-cinnamic acid as an anti-aging
agent capable of slowing down the aging process and
extending life.
Contribution
All the authors equally contributed to the study
concept, data processing and analysis, as well as writing
the manuscript.
Conflict of interest
The authors declare that there is no conflict of interest
regarding the publication of this article.
Критерии авторства
Все авторы внесли равный вклад в создание
исследования, обработку и анализ полученных
результатов, а также в оформлении статьи.
Конфликт интересов
Авторы заявляют об отсутствии конфликта
интересов.
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