Аннотация (русский):
Medicinal plants are sources of natural antioxidants. Acting as reducing agents, these substances protect the human body against oxidative stress and slow down the aging process. We aimed to study the effects of bioactive substances isolated from medicinal plants on the lifespan of Caenorhabditis elegans L. used as a model organism. High-performance liquid chromatography was applied to isolate bioactive substances from the extracts of callus, suspension, and root cultures of meadowsweet (Filipendula ulmaria L.), ginkgo (Ginkgo biloba L.), Baikal skullcap (Scutellaria baicalensis L.), red clover (Trifolium pretense L.), alfalfa (Medicágo sativa L.), and thyme (Thymus vulgaris L.). Their effect on the lifespan of C. elegans nematodes was determined by counting live nematodes treated with their concentrations of 10, 50, 100, and 200 µmol/L after 61 days of the experiment. The results were recorded using IR spectrometry. The isolated bioactive substances were at least 95% pure. We found that the studied concentrations of trans-cinnamic acid, baicalin, rutin, ursolic acid, and magniferin did not significantly increase the lifespan of the nematodes. Naringenin increased their lifespan by an average of 27.3% during days 8–26. Chlorogenic acid at a concentration of 100 µmol/L increased the lifespan of C. elegans by 27.7%. Ginkgo-based kaempferol and quercetin, as well as red clover-based biochanin A at the concentrations of 200, 10, and 100 µmol/L, respectively, increased the lifespan of the nematodes by 30.6, 41.9, and 45.2%, respectively. The bioactive substances produced from callus, root, and suspension cultures of the above medicinal plants had a positive effect on the lifespan of C. elegans nematodes. This confirms their geroprotective properties and allows them to be used as anti-aging agents.

Ключевые слова:
Plants, antioxidants, callus culture, suspension culture, root culture, nematodes, aging

INTRODUCTION
According to the WHO, average life expectancy is
steadily increasing worldwide [1]. Over the last 20 years,
it has grown by 6 years as a result of advances in
science and medicine. However, behind these advances
is an increase in diseases associated with aging, which
has become a serious problem of public health in the
21st century. Aging is a process that affects the entire
human body, in particular its cardiovascular, nervous,
digestive, and immune systems.
The aging process is directly related to oxidative
stress. Age-related diseases cause structural changes
in mitochondria, as well as changes in the functions
of the electron transport chain, which ultimately leads
to oxidative stress. The cardiovascular system is
particularly susceptible to this effect, which explains the
increase in cardiovascular diseases in the elderly [2–5].
Copyright © 2022, Faskhutdinova et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International
License (https://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to
remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.
Research Article Available online at http://jfrm.ru/en
Open Access https://doi.org/10.21603/2308-4057-2022-2-544
https://elibrary.ru/ZVCUUW
Effects of bioactive substances isolated from Siberian
medicinal plants on the lifespan of Caenorhabditis elegans
Elizaveta R. Faskhutdinova1,* , Andrey S. Sukhikh1 , Violeta M. Le1 ,
Varvara I. Minina1 , Mohammed El Amine Khelef2 , Anna I. Loseva1
1 Kemerovo State University , Kemerovo, Russia
2 Moscow State University of Food Production , Moscow, Russia
* e-mail: faskhutdinovae.98@mail.ru
Received 10.02.2022; Revised 10.03.2022; Accepted 05.04.2022; Published online X.X.2022
Abstract:
Medicinal plants are sources of natural antioxidants. Acting as reducing agents, these substances protect the human body against
oxidative stress and slow down the aging process. We aimed to study the effects of bioactive substances isolated from medicinal
plants on the lifespan of Caenorhabditis elegans L. used as a model organism.
High-performance liquid chromatography was applied to isolate bioactive substances from the extracts of callus, suspension, and
root cultures of meadowsweet (Filipendula ulmaria L.), ginkgo (Ginkgo biloba L.), Baikal skullcap (Scutellaria baicalensis L.),
red clover (Trifolium pretense L.), alfalfa (Medicágo sativa L.), and thyme (Thymus vulgaris L.). Their effect on the lifespan of
C. elegans nematodes was determined by counting live nematodes treated with their concentrations of 10, 50, 100, and
200 μmol/L after 61 days of the experiment. The results were recorded using IR spectrometry.
The isolated bioactive substances were at least 95% pure. We found that the studied concentrations of trans-cinnamic acid,
baicalin, rutin, ursolic acid, and magniferin did not significantly increase the lifespan of the nematodes. Naringenin increased
their lifespan by an average of 27.3% during days 8–26. Chlorogenic acid at a concentration of 100 μmol/L increased the lifespan
of C. elegans by 27.7%. Ginkgo-based kaempferol and quercetin, as well as red clover-based biochanin A at the concentrations of
200, 10, and 100 μmol/L, respectively, increased the lifespan of the nematodes by 30.6, 41.9, and 45.2%, respectively.
The bioactive substances produced from callus, root, and suspension cultures of the above medicinal plants had a positive effect
on the lifespan of C. elegans nematodes. This confirms their geroprotective properties and allows them to be used as anti-aging
agents.
Keywords: Plants, antioxidants, callus culture, suspension culture, root culture, nematodes, aging
Funding: The study was financed by the Ministry of Science and Higher Education of the Russian Federation (Minobrnauka)
(project FZSR-2020-0006 “Screening bioactive plant-based substances with geroprotective properties and developing technology
for producing anti-aging nutraceuticals”).
Please cite this article in press as: Faskhutdinova ER, Sukhikh AS, Le VM, Minina VI, Khelef MEA, Loseva AI. Effects
of bioactive substances isolated from Siberian medicinal plants on the lifespan of Caenorhabditis elegans. Foods and Raw
Materials. 2022;10(2):340–352. https://doi.org/10.21603/2308-4057-2022-2-534
Foods and Raw Materials. 2022;10(2)
ISSN 2310-9599 (Print)
ISSN 2308-4057 (Online)
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Faskhutdinova E.R. et al. Foods and Raw Materials. 2022;10(2):340–352
The problem of aging is widely covered by the
free-radical theory of aging developed by Denham
Harman in the 1950s [6]. According to this theory,
the body’s defense mechanisms stop responding to
damage caused by reactive oxygen species, resulting
in the deterioration of cellular homeostasis, energy
imbalance, and mitochondrial insufficiency [7].
The molecules of reactive oxygen species include
nitric oxide, hydrogen peroxide, monoxide radicals,
superoxide anions, tocopherols, ascorbic acid, and
polyphenols [8]. Reactive oxygen species alter cellular
activities such as cell survival, stress responses, and
inflammation. They are also involved in muscle
contractions, regulate vascular tone, as well as
determine bactericidal and bacteriostatic activity [9].
However, their increase leads to oxidative stress,
disrupting the balance of antioxidants and prooxidants
[10]. This causes damage to macromolecules
(lipids, DNA, and proteins) and subsequently to whole
cells, tissues, and organs [4]. Higher concentrations
of reactive oxygen species in the body promote
inflammation, which, in turn, can accelerate the
formation of their molecules [11]. Therefore, it is very
important to maintain a balance between antioxidants
and pro-oxidants.
Antioxidants can react with free radicals and
neutralize them by causing them to decay. The human
body has three lines of defense against oxidative
stress. The first line consists of body enzymes such as
superoxide dismutase, catalase, glutathione peroxidase,
glutathione S-transferase, and glutathione reductase [12].
They prevent cell damage by scavenging free radicals
that cause premature aging and age-related disorders.
The first line also includes non-enzymatic molecules
in the blood plasma such as transferrin, ferritin,
ceruloplasmin, and albumin [13]. These preventative
antioxidants inhibit the formation of new reactive
oxygen species by binding transition metal ions (for
example, copper and iron). The second line of defense is
represented by non-enzymatic antioxidants that provide
intermediate protection against oxidative radicals. The
third line of defense serves to regenerate biomolecules
damaged by oxidative stress [14].
However, protection against oxidative stress should
not be limited to the protective function of the biological
system itself. Noteworthily, one antioxidant molecule
is capable of reacting with only one oxidizing radical.
Therefore, there is a need to replenish antioxidant
molecules, including the use of supplements.
There are three groups of exogenous antioxidants:
mineral elements, nutritional antioxidants (carotenoids,
vitamins E and C), and natural antioxidants
derived from natural sources commonly known as
phytochemicals or phytonutrients [15].
Synthetic antioxidants have been widely used until
recently, but there is some doubt as to their usefulness
and safety. According to some studies, synthetic
antioxidants are ineffective against oxidative stress.
Moreover, their long-term use can lead to diseases such
as skin allergies, gastrointestinal and cardiovascular
diseases, and even increase the risk of cancer [16].
Therefore, there is a need for thorough research into the
safety of synthetic antioxidants.
The main sources of exogenous antioxidants are
fruits, vegetables, cereals, etc. However, modern
research is focused on traditional medicinal plants as a
source of natural antioxidants [17, 18].
Since ancient times, plants have been a source of
many useful substances, including exogenous antioxidants.
These substances act as reducing agents
that scavenge free radicals, protect the body against
oxidative stress, and, as a result, maintain a balance
between oxidants and antioxidants [19]. This is achieved
due to the presence of polyphenols, tocopherols,
carotenoids, ascorbic acid, and macromolecules
(including polysaccharides and peptides), as well as
essential oils [20].
Polyphenols are substances that contain a multiple
number of structural units of phenol [21]. Their
number affects the chemical, biological, and physical
properties of polyphenolic compounds. Polyphenols
are represented by flavonoids, phenolic acids, and
nonflavonoids [22, 23]. Depending on the chemical
structure, flavonoids are classified into flavonols,
flavanones, isoflavones, anthocyanins, and flavan-3-ols.
They are the most abundant class of polyphenols, with
about 8000 compounds identified to date [24]. A couple
of decades ago, researchers significantly increased
their interest in polyphenols due to their beneficial
properties for humans [25]. In particular, polyphenols
curb oxidative stress and related conditions through their
reductive ability to protect cellular components from
oxidative damage caused by free radicals [26].
Model organisms have become an indispensable
part of biological studies that would be impossible to
conduct on humans for ethical or economic reasons [27].
Studies of aging and age-related diseases require an
organism with a relatively short lifespan and clearly
identified genetic factors to ensure reproducibility and
reliability [28]. Nematodes (Caenorhabditis elegans L.),
drosophila (Drosophila melanogaster L.), and yeasts
(Saccharomyces cerevisiae L.) are used to test the effect
of bioactive substances on lifespan [29, 30].
C. elegans is a non-parasitic, free-living nematode
that feeds on various bacteria, primarily Escherichia
coli [31]. This simple multicellular organism up to 1 mm
long is a hermaphrodite capable of self-fertilization [32].
Its hermaphroditic structure contributes to low genetic
variability [33]. C. elegans has a short life cycle (2–
4 days) and remains viable for 20–25 days at 20°C as an
adult [34]. The nematode can be stored in liquid nitrogen
for an almost unlimited amount of time [35].
C. elegans was first used as a model organism for
biological research by Sydney Brenner in the 1965s [36].
Since then, it has been widely used to study aging and
associated oxidative stress, as well as neurodegeneration
and inflammation processes [37].
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C. elegans has a number of advantages that explain
its common uses as a model organism. Firstly, the
nematode is easily cultivated and has a transparent
body, which makes it easy to track the changes
microscopically [33, 38–40]. Secondly, C. elegans has
four organ systems that are the same as in vertebrates
(nervous, digestive, immune, and reproductive), which
allows for reliable and valuable conclusions [41]. Thirdly,
its short lifespan (20–25 days) enables scientists to
conduct rapid experiments aimed to study the effect
of various substances on the lifespan [42]. Finally, the
nematode’s genome is completely deciphered and
easily modified, which facilitates studies of the aging
process [43–46].
Plants of the Siberian Federal Okrug (Russia) are
potential sources of geroprotectors – substances that can
slow down the aging process [47].
Meadowsweet (Filipendula ulmaria L.) is a herbaceous
perennial plant common in Russia and many
European countries [48]. Extracts of this plant
have anticancerous, antioxidant, and anti-inflammatory
activity [49]. This activity is associated with
tannins, phenolic compounds, phenolcarboxylic acids,
catechins, flavonoids, essential oils, and other bioactive
substances contained in the roots and flowers of the
plant [50]. Many previous studies confirm its medicinal
properties [51, 52]. Rutin, one of its phytochemicals,
belongs to the class of natural flavonoids and is known
as quercetin-3-O-rutinoside or vitamin P [53]. It has
antitumorous, anticarcinogenic, and antimicrobial
properties [54, 55].
Ginkgo (Ginkgo biloba L.) is a special medicinal
plant that contains a variety of compounds with a
unique structure due to its phylogenetic divergence
from other plants. Its extract obtained by drying
the leaves is used to treat many neurodegenerative
diseases (memory impairment, dementia, Alzheimer’s
disease) [56]. It is also widely applied as an antiinflammatory,
cardioprotective, and antioxidant
agent [57]. The plant’s active components are
flavonoids, terpenoids, polyphenols, and organic
acids [58]. Quercetin and kaempferol are two of these
beneficial compounds. Quercetin is a flavonoid that
has a positive effect on cardiovascular diseases, breast
cancer, and ischemia [59–62]. Kaempferol is a valuable
component with anticancerous, antitumorous, and antiinflammatory
properties [62].
Baikal skullcap (Scutellaria baicalensis L.) belongs
to the genus Lamiaceae and is still widely used in
Chinese traditional medicine [63]. This plant grows
in China, Russia, Mongolia, Japan, and North Korea.
To date, scientists have identified 126 low-molecular
weight compounds in it, mostly in its root. These
compounds include flavonoids, flavonoid glycosides, and
phenylethanoid glycosides [64]. The most widespread
and studied of them is baicalin, which is used for various
medical purposes [66].
Red clover (Trifolium pretense L.) is one of the
most important representatives of the Leguminosae
family, numbering over 240 species [66]. It has
numerous medicinal properties and therapeutic
effects on respiratory diseases, bacterial and fungal
infections, tumors, and diabetes [67]. The plant is
rich in isoflavones (biochanin A, genistein, trifoside),
flavonoids (quercetin, kaempferol), as well as cinnamic,
caffeic, and chlorogenic acids [68].
Alfalfa (Medicágo sativa L.) is a flowering plant in
the Fabaceae family, which is the largest and most
widespread family in the world. The genus Medicágo
includes 83 species rich in alkaloids, flavonoids,
naphthoquinones, and saponins [69]. Naringenin, one
of its components, is a water-soluble flavonoid of great
value due to its anticancerous, antioxidant, and antiinflammatory
effects [70, 71].
Thyme (Thymus vulgaris L.) is an aromatic perennial
flowering plant belonging to the Lamiaceae family. Its
therapeutic properties are mainly associated with its
essential oil that has antitussive, expectorant, antiseptic,
antimicrobial, and anthelmintic effects [72]. The plant
is traditionally used to treat oral, gastrointestinal, and
urinary tract infections, as well as respiratory diseases
(cough, bronchitis, asthma) [73]. Ursolic acid, one of its
bioactive substances, is a promising agent against cancer,
cardiovascular disease, brain and liver diseases, obesity,
and diabetes [74, 75].
We aimed to study the effect of individual bioactive
substances on the lifespan of the model organism C.
elegans.
STUDY OBJECTS AND METHODS
We used individual bioactive substances isolated
from the extracts of suspension, callus, and root cultures
of Siberian medicinal plants. The extraction parameters
are presented in Table 1.
Table 1 Parameters for obtaining extracts from Siberian medicinal plants
Sample Extraction time, h Temperature, °С Ethanol, %
Callus cultures of meadowsweet (Filipendula ulmaria L.) 5 35 70
Suspension cultures of ginkgo (Ginkgo biloba L.) 6 55 70
Root cultures of Baikal skullcap (Scutellaria baicalensis L.) 5 35 70
Callus cultures of red clover (Trifolium pretense L.) 5 70 60
Callus cultures of alfalfa (Medicágo sativa L.) 3 50 70
Callus cultures of thyme (Thymus vulgaris L.) 4 50 70
Root cultures of sweetvetch (Hedysarum neglectum L.) 6 30 70
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High-performance liquid chromatography (HPLC)
was applied (Shimadzu LC-20 Prominence liquid
chromatograph, Japan) to isolate the following bioactive
substances from the extracts of the above cultures:
1. Rutin from the callus culture extract of meadowsweet
(Filipendula ulmaria) [76];
2. Quercetin from the suspension culture extract of
ginkgo (Ginkgo biloba) [77];
3. Kaempferol from the suspension culture extract of
ginkgo (G. biloba) [77];
4. Baicalin from the root culture extract of Baikal
skullcap (Scutellaria baicalensis) [78];
5. Trans-cinnamic acid from the root culture extract of
Baikal skullcap (S. baicalensis) [79];
6. Chlorogenic acid from the callus culture extract of red
clover (Trifolium pratense) [80];
7. Biochanin A from the callus culture extract of red
clover (T. pratense) [80];
8. Naringenin from the callus culture extract of alfalfa
(Medicágo satíva) [81];
9. Ursolic acid from the callus culture extract of thyme
(Thymus vulgaris) [82]; and
10. Magniferin from the root culture extract of
sweetvetch (Hedysarum neglectum) [83].
To isolate rutin from the callus culture extract
of meadowsweet, the plant’s ethanol extract was
evaporated under vacuum at a temperature under 40°C
on an IKA RV 8 rotary evaporator (IKA, Germany).
After adding deionized water to 1/4 of the concentrate’s
initial volume, evaporation continued until a thick
precipitate was formed. The precipitate was treated
with a chloroform:ethylacetate mixture for 5 min
with vigorous stirring in triplicate. The extracts were
combined and mixed with anhydrous sodium sulfate
(2.0 g per 100 mL of extract). The mixture was kept for
3 h at +4°C and then filtered. The residue containing a
flavonoid fraction was dissolved in 50% ethanol. Then,
50.0 g of activated carbon was added to the mixture
and evaporated until a dry residue was formed. The
adsorbent with the extract residue was transferred to a
shot filter and successively eluted with methanol, water,
7% aqueous phenol, and 15% phenol in methyl alcohol.
The fraction extracted with 7% aqueous phenol was
treated with 100 mL of diethyl ether in triplicate. The
resulting extract was evaporated under vacuum to a
thick precipitate, which was then mixed with 40.0 g
of silica gel (column chromatography grade, Sigma),
dried completely, and transferred to a column (5×6 cm
BioRad) as a suspension in chloroform. The substances
were eluted with a mixture of chloroform:ethanol (80:20)
and evaporated to isolate rutin.
To isolate quercetin and kaempferol from the
suspension culture extract of ginkgo, the extract was
filtered through cellulose filters, diluted with water, and
kept at +4°C for 48 h to filter lipid precipitates. The
extract was then concentrated in a vacuum evaporator
in the presence of sodium chloride (up to 10% by salt
content in solution). Resinous substances were removed
by decantation. Lipophilic substances were purified
by liquid-liquid extraction with n-heptane to isolate
terpenolactones. The aqueous phase was extracted with
n-butanol in triplicate.
The three phases were combined and concentrated
under vacuum until a dry precipitate was formed. The
precipitate was dissolved in a water-alcohol solution
and purified by liquid-liquid extraction with ethyl
acetate. The resulting phase was washed with a sodium
chloride solution and evaporated. The dry residue
was dissolved in acetone containing 40 wt.% of water,
cooled to 10°C, and filtered. Flavonogicosides were
chromatographed on polyamide (Sigma) packed in a
5.3×250 mm chromatographic column on a BioLogic
low-pressure chromatograph (BioRad) using gradient
elution mixtures: chloroform-methanol (100:0 → 60:40)
and then water-ethanol (100:0 → 0:100). The components
were separated and purified by silica gel
rechromatography (Lachema) using an eluent mixture
of chloroform:petroleum ether (30:70), followed by
recrystallization to isolate quercetin and kaempferol.
Baicalin and trans-cinnamic acid were isolated
from the root culture extract of Baikal skullcap by
evaporating the extract under vacuum at a temperature
under 50°C. The evaporated residue was treated with
diethyl ether in triplicate. The resulting ether fraction
was chromatographed on silica gel (mobile phase) in
a n-hexane-acetone gradient (1:0 → 0:1) to isolate
flavonoids and hydroxycinnamic acids. Baicalin and
trans-cinnamic acid were isolated by subsequent
rechromatography on silica gel (mobile phase) with
n-hexane-chloroform (1:0 → 0:1).
To isolate biochanin A from the callus culture
extract of red clover, the ethanol extract was vacuumevaporated
on a rotary evaporator at under 50°C.
Deionized water was added to the precipitate up to 1/4 of
the concentrate’s initial volume to continue evaporation
to a thick precipitate. The precipitate was treated with
n-hexane for 5 min in triplicate and the suspension was
treated ultrasonically. The extracts were filtered through
filter paper and combined. Then, they were evaporated
under vacuum to a thick precipitate. The precipitate was
mixed with 50.0 g of silica gel, dried, and transferred
to a column (5×6 cm BioRad). Then, it was eluted with
a petroleum ether-ethanol mixture (99:1, 98:2, 97:3,
95:5, 93:7, 80:20). Biochanin A was isolated from the
evaporated eluates.
To isolate chlorogenic acid from the callus culture
extract of red clover, the thick precipitate obtained as
described above was treated with diethyl ether to isolate
hydroxycinnamic and coumaric acids. The mixture
was then evaporated to a dry residue and separated on
silica gel (column chromatography grade, Sigma) on a
column (0.65×10 cm BioRad). Then, it was eluted with
isopropyl alcohol:acetic acid:hexane (65:12:23) to isolate
chlorogenic acid.
Naringenin was isolated from the alfalfa extract
as follows. The ethanol extract was evaporated under
vacuum at under 55°C on a rotary evaporator. The
residue was mixed with deionized water added to 1/4 of
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the concentrate’s initial volume to continue vacuum
evaporation to a thick precipitate. The precipitate was
placed on a 5×6 cm BioRad chromatographic column
and eluted with n-hexane to collect 1-mL fractions. The
resulting extracts were evaporated to a thick precipitate,
which was then dissolved in ethanol and fractionated
on LH-20 Sephadex (Aldrich) in toluene. Silica gel was
eluted with isopropyl alcohol:water (40:60) and then
evaporated under vacuum to a thick residue. The residue
was dissolved in ethanol and fractionated on LH-20
Sephadex (Aldrich) in a methanol gradient of 10 → 90%
to isolate naringenin.
Ursolic acid was isolated from the callus culture
extract of thyme. For this, the ethanolic extract was
evaporated under vacuum at under 40°C on a rotary
evaporator. The residue was mixed with deionized
water added to 1/4 of the concentrate’s initial volume
to continue vacuum evaporation to a thick precipitate.
The resulting precipitate was treated three times with
dichloromethane for 5 min with vigorous stirring. The
extracts were combined and mixed with anhydrous
sodium sulfate (20.0 g per liter of extract). The mixture
was kept for 3 h and filtered through a paper filter. The
filtered precipitate was dissolved in ethanol. The ethanol
fraction was passed through an AN-1 anion exchanger
(State Standard 20301-74) and washed with waterethanol
eluents (up to 50% of ethanol). Then, it was
desorbed with 0.1 M hydrochloric acid to isolate ursolic
acid.M
agniferin was isolated from the root culture extract
of sweetvetch. The ethanol extract was evaporated
in a vacuum evaporator at 45°C. The residue was
fractionated on a BioLogic low-pressure chromatograph
(BioRad) using silica gel (column chromatography
grade, Sigma). Petroleum ether-ethyl acetate was used as
an eluent (100:0; 50:1; 20:1; 10:1; 5:1; 2:1; 1:1; and 0:1).
Methanol was fed to the column to desorb gallic acid,
resulting in nine 300-mL fractions collected. A crude
crystal of magniferin was obtained from fraction 3.
Then, it was recrystallized from a mixture of petroleum
ether:acetone (20:1) and purified by rechromatography
on CL6B Sepharose (Sigma – Aldrich) using a BioLogic
low-pressure chromatograph (BioRad) to isolate pure
mangiferin.
All the isolated bioactive substances were at least
95% pure. Their IR spectra were registered on an
SF-2000 instrument (OKB Spektr, Russia).
Further, we analyzed the effect of bioactive
substance concentrations on the lifespan of wild-type
Caenorhabditis elegans nematodes (strain N2 Bristol,
www.wormbook.org). Our study consisted of five stages
described below.
Cultivation of nematodes on solid agar. Obtaining
an Escherichia coli bacterial culture. E. coli OP50
was seeded on Petri dishes with a Lysogeny broth
(L-broth) solid medium (HiMedia Laboratories, India).
Then, under sterile conditions, one bacterial colony was
selected and placed in 5–10 mL of L-broth (HiMedia
Laboratories, India) to incubate at 37°C overnight with
vigorous stirring. After that, the culture was transferred
to a refrigerator and stored at +4°C.
Inoculating E. coli OP50 on NGM agar plates. 50
μL of the E. coli OP50 overnight culture was placed in
the center of a 100-mm Petri dish. Using a sterile glass
rod, the drop was distributed over the center of the dish
in the shape of a square, without touching the walls, and
incubated at 37°C for a day. After incubation, the dishes
were wrapped in parafilm and stored in the refrigerator
for several weeks.
Preparing NGM agar plates. After autoclaving, the
sterile NGM agar was cooled to 55°C in a water bath.
Then, the cooled nutrient medium was mixed with 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 K3PO4 buffer.
After thorough mixing, it was poured into sterile Petri
dishes, 20 mL each. To ensure the absence of bacterial
contamination, the dishes were left for 2–3 days at room
temperature.
Transferring nematodes to new NGM agar
dishes. The nematodes were transferred in two ways:
with a loop and with 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 NGM Petri dish with agar. The
second method involved cutting a 0.5×0.5 cm piece of
agar containing a nematode with a sterile scalpel and
transferring it to the center of the dish surface down.
The dishes were incubated at 20°C.
Nematode synchronization. 5–10 mL of sterile
water was pipetted on the surface of the dish containing
a nematode until its eggs were completely attached
to the agar. The liquid from the Petri dish was placed
in a 50 mL tube and centrifuged for 2 min (1200 rpm).
Then, the supernatant was removed and the precipitate
was washed with 10 mL of distilled water to repeat
centrifugation under the above conditions.
After repeated centrifugation, the supernatant was
removed and the precipitate was mixed with 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. The
mixture was thoroughly vortexed (Biosan, Latvia) for
10 min with 2 min intervals to observe the hydrolysis
of nematodes under an Axio Observer Z1 microscope
(Karl Zeiss, Germany). At the end of the process, 5 mL
of M9 medium was added to neutralize the reaction. The
resulting mixture was centrifuged for 2 min (2500 rpm).
After that, the supernatant was removed and the
precipitate was mixed with 10 mL of sterile water to
repeat the washing and centrifugation three times. Then,
the precipitate was washed with 10 mL of S-medium
and the supernatant was removed. Finally, 10 mL of
S-medium was added and the test tube with nematode
eggs was placed on a slow shaker for a day at room
temperature for the complete transfer of the nematodes
to the L1 stage.
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Cultivation of nematodes in a liquid medium.
After the nematodes passed to the L1 stage, an overnight
bacterial culture of E. coli OP50 was added to the
S-medium. The culture had previously been washed and
resuspended in the S-medium to a bacterial concentration
of 0.5 mg/mL. Then, 120 μL amounts of the suspension
containing the bacteria and nematodes were poured into
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
was poured into each well of the plate to prevent the
nematodes from reproduction and left for a day at 20°C.
At the end of incubation, the nematodes entered the L4
stage. Then, 15-μL amounts of the bioactive substances
under study were added to the wells in accordance with
the experiment plan.
Preparation of bioactive substances. Stock
solutions of bioactive substances were prepared in
dimethyl sulfoxide at a concentration of 10 mmol/L.
The substances were tested by diluting stock solutions
in sterile distilled water to concentrations of 2000,
1000, 500, and 100 μM. Each well was filled with 15 μL
of freshly prepared stock solutions so that working
concentrations of each bioactive substance reached 2000,
1000, 500, and 100 μmol/L, respectively. The stocks
were stored at 4°C.
Effects of bioactive substances on nematode
lifespan. The effect of bioactive substances at
concentrations of 0, 10, 50, 100, and 200 μmol/L on the
lifespan of C. elegans was determined by the number
of nematodes surviving in the presence of the tested
substances. The experiment was carried out in 6-fold
repetitions using 96-well plates and a liquid S-medium
for nematode cultivation. The numbers of live and dead
nematodes were counted every 4–7 days during the
61-day experiment. The experiment was considered
completed when there were no live nematodes left in the
control group.
Each concentration of bioactive substances was
studied in 6-fold repetitions. Statistical data were
analyzed in the Microsoft Office Excel 2007. Statistical
analysis was performed using a paired Student’s t-test
for each pair of interests. Differences were considered
statistically significant at P < 0.05.
RESULTS AND DISCUSSION
The effects of the bioactive substances obtained
from the extracts of suspension, callus, and root
cultures of Siberian medicinal plants on the lifespan
of Caenorhabditis elegans nematodes are graphically
presented in Fig. 1.
As can be seen in Fig. 1a, rutin, which was isolated
from the extract of meadowsweet callus culture, did
not significantly increase the lifespan of C. elegans
nematodes. At concentrations of 50 and 100 μmol/L, it
had a positive effect from day 8 to day 34, but then the
number of surviving nematodes approached the control.
Its greatest effect was observed at a concentration of
50 μmol/L on day 13, with the survival rate of 32.3%
(15.3% higher than in the control group).
Quercetin, which was obtained from the suspension
culture extract of ginkgo, had a significant effect on
the lifespan of C. elegans nematodes at concentrations
of 10 and 100 μmol/L (Fig. 1b). The proportion of
surviving nematodes was 32.6–4.6% from day 8 to day
45 of the experiment. On day 8, all the concentrations
of quercetin had a positive effect on the lifespan. The
proportions of surviving nematodes treated with 10, 50,
100 and 200 μmol/L of this bioactive substance were
72.9, 74.0, 67.5, and 63.6%, respectively (higher than
in the control nematodes by 41.9, 43.0, 36.5, and 32.6%,
respectively).
Of special interest was kaempferol obtained from
the suspension culture extract of ginkgo (Fig. 1c). At a
concentration of 50 μmol/l, this substance increased
the lifespan of nematodes throughout the experiment
(except for 3 days), compared to the control. The
nematode population was active, reaching 10.3% on
day 61. We also observed kaempferol’s positive effect
at a concentration of 10 μmol/L in the period of 8 to 61
days. The maximum proportion of surviving nematodes
treated with this concentration was registered on day 8
at 48.6%, which was 17.6% higher than in the control
group. However, the greatest increase in the nematode
lifespan was provided by a concentration of 200 μmol/L
on day 8, with the survival rate of 61.6% (by 30.6%
higher than in the control nematodes).
Baicalin was produced from the root culture of
Baikal skullcap (Fig. 1d). At concentrations of 10, 100,
and 200 μmol/L, it increased the lifespan of nematodes
from day 8 to day 13. After that period, the number of
surviving nematodes exposed to 200 μmol/L baicalin
became lower than in the control group. During
days 13–17, their lifespan increased only at baicalin’s
concentrations of 10 and 100 μmol/L. From day 17 until
the end of the experiment, the number of surviving
nematodes treated with 10, 50, and 200 μmol/L baicalin
was greater than in the control group. Noteworthily,
the end of the experiment saw greater proportions
of surviving nematodes treated with baicalin at all
concentrations (10, 50, 100, and 200 μmol/L) than that of
the control (by 4.3, 6.7, 2.3, and 2.1%, respectively).
Trans-cinnamic acid was isolated from the root
culture extract of Baikal skullcap (Fig. 1e). As we can
see, on day 8, its concentrations of 10, 50, 100, and
200 μmol/L increased the lifespan of nematodes by 18.1,
26.3, 24.1, and 36.6%, respectively. During days 13–34,
the concentration of 200 μmol/L had no positive
effect on the lifespan of nematodes, unlike the other
concentrations. However, from day 34 to the end of the
experiment, trans-cinnamic acid at all concentrations
increased the percentage of surviving nematodes. The
greatest increase in the lifespan was observed in the
nematodes treated with 50 μmol/L of this bioactive
substance (9.8%).
Chlorogenic acid obtained from the callus culture
extract of red clover showed a generally positive effect
346
Faskhutdinova E.R. et al. Foods and Raw Materials. 2022;10(2):340–352
Figure 1 Beginning. Effects of bioactive substances isolated from the extracts of suspension, callus, and root cultures
of medicinal plants on the lifespan of Caenorhabditis elegans nematodes: (a) rutin from the suspension culture extract of
meadowsweet; (b) quercetin from the suspension culture extract of ginkgo; (c) kaempferol from the suspension culture extract of
ginkgo; (d) baicalin from the root culture extract of Baikal skullcap; (e) trans-cinnamic acid from the root culture extract of Baikal
skullcap; (f) chlorogenic acid from the callus culture extract of red clover; (g) biochanin A from the callus culture extract red
clover; (h) naringenin from the callus culture extract of alfalfa; (i) ursolic acid from the callus culture extract of thyme;
(j) magniferin from the root culture extract of sweetvetch
a b
a b
c d
0 3 8 13 17 20 26 30 34 45 55 61
Lifespan, days
10 мкмоль/л 50 мкмоль/л 200 мкмоль/л
100 мкмоль/л Контроль
20.0
70,0
20 26 30 34 45 55 61
Surviving nematodes , %
Lifespan, days
10 мкмоль/50 мкмоль/200 100 Контроль
0 3 8 13 17 20 26 30 34 45 55 61
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
30 34 45 55 61
Surviving nematodes , %
10 50 200 Контроль
100 c d
a b
c d
0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 Surviving nematodes , %
Lifespan, days
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
8 13 17 20 26 30 34 Surviving nematodes , %
Lifespan, days
a b
c d
0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 Surviving nematodes , %
Lifespan, days
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 Surviving nematodes , % Lifespan, days
a b
c d
0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes , %
Lifespan, days
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes , %
Lifespan, days
a b
c d
0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes , %
Lifespan, days
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes , %
Lifespan, days
%
a c 0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/50 мкмоль/200 мкмоль/100 мкмоль/Контроль
a c 0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
a c 0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
Surviving nematodes , %
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
Surviving nematodes , %
a b
c d
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л 50 мкмоль/л 200 мкмоль/л
100 мкмоль/л Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 Surviving nematodes , %
Lifespan, days
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80,0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 Surviving nematodes , % Lifespan, days
a b
c d
0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
0 3 8 13 17 20 26 30 34 45 55 Surviving nematodes , % Lifespan, days
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 Surviving nematodes , %
Lifespan, days
a b
c d
0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes , %
Lifespan, days
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes , %
Lifespan, days
a b
c d
0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes , %
Lifespan, days
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes , %
Lifespan, days
a b
0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
13 17 20 26 30 34 45 55 Surviving nematodes , %
Lifespan, days
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
Surviving nematodes, %
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
Surviving nematodes , %
a b
0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
13 17 20 26 30 34 45 55 61
Surviving nematodes , %
Lifespan, days
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
Surviving nematodes, %
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
Surviving nematodes , %
a b
0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
13 17 20 26 30 34 45 55 61
Surviving nematodes , %
Lifespan, days
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61 Surviving nematodes, %
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
Surviving nematodes , %
e f
60.0
70.0
80.0
90.0
100.0
a c 0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/50 мкмоль/200 100 Контроль
a b
c d
0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 Surviving nematodes , %
Lifespan, days
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
100,0
0 3 8 13 17 20 26 30 34 Surviving nematodes , %
Lifespan, days
a b
c d
0 3 8 13 17 20 26 30 34 45 55 61
Lifespan, days
10 мкмоль/л 50 мкмоль/л 200 мкмоль/л
100 мкмоль/л Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes , %
Lifespan, days
10 50 200 100 Контроль
0 3 8 13 17 20 26 30 34 45 55 61
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
60.0
70.0
80.0
90.0
100.0
30 34 45 55 61
Surviving nematodes , Lifespan, days
10 50 200 Контроль
100 a c 0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/50 мкмоль/200 100 Контроль
a c 0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/50 мкмоль/200 мкмоль/100 мкмоль/Контроль
a c 0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
a c 0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
Surviving nematodes , %
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
Surviving nematodes , %
a c 0.0
10.0
20,0
30.0
40.0
50.0
0 3 8 13 17 20 26 30 34 45 55 61
nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/50 мкмоль/200 100 Контроль
e f
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 Surviving nematodes, %
Lifespan, days
a b
0.0
10.0
20,0
30.0
40.0
50.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
0 3 8 13 17 20 26 30 34 45 Surviving nematodes , %
Lifespan, days
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 Surviving nematodes , %
e f
0 3 8 13 17 20 26 30 34 45 55 61
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, Lifespan, days
10 мкмоль/50 мкмоль/200 Контроль
100 a 20,0
90.0
100.0
26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
10 мкмоль/50 мкмоль/200 100 Контроль
a 0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
10 мкмоль/50 мкмоль/200 100 Контроль
a 0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
Surviving nematodes, %
10 мкмоль/50 мкмоль/200 мкмоль/100 мкмоль/Контроль
a 0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
Surviving nematodes, %
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
a 0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
Surviving nematodes , %
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
Surviving nematodes , %
347
Faskhutdinova E.R. et al. Foods and Raw Materials. 2022;10(2):340–352
Figure 1g shows the effect of biochanin A isolated
from red clover callus culture. As we can see, the
best survival rate was provided by this substance
at 100 μmol/L. Unlike the other concentrations, this
concentration had a positive effect on C. elegans
throughout the entire experiment. Days 8–20 saw the
highest survival rates, with the greatest increase in
lifespan occurring on day 13 (by 45.2% compared to
the control). Noteworthily, 200 μmol/L of biochanin
A had a negative effect on the survival and lifespan
of C. elegans almost throughout the experiment,
except for the very end. On day 61, the proportion of
surviving nematodes increased by 1.8% and amounted
to 2.5% (compared to 0.7% in the control group). The
concentrations of 10 and 50 μmol/L increased the
on the lifespan of C. elegans (Fig. 1f). As can be seen,
100 μmol/L of this substance increased the survival of
nematodes throughout the experiment, with the greatest
increase (by 40.1%) on day 8. The other concentrations
showed varying survival rates. Days 8–13 saw greater
lifespans in the nematodes exposed to chlorogenic
acid at all four concentrations. During days 13–26,
increased lifespan was provided by the concentrations
of 10, 50, and 100 μmol/L. The maximum survival
rate was observed on day 20 (27.7%) in the nematodes
treated with 100 μmol/L of chlorogenic acid. From day
26 to day 34, the concentration of 200 μmol/L had no
effect on the lifespan of C. elegans. However, from day
45 to the end of the experiment, chlorogenic acid had a
positive effect again at all its concentrations.
Figure 1 Ending. Effects of bioactive substances isolated from the extracts of suspension, callus, and root cultures of
medicinal plants on the lifespan of Caenorhabditis elegans nematodes: (a) rutin from the suspension culture extract of
meadowsweet; (b) quercetin from the suspension culture extract of ginkgo; (c) kaempferol from the suspension culture extract of
ginkgo; (d) baicalin from the root culture extract of Baikal skullcap; (e) trans-cinnamic acid from the root culture extract of Baikal
skullcap; (f) chlorogenic acid from the callus culture extract of red clover; (g) biochanin A from the callus culture extract red
clover; (h) naringenin from the callus culture extract of alfalfa; (i) ursolic acid from the callus culture extract of thyme;
(j) magniferin from the root culture extract of sweetvetch
g h
i j
0.0
10.0
0 3 Surviving nematodes, a c 0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/50 мкмоль/200 мкмоль/100 мкмоль/Контроль
a c 0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
a c 0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
Surviving nematodes , %
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
Surviving nematodes , %
a c 20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
8 13 17 20 26 30 34 45 55 61
%
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/50 мкмоль/200 100 Контроль
g h
20.0
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 Surviving nematodes, %
Lifespan, days
g h
0 3 8 13 17 20 26 30 34 45 55 61
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0.0
10.0
20.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, Lifespan, days
10 50 200 100 Контроль
a b
c d
0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 Surviving nematodes , %
Lifespan, days
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 Surviving nematodes , % Lifespan, days
a b
c d
0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes , %
Lifespan, days
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes , %
Lifespan, days
a b
c d
0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes , %
Lifespan, days
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes , %
Lifespan, days
i j
Figure 1 Effects of bioactive substances isolated from the extracts of suspension, callus, and root cultures of medicinal plants on th e nematodes: a – rutin from the suspension culture extract of meadowsweet; b – quercetin from the suspension culture extract of ginkgo; c – kaempferol extract of ginkgo; d – baicalin from the root culture extract of Baikal skullcap; e – trans-cinnamic acid from the root culture extract of Baikal callus culture extract of red clover; g – biochanin A from the callus culture extract red clover; h – naringenin from the callus culture extract of culture extract of thyme; j – magniferin from the root culture extract of sweetvetch
0.0
10.0
20.0
30.0
40.0
50.0
60.0
Surviving nematodes, %
Lifespan, days
10 мкмоль/л 200 мкмоль/л 100 мкмоль/л Контроль
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 Surviving nematodes, %
Lifespan, days
a b
c d
0.0
20,0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
17 20 26 30 34 45 Surviving nematodes , %
Lifespan, days
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 Surviving nematodes , %
Lifespan, days
i j
1 Effects of bioactive substances isolated from the extracts of suspension, callus, and root cultures of medicinal plants on th e lifespan of Caenorhabditis nematodes: a – rutin from the suspension culture extract of meadowsweet; b – quercetin from the suspension culture extract of ginkgo; c – kaempferol from the suspension ginkgo; d – baicalin from the root culture extract of Baikal skullcap; e – trans-cinnamic acid from the root culture extract of Baikal skullcap; f – chlorogenic culture extract of red clover; g – biochanin A from the callus culture extract red clover; h – naringenin from the callus culture extract of alfalfa; i – ursolic acid culture extract of thyme; j – magniferin from the root culture extract of sweetvetch
0 3 8 13 17 20 26 30 34 45 55 61
Lifespan, days
10 мкмоль/л 200 мкмоль/л 100 мкмоль/л Контроль
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
10 50 200 100 Контроль
a c 0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/50 мкмоль/200 100 Контроль
a c 0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/50 мкмоль/200 мкмоль/100 мкмоль/Контроль
a c 0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
a c 0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
Surviving nematodes , %
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
Surviving nematodes , %
a c 0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/50 мкмоль/200 100 Контроль
0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 Surviving nematodes , %
Lifespan, days
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 Surviving nematodes , %
Lifespan, days
a c 0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 Surviving nematodes , %
Lifespan, days
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 Surviving nematodes , %
Lifespan, days
a c 0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 Surviving nematodes , %
Lifespan, days
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 Surviving nematodes , %
Lifespan, days
a b
c d
0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes , %
Lifespan, days
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes , %
Lifespan, days
a b
c d
0.0
10.0
20,0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 μmol/L 50 μmol/L 200 μmol/L
100 μmol/L control
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 Surviving nematodes , %
Lifespan, days
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 45 55 61
Surviving nematodes, %
Lifespan, days
10 мкмоль/л
50 мкмоль/л
200 мкмоль/л
100 мкмоль/л
Контроль
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 3 8 13 17 20 26 30 34 Surviving nematodes , %
Lifespan, days
348
Faskhutdinova E.R. et al. Foods and Raw Materials. 2022;10(2):340–352
survival of nematodes during days 13–45 by an average
of 8.4 and 9.1%, respectively. Both concentrations
provided maximum lifespan increases during days
13–20 and had a weaker effect towards the end of the
experiment. From day 26 to day 61, biochanin A at 10,
50, and 200 μmol/L had no significant effect on the
lifespan of nematodes compared to the control group.
Naringenin was isolated from the extract of alfalfa
callus culture. As shown by Fig. 1h, its concentration
of 100 μmol/L had the greatest effect on the lifespan
of nematodes compared to the other concentrations,
especially during days 8–26. During that period,
the survival of nematodes increased by an average
of 27.3% compared to the control group, with
maximum survival on day 13 (by 35.4%). The other
concentrations of naringenin (10, 50, and 200 μmol/L)
did not have a significant effect on the survival or
lifespan of nematodes.
Ursolic acid was isolated from thyme callus
culture. According to Fig. 1i, it had no significant
effect on the lifespan of nematodes at all its
concentrations. The greatest increase in survival (by
14.1%) was observed on day 8 in the nematodes treated
with 100 μmol/L of ursolic acid.
Similarly, we found no positive effect in magniferin
obtained from the root culture extract of sweetvetch (Fig.
1j). Moreover, its concentrations of 100 and 200 μmol/L
reduced the proportion of surviving nematodes from
day 13 to day 55 of the experiment. The greatest
increases in the lifespan of nematodes were observed
at magniferin concentrations of 10 and 50 μmol/L on
day 8, amounting to 19.3 and 24.2%, respectively. At
the end of the experiment, the longest lifespan was
demonstrated by the nematodes exposed to 100 μmol/L
of magniferin (1.4%).
CONCLUSION
Having applied HPLC methods, we isolated the
following bioactive substances from the extracts of
callus, suspension, and root cultures of medicinal plants
growing in the Siberian Federal Okrug: rutin – from
the callus culture extract of meadowsweet (Filipendula
ulmaria L.); quercetin – from the suspension culture
extract of ginkgo (Ginkgo biloba L.); kaempferol – from
the suspension culture extract of ginkgo (G. biloba);
baicalin – from the root culture extract of Baikal
skullcap (Scutellaria baicalensis L.); trans-cinnamic
acid – from the root culture extract of Baikal skullcap
(S. baicalensis); chlorogenic acid – from the callus
culture extract of red clover (Trifolium pretense L.);
biochanin A – from the callus culture extract of red
clover (T. pratense); naringenin – from the callus
culture extract of alfalfa (Medicágo sativa L.); ursolic
acid – from the callus culture extract of thyme (Thymus
vulgaris L.); and magniferin – from the root culture
extract of sweetvetch (Hedysarum neglectum L.). All
the bioactive substances were at least 95% pure and
were registered using IR spectroscopy on an SF-2000
instrument (OKB Spektr, Russia).
We determined the effect of the above bioactive
substances at concentrations of 10, 50, 100, and
200 μmol/L on the lifespan of Caenorhabditis elegans
nematodes, which are widely used as model organisms
to study the aging process. We used 96-well plates for
the experiment that lasted 61 days. Surviving nematodes
were counted every 4–7 days and the experiment
was considered completed when there were no live
nematodes left in the control group. Stock solutions of
the following bioactive substances were prepared for
the experiment: rutin, quercetin, kaempferol, baicalin,
trans-cinnamic acid, chlorogenic acid, biochanin A,
naringenin, ursolic acid, and magniferin.
Trans-cinnamic acid, baicalin, rutin, ursolic acid, and
magniferin did not significantly increase the lifespan of the
nematodes.
Chlorogenic acid and naringenin had a little
effect on the lifespan of nematodes, while quercetin,
kaempferol, and biochanin A demonstrated their high
survival.
Noteworthily, the greatest proportions of surviving
nematodes treated with various concentrations of
bioactive substances were recorded on days 8 to 13
for all the experimental samples. Then, the lifespan of
C. elegans decreased and their survival rates
approached those of the control group.
Thus, 200 μmol/L of kaempferol, 10 μmol/L of
quercetin (both obtained from ginkgo suspension
culture extract), and 100 μmol/L of biochanin A
(obtained from red clover callus culture extract)
increased the lifespan of C. elegans nematodes by
30.6, 41.9, and 45.2%, respectively, compared to the
control (days 8 and 13). These results suggest that
the mentioned bioactive substances can be effectively
used as anti-aging agents.
CONTRIBUTION
All the authors are equally responsible for the research
results and the manuscript.
CONFLICT OF INTEREST
The authors declare no conflict of interest.

1. WHO: People living longer and healthier lives but COVID-19 threatens to throw progress off track [Internet]. [cited 2022 Jan 15]. Available from: https://www.who.int/news/item/13-05-2020-people-living-longer-and-healthier-lives-but-covid-19-threatens-to-throw-progress-off-track

2. Bosch-Morell F, Villagrasa V, Ortega T, Acero N, Muñoz-Mingarro D, González-Rosende M, et al. Medicinal plants and natural products as neuroprotective agents in age-related macular degeneration. Neural Regeneration Research. 2020;15(12):2207-2216. https://doi.org/10.4103/1673-5374.284978

3. Elfawy HA, Das B. Crosstalk between mitochondrial dysfunction, oxidative stress, and age related neurodegenerative disease: Etiologies and therapeutic strategies. Life Sciences. 2019;218:165-184. https://doi.org/10.1016/j.lfs.2018.12.029

4. Tan BL, Norhaizan ME, Liew W-P-P, Rahman HS. Antioxidant and oxidative stress: A mutual interplay in age-related diseases. Frontiers in Pharmacology. 2018;9. https://doi.org/10.3389/fphar.2018.01162

5. Prosekov AYu, Dyshlyuk LS, Milentyeva IS, Sykhikh SA, Babich OO, Ivanova SA, et al. Antioxidant and antimicrobial activity of bacteriocin-producing strains of lactic acid bacteria isolated from the human gastrointestinal tract. Progress in Nutrition. 2017;19(1):67-80. https://doi.org/10.23751/pn.v19i1.5147

6. Harman D. Aging: A theory based on free radical and radiation chemistry. Journal of Gerontology. 1956;11(3):298-300. https://doi.org/10.1093/geronj/11.3.298

7. McGuire PJ. Mitochondrial dysfunction and the aging immune system. Biology. 2019;8(2). https://doi.org/10.3390/biology8020026

8. Dębowska K, Smulik-Izydorczyk R, Pięta J, Adamus J, Michalski R, Sikora A. Oxidation of the selected probes for detection of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in aqueous solutions of nitric oxide donors. Free Radical Biology and Medicine. 2018;120. https://doi.org/10.1016/j.freeradbiomed.2018.04.258

9. Jakubczyk K, Dec K, Kałduńska J, Kawczuga D, Kochman J, Janda, K. Reactive oxygen species - sources, functions, oxidative damage. Polski merkuriusz lekarski: organ Polskiego Towarzystwa Lekarskiego. 2020;48(284):124-127.

10. He L, He T, Farrar S, Ji L, Liu T, Ma X. Antioxidants maintain cellular redox homeostasis by elimination of reactive oxygen species. Cellular Physiology and Biochemistry. 2017;44(2):532-553. https://doi.org/10.1159/000485089

11. van de Lagemaat EE, de Groot LCPGM, van den Heuvel EGHM. Vitamin B12 in relation to oxidative stress: A systematic review. Nutrients. 2019;11(2). https://doi.org/10.3390/nu11020482

12. Singh A, Kukreti R, Saso L, Kukreti S. Oxidative stress: A key modulator in neurodegenerative diseases. Molecules. 2019;24(8). https://doi.org/10.3390/molecules24081583

13. Warraich UA, Hussain F, Kayani HUR. Aging - Oxidative stress, antioxidants and computational modeling. Heliyon. 2020;6(5). https://doi.org/10.1016/j.heliyon.2020.e04107

14. Mirończuk-Chodakowska I, Witkowska AM, Zujko ME. Endogenous non-enzymatic antioxidants in the human body. Advances in Medical Sciences. 2018;63(1):68-78. https://doi.org/10.1016/j.advms.2017.05.005

15. Santos AL, Sinha S, Lindner AB. The good, the bad, and the ugly of ROS: New insights on aging and aging-related diseases from eukaryotic and prokaryotic model organisms. Oxidative Medicine and Cellular Longevity. 2018;2018. https://doi.org/10.1155/2018/1941285

16. Liu R, Mabury SA. Synthetic phenolic antioxidants: A review of environmental occurrence, fate, human exposure, and toxicity. Environmental Science and Technology. 2020;54(19):11706-11719. https://doi.org/10.1021/acs.est.0c05077

17. Asyakina LK, Fotina NV, Izgarysheva NV, Slavyanskiy AA, Neverova OA. Geroprotective potential of in vitro bioactive compounds isolated from yarrow (Achilleae millefolii L.) cell cultures. Foods and Raw Materials. 2021;9(1):126-134. https://doi.org/10.21603/2308-4057-2021-1-126-134

18. Gadouche L, Zidane A, Zerrouki K, Azouni K, Bouinoune S. Cytotoxic effect of Myrtus communis, Aristolochia longa, and Calycotome spinosa on human erythrocyte cells. Foods and Raw Materials. 2021;9(2):379-386. https://doi.org/10.21603/2308-4057-2021-2-379-386

19. Hassan W, Noreen H, Rehman S, Gul S, Amjad Kamal M, Kamdem JP, et al. Oxidative stress and antioxidant potential of one hundred medicinal plants. Current Topics in Medicinal Chemistry. 2017;17(12):1336-1370. https://doi.org/10.2174/1568026617666170102125648

20. Song R, Wu Q, Yun Z, Zhao L. Advances in antioxidative bioactive macromolecules. IOP Conference Series: Earth and Environmental Science. 2020;512(1). https://doi.org/10.1088/1755-1315/512/1/012094

21. Nascimento-Souza MA, de Paiva PG, Pérez-Jiménez J, do Carmo Castro Franceschini S, Ribeiro AQ. Estimated dietary intake and major food sources of polyphenols in elderly of Viçosa, Brazil: a population-based study. European Journal of Nutrition. 2018;57(2):617-627. https://doi.org/10.1007/s00394-016-1348-0

22. Fraga CG, Croft KD, Kennedy DO, Tomás-Barberán FA. The effects of polyphenols and other bioactives on human health. Food and Function. 2019;10(2):514-528. https://doi.org/10.1039/C8FO01997E

23. Majidinia M, Bishayee A, Yousefi B. Polyphenols: Major regulators of key components of DNA damage response in cancer. DNA Repair. 2019;82. https://doi.org/10.1016/j.dnarep.2019.102679

24. Abdel-Moneim AME, Shehata AM, Alzahrani SO, Shafi ME, Mesalam NM, Taha AE, et al. The role of polyphenols in poultry nutrition. Journal of Animal Physiology and Animal Nutrition. 2020;104(6):1851-1866. https://doi.org/10.1111/jpn.13455

25. Tungmunnithum D, Thongboonyou A, Pholboon A, Yangsabai A. Flavonoids and other phenolic compounds from medicinal plants for pharmaceutical and medical aspects: An overview. Medicines. 2018;5(3). https://doi.org/10.3390/medicines5030093

26. Papaevgeniou N, Chondrogianni N. Anti-aging and anti-aggregation properties of polyphenolic compounds in C. elegans. Current Pharmaceutical Design. 2018;24(19):2107-2120. https://doi.org/10.2174/1381612824666180515145652

27. Chaudhary MK, Rizvi SI. Invertebrate and vertebrate models in aging research. Biomedical Papers. 2019;163(2):114-121. https://doi.org/10.5507/bp.2019.003

28. Ye Y, Gu Q, Sun X. Potential of Caenorhabditis elegans as an antiaging evaluation model for dietary phytochemicals: A review. Comprehensive Reviews in Food Science and Food Safety. 2020;19(6):3084-3105. https://doi.org/10.1111/1541-4337.12654

29. Reiss AP, Rankin CH. Gaining an understanding of behavioral genetics through studies of foraging in Drosophila and learning in C. elegans. Journal of Neurogenetics. 2021;35(3):119-131. https://doi.org/10.1080/01677063.2021.1928113

30. Douglas AE. The Drosophila model for microbiome research. Lab Animal. 2018;47(6):157-164. https://doi.org/10.1038/s41684-018-0065-0

31. Markaki M, Tavernarakis N. Caenorhabditis elegans as a model system for human diseases. Current Opinion in Biotechnology. 2020;63:118-125. https://doi.org/10.1016/j.copbio.2019.12.011

32. Mack HID, Heimbucher T, Murphy CT. The nematode Caenorhabditis elegans as a model for aging research. Drug Discovery Today: Disease Models. 2018;27:3-13. https://doi.org/10.1016/j.ddmod.2018.11.001

33. Salzer L, Witting M. Quo vadis Caenorhabditis elegans metabolomics - A review of current methods and applications to explore metabolism in the nematode. Metabolites. 2021;11(5). https://doi.org/10.3390/metabo11050284

34. Martel J, Wu C-Y, Peng H-H, Ko Y-F, Yang, H-C, Young, JD, et al. Plant and fungal products that extend lifespan in Caenorhabditis elegans. Microbial Cell. 2020;7(10):255-269. https://doi.org/10.15698/mic2020.10.731

35. Elkabti AB, Issi L, Rao RP. Caenorhabditis elegans as a model host to monitor the Candida infection processes. Journal of Fungi. 2018;4(4). https://doi.org/10.3390/jof4040123

36. Ayuda-Durán B, González-Manzano S, González-Paramás AM, Santos-Buelga C. Caernohabditis elegans as a model organism to evaluate the antioxidant effects of phytochemicals. Molecules. 2020;25(14). https://doi.org/10.3390/molecules25143194

37. Van Pelt KM, Truttmann MC. Caenorhabditis elegans as a model system for studying aging-associated neurodegenerative diseases. Translational Medicine of Aging. 2020;4:60-72. https://doi.org/10.1016/j.tma.2020.05.001

38. Bouyanfif A, Jayarathne S, Koboziev I, Moustaid-Moussa N. The nematode Caenorhabditis elegans as a model organism to study metabolic effects of ω-3 polyunsaturated fatty acids in obesity. Advances in Nutrition. 2019;10(1):165-178. https://doi.org/10.1093/advances/nmy059

39. Wang C, Xia C, Zhu Y, Zhang H. Innovative fluorescent probes for in vivo visualization of biomolecules in living Caenorhabditis elegans. Cytometry Part A. 2021;99(6):560-574. https://doi.org/10.1002/cyto.a.24325

40. Sandhof CA, Hoppe SO, Tittelmeier J, Nussbaum-Krammer C. C. elegans models to study the propagation of prions and prion-like proteins. Biomolecules. 2020;10(8). https://doi.org/10.3390/biom10081188

41. Hunt PR. The C. elegans model in toxicity testing. Journal of Applied Toxicology. 2017;37(1):50-59. https://doi.org/10.1002/jat.3357

42. Martel J, Wu C-Y, Peng H-H, Ko Y-F, Yang H-C, Young JD, et al. Plant and fungal products that extend lifespan in Caenorhabditis elegans. Microbial Cell. 2020;7(10):255-269. https://doi.org/10.15698/mic2020.10.731

43. Backes C, Martinez-Martinez D, Cabreiro F. C. elegans: A biosensor for host-microbe interactions. Lab Animal. 2021;50(5):127-135. https://doi.org/10.1038/s41684-021-00724-z

44. Ma L, Zhao Y, Chen Y, Cheng B, Peng A, Huang K. Caenorhabditis elegans as a model system for target identification and drug screening against neurodegenerative diseases. European Journal of Pharmacology. 2018;819:169-180. https://doi.org/10.1016/j.ejphar.2017.11.051

45. Liang JJH, McKinnon IA, Rankin CH. The contribution of C. elegans neurogenetics to understanding neurodegenerative diseases. Journal of Neurogenetics. 2020;34(3-4):527-548. https://doi.org/10.1080/01677063.2020.1803302

46. Keith Blackwell T, Sewell AK, Wu Z, Han M. TOR signaling in Caenorhabditis elegans development, metabolism, and aging. Genetics. 2019;213(2):329-360. https://doi.org/10.1534/genetics.119.302504

47. Fedorova AM, Dmitrieva AI, Dyshlyuk LS. Cultivation of wild medicinal plants of the SFD in vitro to accumulate the potential geroprotectors. Scientific Works of the North Caucasus Federal Scientific Center of Horticulture, Viticulture, Winemaking. 2020;30:134-138. (In Russ.). https://doi.org/10.30679/2587-9847-2020-30-134-138

48. Baranenko D, Bespalov V, Nadtochii L, Shestopalova I, Chechetkina A, Lepeshkin A, et al. Development of encapsulated extracts on the basis of meadowsweet (Filipendula ulmaria) in the composition of functional foods with oncoprotective properties. 2019;17(5):1829-1838. https://doi.org/10.15159/ar.19.155

49. Bespalov VG, Alexandrov VA, Vysochina GI, Kostikova VA, Semenov AL, Baranenko DA. Inhibitory effect of Filipendula ulmaria on mammary carcinogenesis induced by local administration of methylnitrosourea to target organ in rats. Anti-Cancer Agents in Medicinal Chemistry. 2018;18(8):1177-1183. https://doi.org/10.2174/1871520618666180402125913

50. Shaldayeva TM, Vysochina GI, Kostikova VA. Phenolic compounds and antioxidant activity of some species of the genus Filipendula Mill. (Rosaceae). Proceedings of Voronezh State University. Series: Chemistry. Biology. Pharmacy. 2018;(1):204-212. (In Russ.).

51. Kurkin VA, Sazanova KN, Zaitseva EN, Sharipova SKh, Pravdivtseva OE, Avdeeva EV, et al. Antidepressant activity of flavonoids and thick extract from Filipendula ulmaria fruit. Pharmaceutical Chemistry Journal. 2020;54(8):797-799. https://doi.org/10.1007/s11094-020-02276-x

52. Adamczak A, Ożarowski M, Karpiński TM. Antibacterial activity of some flavonoids and organic acids widely distributed in plants. Journal of Clinical Medicine. 2020;9(1). https://doi.org/10.3390/jcm9010109

53. Satari A, Ghasemi S, Habtemariam S, Asgharian S, Lorigooini Z. Rutin: A flavonoid as an effective sensitizer for anticancer therapy; insights into multifaceted mechanisms and applicability for combination therapy. Evidence-based Complementary and Alternative Medicine. 2021;2021. https://doi.org/10.1155/2021/9913179

54. Wang C, Shang S, Zheng X, Lei P, Han J, Yuan L, et al. Fluorescent sensors based on Cu-doped carbon quantum dots for the detection of rutin. Journal of the Brazilian Chemical Society. 2019;30(5):988-996. https://doi.org/10.21577/0103-5053.20180245

55. Negahdari R, Bohlouli S, Sharifi S, Maleki Dizaj S, Rahbar Saadat Y, Khezri K, et al. Therapeutic benefits of rutin and its nanoformulations. Phytotherapy Research. 2021;35(4):1719-1738. https://doi.org/10.1002/ptr.6904

56. Singh SK, Srivastav S, Castellani RJ, Plascencia-Villa G, Perry G. Neuroprotective and antioxidant effect of Ginkgo biloba extract against AD and other neurological disorders. Neurotherapeutics. 2019;16(3):666-674. https://doi.org/10.1007/s13311-019-00767-8

57. Priyanka A, Sindhu G, Shyni GL, Preetha Rani MR, Nisha VM, Raghu KG. Bilobalide abates inflammation, insulin resistance and secretion of angiogenic factors induced by hypoxia in 3T3-L1 adipocytes by controlling NF-κB and JNK activation. International Immunopharmacology. 2017;42:209-217. https://doi.org/10.1016/j.intimp.2016.11.019

58. Eisvand F, Razavi BM, Hosseinzadeh H. The effects of Ginkgo biloba on metabolic syndrome: A review. Phytotherapy Research. 2020;34(8):1798-1811. https://doi.org/10.1002/ptr.6646

59. Patel RV, Mistry BM, Shinde SK, Syed R, Singh V, Shin H-S. Therapeutic potential of quercetin as a cardiovascular agent. European Journal of Medicinal Chemistry. 2018;155:889-904. https://doi.org/10.1016/j.ejmech.2018.06.053

60. Li X, Zhou N, Wang J, Liu Z, Wang X, Zhang Q, et al. Quercetin suppresses breast cancer stem cells (CD44+/CD24−) by inhibiting the PI3K/Akt/mTOR-signaling pathway. Life Sciences. 2018;196:56-62. https://doi.org/10.1016/j.lfs.2018.01.014

61. Bahadır HM, Sarıgöz T, Topuz Ö, Sevim Y, Ertan T, Sarıcı İŞ. Protective effects of quercetin on hepatic ischemia-reperfusion injury. Istanbul Medical Journal. 2018;19(1):47-51. https://doi.org/10.5152/imj.2017.72325

62. Kashyap D, Sharma A, Tuli HS, Sak K, Punia S, Mukherjee TK. Kaempferol - A dietary anticancer molecule with multiple mechanisms of action: Recent trends and advancements. Journal of Functional Foods. 2017;30:203-219. https://doi.org/10.1016/j.jff.2017.01.022

63. Zhao T, Tang H, Xie L, Zheng Y, Ma Z, Sun Q, et al. Scutellaria baicalensis Georgi.(Lamiaceae): a review of its traditional uses, botany, phytochemistry, pharmacology and toxicology. Journal of Pharmacy and Pharmacology. 2019;71(9):1353-1369. https://doi.org/10.1111/jphp.13129

64. Wang Z-L, Wang S, Kuang Y, Hu Z-M, Qiao X, Ye M. A comprehensive review on phytochemistry, pharmacology, and flavonoid biosynthesis of Scutellaria baicalensis. Pharmaceutical Biology. 2018;56(1):465-484. https://doi.org/10.1080/13880209.2018.1492620

65. Liao H, Ye J, Gao L, Liu Y. The main bioactive compounds of Scutellaria baicalensis Georgi. for alleviation of inflammatory cytokines: A comprehensive review. Biomedicine and Pharmacotherapy. 2021;133. https://doi.org/10.1016/j.biopha.2020.110917

66. Antonescu A-I, Miere F, Fritea L, Ganea M, Zdrinca M, Dobjanschi L, et al. Perspectives on the combined effects of Ocimum basilicum and Trifolium pratense extracts in terms of phytochemical profile and pharmacological effects. Plants. 2021;10(7). https://doi.org/10.3390/plants10071390

67. Egan LM, Hofmann RW, Seguin P, Ghamkhar K, Hoyos-Villegas V. Pedigree analysis of pre-breeding efforts in Trifolium spp. germplasm in New Zealand. BMC Genet. 2020;21(1). https://doi.org/10.1186/s12863-020-00912-9

68. Kubes J, Skalicky M, Tumova L, Martin J, Hejnak V, Martinkova J. Vanadium elicitation of Trifolium pretense L. cell culture and possible pathways of produced isoflavones transport across the plasma membrane. Plant Cell Reports. 2019;38(5):657-671. https://doi.org/10.1007/s00299-019-02397-y

69. Wang G, Wang J, Liu W, Nisar MF, El-Esawi MA, Wan C. Biological activities and chemistry of triterpene saponins from Medicago species: An update review. Evidence-based Complementary and Alternative Medicine. 2021;2021. https://doi.org/10.1155/2021/6617916

70. Sharma P, Kumar V, Guleria P. Naringin: Biosynthesis and pharmaceutical applications. Indian Journal of Pharmaceutical Sciences. 2019;81(6): 988-999. https://doi.org/10.36468/pharmaceutical-sciences.596

71. Bhia M, Motallebi M, Abadi B, Zarepour A, Pereira-Silva M, Saremnejad F, et al. Naringenin nano-delivery systems and their therapeutic applications. Pharmaceutics. 2021;13(2). https://doi.org/10.3390/pharmaceutics13020291

72. Salehi B, Mishra AP, Shukla I, Sharifi-Rad M, Contreras MDM, Segura-Carretero A, et al. Thymol, thyme, and other plant sources: Health and potential uses. Phytotherapy Research. 2018;32(9):1688-1706. https://doi.org/10.1002/ptr.6109

73. Abbaszadeh S, Teimouri H, Farzan B. An ethnobotanical study of medicinal plants with antianxiety and antidepressant effects in Shahrekord. Egyptian Journal of Veterinary Sciences. 2019;50(1):81-87.

74. Seo DY, Lee SR, Heo J-W, No M-H, Rhee BD, Ko KS, et al. Ursolic acid in health and disease. Korean Journal of Physiology and Pharmacology. 2018;22(3):235-248. https://doi.org/10.4196/kjpp.2018.22.3.235

75. Mlala S, Oyedeji AO, Gondwe M, Oyedeji OO. Ursolic acid and its derivatives as bioactive agents. Molecules. 2019;24(15). https://doi.org/10.3390/molecules24152751

76. Bijttebier S, Van Der Auwera A, Voorspoels S, Noten B, Hermans N, Pieters L, et al. A first step in the quest for the active constituents in Filipendula ulmaria (meadowsweet): Comprehensive phytochemical identification by liquid chromatography coupled to quadrupole-orbitrap mass spectrometry. Planta Medica. 2016;82(6):559-572. https://doi.org/10.1055/s-0042-101943

77. Budeč M, Bošnir J, Racz A, Lasić D, Brkić D, Mosović Ćuić A, et al. Verification of authenticity of Ginkgo biloba L. Leaf extract and its products present on the Croatian market by analysis of quantity and ratio of ginkgo flavone glycosides (quercetin, kaempferol and isorhamnetin) to terpene trilactones to the effect of unmasking counterfeit drugs endangering patient health. Acta Clinica Croatica. 2019;58(4):672-692. https://doi.org/10.20471/acc.2019.58.04.15

78. Boyko NN, Pisarev DI, Zhilyakova ET, Maljutina AYu, Novikov OO, Bocharnikova MA. Study of baicalin hydrolysis kinetics in the process of its extraction from Scutellaria baicalensis Georgi roots. Pharmacy and Pharmacology. 2019;7(3):129-137. (In Russ.). https://doi.org/10.19163/2307-9266-2019-7-3-129-137

79. Lay H-L, Chen C-C, Chiang S-T. Simultaneous analysis of nine components in “Byi-Liang-Tang” preparation by high performance liquid chromatography. Journal of Food and Drug Analysis. 2004;12(2):115-119. https://doi.org/10.38212/2224-6614.2654

80. Dyshlyuk LS, Fedorova AM, Loseva AI, Eremeeva NI. Callus cultures of Thymus vulgaris and Trifolium pratense as a source of geroprotectors. Food Processing: Techniques and Technology. 2021;51(2):423-432. https://doi.org/10.21603/2074-9414-2021-2-423-432

81. Chen XM, Wang FF, Wang YQ, Li XL, Wang AR, Wang CL, et al. Discrimination of the rare medicinal plant Dendrobium officinale based on naringenin, bibenzyl, and polysaccharides. Science China Life Sciences. 2012;55(12):1092-1099. https://doi.org/10.1007/s11427-012-4419-3

82. Shimada A, Ueno H, Inagaki M. Glutaminase inhibitory activities of pentacyclic triterpenes isolated from Thymus vulgaris L. Natural Product Research. 2021;36(11):1864-2868. https://doi.org/10.1080/14786419.2021.1921766

83. Fedorova YuS, Sukhikh AS, Suslov NI, Zakhar YuV, Soboleva OM. Chromatography of hedysarum extracts on modified Sepharose CL sorbents. Fundamental and Clinical Medicine. 2019;4(3):61-67. (In Russ.). https://doi.org/10.23946/2500-0764-2019-4-3-61-67