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Archives • 2021 • vol.2 • 167-178
http://pharmacologyonline.silae.it
ISSN: 1827-8620
STUDY OF THE HYPOGLYCEMIC EFFECT OF THE EXTRACT FROM THE
TUBERS OF STACHYS SIEBOLDII MIQ.
Slobodianiuk, Liudmyla 1*; Budniak, Liliia 2; Marchyshyn, Svitlana 1; Berdey, Ihor 2; Slobodianiuk,
Olha 3
1Department of Pharmacognosy and Medical Botany, I. Horbachevsky Ternopil National Medical
University, Maidan Voli 1, 46001 Ternopil, Ukraine
2Department of Pharmacy Management, Economics and Technology, I. Horbachevsky Ternopil National
Medical University, Maidan Voli 1, 46001 Ternopil, Ukraine
3Department of Developmental Psychology and Counseling, Ternopil Volodymyr Hnatiuk National
Pedagogical University, 2 Maxyma Kryvonosa str., 46018 Ternopil, Ukraine
* husaklv@tdmu.edu.ua
Abstract
The prevalence of diabetes mellitus is becoming an epidemic. The variety of etiological factors
contributes to the fact that types 2 of diabetes mellitus occur among different age groups and among
different segments of the population. Particular attention deserves the different medicinal plants such
will have more biologically active substances that will influence all links of the pathogenetic mechanism
of development of diabetes mellitus and its complications. Of great interest in this regard is one of the
oldest vegetable and medicinal plants Stachys sieboldii MIQ. The study of the hypoglycemic effect of
the extract from the tubers of Stachys sieboldii was conducted on a model of dexamethasone insulin
resistance caused in rats weighing 180–230 g by subcutaneous administration of dexamethasone at a
dose of 4 mg/kg for 4 days. The most pronounced hypoglycemic effect of the studied extract was
found in doses of 25 and 50 mg/kg and with increasing dose, the activity of the test sample decreased.
In terms of expressiveness of the hypoglycemic effect, the extract from the tubers of Stachys sieboldii
at a dose of 25 mg/kg significantly prevailed, and at a dose of 50 mg/kg was not inferior to the
reference drug the official herbal mixture “Arfazetin”. The results obtained are an experimental
rationale for extending the indications of use of the extract from the tubers of Stachys sieboldii.
Keywords: extract from the tubers of Stachys sieboldii, hypoglycemic effect, diabetes mellitus,
dexamethasone, insulin
PhOL Slobodianiuk, et al. 168 (pag 167-178)
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ISSN: 1827-8620
Introduction
Diabetes mellitus is one of the most problems of
the world, which requires instant solutions, as the
epidemiological situation is alarming. The number of
patients is increasing rapidly each year, leading to
increased disability and mortality due to the
development of angiopathies [1-3]. Modern
pharmacotherapy increasingly takes into account
the centuries-old experience of folk medicine with
the use of phytopreparations as monotherapy and
in combination with synthetic drugs [4, 5]. The
advent of synthetic drugs, which mostly simulate
the biologically active substances of plants, has not
reduced the role of natural drugs [6, 7]. This is quite
justified because phytotherapy has a number of
advantages over traditional therapy with synthetic
drugs, namely, it is low-toxic, has a mild
pharmacological effect, and can be used for a long
period of time without significant side effects [8, 9].
Medicinal plants are unique sources of healing
compounds – biologically active substances that are
used both for the prevention and treatment of
different diseases of the human body [10-13]. Of
great interest in this regard is one of the oldest
vegetable and medicinal plants – Japanese artichoke
(Stachys sieboldii MIQ), also known as stachys or
Chinese artichoke. According to traditional oriental
medicine, the root tubers of Japanese artichoke
help improve digestion and have a healing effect in
diabetes and hypertension [14].
Japanese artichoke has long been used in Chinese
and Tibetan folk medicine in the treatment of
tuberculosis, hypertension, ischemic stroke, senile
dementia, and various gastrointestinal diseases.
Biologically active substances contained in root
tubers have a positive effect on carbohydrate and
lipid metabolism, lower blood pressure, cholesterol
[15, 16]. Biologically active substances of stachys
manifest a wide range of pharmacological
properties in the complete absence of toxicity. In
folk medicine, its tubers are used as a hypoglycemic,
anticoagulant, antihypertensive, antiulcer and
sedative agent [17, 18]. It lowers cholesterol,
regulates metabolic processes and strengthens the
immune system [19, 20]. In the literature there is
information about the antimicrobial and antitumor
activity of stachys [15].
Hiroaki Nishimura et al. have singled out from the
leaves of Stachys sieboldii on the basis of chemical
and spectral analysis of the structure of three new
glycosides, called stachysosides A, B and C, they
were identified as 2- (3,4-dihydroxyphenyl) ethyl O-
α-l-arabinopyranosyl- (1 →2) -α-1-rhamnopyranosyl-
(1→3)-4-OE-caffeoyl-β-d-glucopyranoside
(stachysosides A); 2-(3,4-dihydroxyphenyl)ethyl O-α-
1-arabinopyranosyl-(1→2)-α-1-rhamnopyranosyl-
(1→3)-4-OE-feruloyl-β-d-glucopyranoside
(stachysosides B)and 2-(3-hydroxy-4-
methoxyphenyl)ethyl O-α-1-arabinopyranosyl-(1→2)-
α-1-rhamnopyranosyl-(1→3)-4-OE-feruloyl-β-d-
glucopyranoside (stachysosides C) [21]. These
compounds have been shown to provide Stachys
sieboldii with antioxidant activity because they
inhibit hyaluronidase activity [18].
Japanese scientists have found that methanolic
tuber extract of Japanese artichoke, which contains
glycosides, including acteoside and stachysosides C,
significantly inhibits induced mortality from
potassium cyanide poisoning in mice [22]. This
extract inhibits hyaluronidase activity, has anti-
inflammatory action, and is effective in kidney
disease [17].
The antimicrobial activity of methanolic and
ethanolic extracts from the leaves, herb and tubers
of Japanese artichoke was studied. It was found
that methanol extract from the leaves and tubers
and ethanol extract from the tubers of Stachys
sieboldii show a pronounced antibacterial effect on
the culture of Salmonella typhimurium [23].
According to literature sources, the tubers of
Japanese artichoke contain up to 30.5 % dry matter,
of which up to 2.2 % is protein, up to 1.7 % – amides,
up to 0.2 % – fats, up to 19 % – carbohydrates.
Carbohydrates contain sugars – 1.8 %, fiber – 2.1 %,
pectin – 1.9 %. It should be noted that carbohydrates
contain a rare tetrasaccharide – stachyose, which is
similar in composition and properties to inulin and
has an insulin-like effect [24, 25].
Due to the lack of enzymes capable to hydrolyze
stachyose, in the human body, this substance is not
exposed to digestive enzymes and is not absorbed
in the upper gastrointestinal tract. Stachyose
PhOL Slobodianiuk, et al. 169 (pag 167-178)
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ISSN: 1827-8620
reaches the large intestine in unchanged form,
where fermentation occurs with the formation of
monosaccharide residues – α-galactose, β-fructose
and α-glucose [26].
Stachyose, like insulin (a hormone of the
pancreas), provides active absorption of
carbohydrates by organs and tissues.
Demonstrating such a biological effect, Japanese
artichoke becomes an indispensable medicinal plant
in diabetes mellitus, which is often complicated by
coronary heart disease, arrhythmia, hypertension,
pulmonary tuberculosis with the formation of
caverns, fatty liver disease, pyelonephritis,
neuralgia, severe visual disturbances, ulcers,
gangrene of the lower extremities, furunculosis of
the skin, etc. [27].
Therefore, the pharmacological study of
hypoglycemic action of the studied plant is relevant
in order to create domestic new hypoglycemic drugs
based on the tubers of Stachys sieboldii, which,
according to the literature, contains a
tetrasaccharide stachyose, which has an insulin-like
effect [25].
Methods
Plant Materials
Tubers of Chinese artichoke (S. sieboldii Miq.)
were collected on research grounds of Educational
and Scientific Centre “Institute of Biology and
Medicine”, Taras Shevchenko National University of
Kyiv in November 2017. The tubers was dried using
conventional methods and then stored in paper
bags in dry place [28-30]. A voucher specimen was
deposited in the laboratory herbarium of the
Department of Pharmacognosy and Medical Botany
(TSMU, Ternopil, Ukraine) [31, 32].
Preparation of extract
The dried sample was powdered by a pin crusher
and the powders were extracted 3 times with 70%
ethanol. 70% ethanolic extract was filtered through
filter paper (100 mm; Whatman, Maidstone, UK) and
evaporated using a vacuum rotary evaporator [33,
34].
Animal models
The experiments were performed on 50 white
Wistar rats weighing 180–230 g. All animals were
kept on a standard I. Horbachevsky Ternopil
National Medical University (TNMU), vivarium diet
[35]. The animals were kept in room having
temperature 22 ± 2 º C, and relative humidity of 44-
55 % under 12/12 hour light and dark cycle with
standard laboratory diet and water given ad libitum
[36].
Pharmacological studies have been conducted in
accordance with the rules and requirements of the
“General Principles for the Work on Animals”
approved by the I National Congress on Bioethics
(Kyiv, Ukraine, 2001 and agreed with the provisions
of the “European Convention for the Protection of
Vertebrate Animals used for Experimental and other
Scientific Purposes” (Council of Europe No 123,
Strasbourg 1985), and the Law of Ukraine “On the
Protection of Animals from Cruelty” of 26.02.2006
[37-40]. The removal of animals from the
experiment was carried out under light inhalation
(ether) anesthesia by decapitation.
Study of the hypoglycemic effect of the extract
from the tubers of Stachys sieboldii
The study of the hypoglycemic effect of the
extract from the tubers of Japanese artichoke was
conducted on a model of dexamethasone insulin
resistance caused in rats weighing 180–230 g by
subcutaneous administration of dexamethasone at
a dose of 4 mg/kg for 4 days. According to the
literature, the administration of glucocorticoids in
high doses leads to the development of moderate
basal hyperglycemia, increased concentrations of
insulin and free fatty acids in the serum of rats. In
addition, they show a decrease in the sensitivity of
peripheral tissues to insulin and impaired glucose
tolerance [41, 42]. On the third day, the level of
basal glycemia was determined, which allowed
randomization of animals into groups by random
sampling using glucose levels as the main sign of
distribution.
The studied drugs were administered in the
therapeutic mode, the extract from the tubers of
Stachys sieboldii in doses of 25, 50 and 100 mg/kg,
the reference drug was an aqueous extract of the
official herbal mixture “Arfazetin” (Viola, Ukraine) –
at a dose of 9 ml/kg. It was started 4 days before the
PhOL Slobodianiuk, et al. 170 (pag 167-178)
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ISSN: 1827-8620
last dexamethasone injection and continued for 4
consecutive days. Control animals received distilled
water in a similar manner.
On the 7th day of the experiment, the level of
basal glycemia in animals was determined and the
dynamics of hypoglycemic action of the extract
from the tubers of Stachys sieboldii (STE) was
studied. Glucose content was determined before
the administration of Japanese artichoke extract
(0 h) and 1, 2 and 4 hours after. An Intraperitoneal
glucose tolerance test was performed on the 8th
day of the experiment (only 5 days of administration
of the studied drugs).
Blood glucose was determined by the fasting
glucose oxidase method in the morning in blood
samples obtained from the tail vein of rats using
Phyllis-diagnostics biochemical kits according to the
instructions.
Intraperitoneal glucose tolerance test was
performed in the morning on an empty stomach
loading with glucose solution (3 g/kg) [42]. The
studied agents were administered intragastrically 1
hour before intraperitoneal administration of 40 %
glucose solution at a dose of 3 g/kg. The blood
glucose content of the animals was determined
immediately after administration of the studied
drugs (0 min) and 15, 45, 60 and 90 min after
carbohydrate loading.
The dynamics of hypoglycemic action were
assessed within 4 hours: the initial level was
determined, 1, 2, 3 and 4 hours after administration
of STE in different doses and the official herbal
mixture “Arfazetin”.
The intensity of lipid peroxidation processes and
the state of the antioxidant system were evaluated
to characterize the total effectiveness of STE in this
model. The content of substances that react with
thiobarbituric acid (TBA reactants, TBA-R), which are
the end products of degradation of unsaturated
fatty acids of membrane phospholipids and catalase
activity, an important enzyme of antioxidant system
was determined in the serum of experimental
animals on the background of diabetes. The content
of TBA reactants was determined by I. D. Stalna and
T. G. Garishvili methods [43].
The obtained experimental data were processed
by the methods of variation statistics (arithmetic
mean and its standard error were calculated). For
multiple comparisons of data with normal
distribution, parametric one-way analysis of variance
ANOVA was performed and Newman-Keuls method
was used, and the data were presented as mean (M)
and mean error (m). In other cases, comparisons of
samples using the Mann-Whitney test were used.
Differences between experimental groups were
considered statistically significant at p ≤ 0.05. A
standard package of statistical programs Statistica,
v. 6.0 (StatSoft inc. USA) was used to perform
mathematical calculations.
Results and Discussion
Determination of the dynamics of the
hypoglycemic effect of the extract from the tubers
of Stachys sieboldii (STE) showed that a single
injection in rats with diabetes, the studied agent
showed a stable moderate hypoglycemic effect,
which lasted for 4 h (Table 1). The studied extract
showed the greatest hypoglycemic effect at a dose
of 25 mg/kg. The activity of the studied agent
averaged 19 %. At doses of 50 and 100 mg/kg, the
extract almost did not reduce the level of basal
glucose, its activity averaged 8–10 %. The official
herbal mixture “Arfazetin” showed a similar
dynamics of hypoglycemic action, but in terms of
expressiveness was inferior to the extract of Stachys
sieboldii at a dose of 25 mg/kg (Table 1).
According to the data obtained, four-fold
administration of dexamethasone to rats at a dose
of 4 mg/kg led to the development of moderate
hyperglycemia, which lasted for the next 4 days.
Basal glucose level in animals after dexamethasone
injections increased statistically significantly to 7.89
mmol/l compared with 4.48 mmol/l in intact animals
(Fig. 1).
Therapeutic administration of the studied extract
in doses of 25 and 50 mg/kg contributed to reduce
the level of basal glycemia to the values of intact
animals (Fig. 1). Increasing the dose of the extract to
100 mg/kg led to a decrease in efficiency: the level of
basal glucose, although statistically significantly
lower than in the control pathology group, did not
reach the level of intact animals. A similar trend was
observed in the group of animals receiving the
PhOL Slobodianiuk, et al. 171 (pag 167-178)
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ISSN: 1827-8620
reference drug the official herbal mixture
“Arfazetin” at a dose of 9 ml/kg (Fig. 1).
At the end of the experiment, an intraperitoneal
glucose tolerance test was performed (Table 2), the
results of which indicated a violation of glucose
utilization processes (Table 2). The glycemic
response of rats from the control pathology group
to the carbohydrate load was characterized by a
slowing of the decrease in glucose level during the
whole observation period. The blood glucose level
of animals from the control pathology group
remained statistically higher than in intact animals at
90 min of the test (Table 2). It was established that
the inhibitory effect of glucocorticoids on the
secretory activity of pancreatic β-cells is associated
with the inactivation of mitochondrial FAD
glycerophosphate dehydrogenase, an enzyme that
plays a key role in glucose-stimulated insulin
secretion.
Therefore, the obtained data indicate the initial
disruption of glucose utilization processes due to
the administration of excessive doses of
dexamethasone.
When using the extract from the STE in doses of
25 and 50 mg/kg, the glycemic curve did not differ
from that in intact animals, indicating the
restoration of glucose utilization process and
carbohydrate metabolism in general (Table 2). The
effectiveness of the agent decreased when
increasing the dose of the extract to 100 mg/kg.
The dynamics of glycemia during the
intraperitoneal glucose tolerance test of the official
herbal mixture “Arfazetin” was the same as that
under the action of the studied extract, but less
pronounced (Table 2).
Determination of the main values of the markers
of the state of the lipid peroxidation
system/antioxidant system of the body showed that
under conditions of dexamethasone diabetes in the
blood of rats statistically significantly increases the
level of TBA-R. At the same time, the activity of
catalase – an enzyme of the antioxidant system,
showed a clear tendency to decrease. The absence
of a statistically significant difference between the
experimental groups is explained by the large
variability of intragroup values of this indicator.
Thus, the analysis of the obtained data allows
stating the imbalance of pro/antioxidant processes
in the lipid peroxidation/antioxidant system of the
rats’ body under the conditions of diabetes mellitus
caused by dexamethasone (Table 3).
The administration of the studied extract at doses
of 25 and 50 mg/kg led to the normalization of
processes in the system of lipid
peroxidation/antioxidant system of the body.
Increasing the dose of STE to 100 mg/kg contributed
to excessive activation of catalase, due to which
there was a significant decrease in the level of TBA-
R compared with both control pathology and
physiological values of intact animals (Table 3). It
should be noted that a reference drug the official
herbal mixture “Arfazetin” showed the least
pronounced antioxidant effect of the studied drugs.
Thus, the model of dexamethasone diabetes has
pronounced hypoglycemic and antioxidant
properties of the extract from the tubers of Stachys
sieboldii. The most pronounced hypoglycemic effect
of the studied STE was shown in doses of 25 and 50
mg/kg, with increasing dose the activity of the
studied agent decreased. In terms of expressiveness
of the hypoglycemic effect, the studied extract at a
dose of 25 mg/kg significantly prevails, and at a dose
of 50 mg/kg is not inferior to the reference drug the
official herbal mixture “Arfazetin”.
Conclusions
For the first time, the screening study of the
hypoglycemic activity of extract of the Stachys
sieboldii tubers used in folk medicine for the
prevention and treatment of diabetes mellitus type
2 was conducted. The model of dexamethasone
diabetes has pronounced hypoglycemic and
antioxidant properties of the extract from the
tubers of Stachys sieboldii. The most pronounced
hypoglycemic effect of the studied extract was
found in doses of 25 and 50 mg/kg and with
increasing dose, the activity of the test sample
decreased. In terms of expressiveness of the
hypoglycemic effect, the EST at a dose of 25 mg/kg
significantly prevailed, and at a dose of 50 mg/kg
was not inferior to the reference drug the official
herbal mixture “Arfazetin”. The antihyperglycemic,
hypolipidemic and antioxidant effects make these
extract of Stachys sieboldii tubers promising tools
PhOL Slobodianiuk, et al. 172 (pag 167-178)
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ISSN: 1827-8620
for prevention and treatment of diabetes and its
complications.
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PhOL Slobodianiuk, et al. 175 (pag 167-178)
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ISSN: 1827-8620
Table 1. Dynamics of hypoglycemic action of Stachys sieboldii extract on a model of dexamethasone diabetes,
n=6 (M±m)
Group of
animals
Term of observation
0 h
1 h
2 h
4 h
mmol/l
mmol/l
% decrease
mmol/l
% decrease
mmol/l
% decrease
Intact control
4.48±
0.06
4.23±
0.16
5.70±3.21
4.04±
0.10
9.84±2.41
4.15±
0.16
7.32±3.46
Control
pathology
7.87±
0.34*
7.12±
0.20*
8.94±3.34
7.31±
0.35*
6.98±2.76
7.41±
0.41*
5.81±3.03
STE,
25 mg/kg
7.26±
0.47*
6.04±
0.12**
15.04±
5.59
5.73±
0.21*/**
20.13±
3.17*/**
5.59±
0.20**
22.12±
3.09*/**
STE,
50 mg/kg
7.54±
0.40*
7.08±
0.22#
5.52±
2.77
6.86±
0.21#
8.41±
2.69
6.65±
0.20#
11.31±
2.60
STE,
100 mg/kg
7.52±
0.38*
6.97±
0.26#
6.87±
2.27#
6.84±
0.28#
8.76±
2.05#
6.50±
0.27#
13.36±
2.01#
Arfazetin,
9 ml/kg
7.96±
0.20*
7.06±
0.29#
10.82±
4.73#
7.42±
0.36#
6.96±
2.77#
7.20±
0.26#
8.58±
2.81#
Notes: * − the differences are statistically significant for the group of intact control, р<0.05;
** − the differences are statistically significant for the group of control pathology, р<0.05;
# − the differences are statistically significant for the group of STE (25 mg/kg), р<0,05;
n – number of animals in a group;
STE – extract from the tubers of Stachys sieboldii.
PhOL Slobodianiuk, et al. 176 (pag 167-178)
http://pharmacologyonline.silae.it
ISSN: 1827-8620
Figure 1. Dynamics of basal glycemia in rats on the background of the administration of extract of the Stachys
sieboldii tubers in comparison with the official herbal mixture “Arfazetin” in the model of dexamethasone
diabetes, n = 6
Notes: IC – intact control;
CP – control pathology (dexamethasone diabetes);
STE – thick extract from the tubers of Stachys sieboldii + dexamethasone diabetes;
Arfazetin – the official herbal mixture “Arfazetin” + dexamethasone diabetes;
* − the differences are statistically significant for the group of intact control, р<0.05;
** − the differences are statistically significant for the group control pathology, р<0 .05;
n – number of animals in a group.
4.48
3.58
7.87*
5.98*
7.26*
4.38**
7.54*
4.02**
7.52*
4.89*/**
7.96*
4.81*/**
0
1
2
3
4
5
6
7
8
9
10
4 доба (введення дексаметазону)
8 доба (лікування ГЕ)
Glucose, mmol/l
Ba sa l glycemia, m mol/l
ІК
КП
ГЕ, 25 мг/кг
ГЕ, 50 мг/кг
ГЕ, 100 мг/кг
Арфазетин, 9 мл/кг
4th day (administration of dexamethasone)
8th day (treatment with TE)
IC
CP
STE 25 mg/kg
STE 50 mg/kg
STE 100 mg/kg
Arfazetin 9 ml/kg
PhOL Slobodianiuk, et al. 177 (pag 167-178)
http://pharmacologyonline.silae.it
ISSN: 1827-8620
Table 2. Influence of Stachys sieboldii extract on glycemic dynamics in glucose tolerance test in animals with
dexamethasone-induced insulin resistance, n = 6 (M ± m)
Group of animals
Term of observation
0 min
15 min
45 min
60 min
90 min
Intact control
3.58±
0.14
10.72±
0.43
8.50±
0.34
6.38±
0.25
3.27±
0.13
Control pathology
5.98±
0.35*
13.65±
1.05
11.63±
1.28
8.77±
0.54*
5.77±
0.39*
STE,
25 mg/kg
4.38±
0.20**
7.74±
1.03**
7.79±
0.17**
5.72±
0.06**
4.12±
0.21**
STE,
50 mg/kg
4.02±
0.20**
12.78±
0.49
10.39±
1.02
7.71±
0.71**
4.63±
0.32**
STE,
100 mg/kg
4.89±
0.15*/**
11.86±
1.03
6.96±
0.49**
6.01±
0.52**
5.97±
0.42*
Arfazetin,
9 ml/kg
4.81±
0,15*/**
12.11±
1.16
9.37±
0.69
6.33±
0.41**
6.06±
0.42*
Notes: * − the differences are statistically significant for the group of intact control, р<0.05;
** − the differences are statistically significant for the group of control pathology, р<0.05;
# − the differences are statistically significant for the group of extract from the tubers of Stachys sieboldii
(dose of 25 mg/kg), р<0.05;
n – number of animals in a group;
STE – extract from the tubers of Stachys sieboldii.
PhOL Slobodianiuk, et al. 178 (pag 167-178)
http://pharmacologyonline.silae.it
ISSN: 1827-8620
Table 3. Influence of Stachys sieboldii extract on the state of pro/antioxidant processes in the LPO/AOS under
conditions of dexamethasone diabetes in rats, n = 6 (M ± m)
Group of animals
Values
TBA-R
Catalase
Intact control
1.20±0.05
19.52±2.55
Control pathology
1.96±0.10*
13.70±0.67*
STE, 25 mg/kg
1.29±0.08**# α
17.23±1.18*/**
STE, 50 mg/kg
0.82±0.02**# α
19.82±1.97**# α
STE, 100 mg /kg
0.78±0.07*/**#α
23.65±4.46*/**# α
Arfazetin, 9 ml/kg
1.48±0.03**#
17.27±2.01*/**
Notes: * − the differences are statistically significant for the group of intact control, р<0.05;
** − the differences are statistically significant for the group of control pathology, р<0.05;
# − the differences are statistically significant for the group of STE (dose of 25 mg/kg), р<0.05;
α − the differences are statistically significant for the group of reference drug the official herbal mixture
“Arfazetin”, р<0.05;
n – number of animals in a group;
STE – extract from the tubers of Stachys sieboldii.
