INHIBITION OF PANCREATIC α-AMYLASE BY WATER EXTRACTS OF SOME HERBAL MIXTURES

Abstract

Diabetes mellitus is an important social and medical problem, as it causes the development of dangerous complications that lead to disability and mortality. This disease is characterized by a multi-vector pathogenesis that requires a comprehensive approach to treatment. Inhibition of pancreatic α-amylase activity is an important mechanism in the prevention and treatment of type 2 diabetes. The aim of our research was to study an inhibitory α-amylase activity of the herbal mixtures, which are used in folk medicine for the prevention and treatment of diabetes mellitus type 2 in Ukraine and with established hypoglycemic, hypolipidemic, antioxidant, hepatoprotective, pancreatoprotective activity in pharmacological study in vivo and the defined phytochemical composition that determines such pharmacodynamics. During the study of antidiabetic activity in vitro it was established the α-amylase IC50 was 699.49 µg/mL of the sample 1, 758.15 µg/mL of the sample 2, 781.76 µg/mL of the sample 3, 700.17 µg/mL of the sample 4 and 646.52 µg/mL of the sample 5. The present study showed a high inhibitory activity of herbal mixtures to pancreatic α-amylase, which suggests the effectiveness of the studied herbal mixtures for the prevention and treatment of type 2 diabetes

Full text

Archives • 2021 • vol.2 • 1443-1449
http://pharmacologyonline.silae.it
ISSN: 1827-8620
INHIBITION OF PANCREATIC α-AMYLASE BY WATER EXTRACTS OF SOME
HERBAL MIXTURES
Savych, Alona*1, Marchyshyn, Svitlana1, Milian, Ivanna2
1Department of Pharmacognosy with Medical Botany, I. Horbachevsky Ternopil National Medical
University, Ukraine
2 Department of General Chemistry, I. Horbachevsky Ternopil National Medical University, Ukraine
*alonasavych@gmail.com
Abstract
Diabetes mellitus is an important social and medical problem, as it causes the development of
dangerous complications that lead to disability and mortality. This disease is characterized by a multi-
vector pathogenesis that requires a comprehensive approach to treatment. Inhibition of pancreatic α-
amylase activity is an important mechanism in the prevention and treatment of type 2 diabetes.
The aim of our research was to study an inhibitory α-amylase activity of the herbal mixtures, which
are used in folk medicine for the prevention and treatment of diabetes mellitus type 2 in Ukraine and
with established hypoglycemic, hypolipidemic, antioxidant, hepatoprotective, pancreatoprotective
activity in pharmacological study in vivo and the defined phytochemical composition that determines
such pharmacodynamics.
During the study of antidiabetic activity in vitro it was established the α-amylase IC50 was 699.49
µg/mL of the sample 1, 758.15 µg/mL of the sample 2, 781.76 µg/mL of the sample 3, 700.17 µg/mL of the
sample 4 and 646.52 µg/mL of the sample 5.
The present study showed a high inhibitory activity of herbal mixtures to pancreatic α-amylase,
which suggests the effectiveness of the studied herbal mixtures for the prevention and treatment of
type 2 diabetes
Keywords: diabetes mellitus, herbal mixtures, α-amylase activity, acarbose

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Introduction
Diabetes mellitus is a global social problem in the
field of health care, due to rapid spread of this
disease and the development of serious
complications such as micro- and
macroangiopathies, which significantly reduce the
quality and life expectancy of patients [1]. According
to the official information of International Diabetes
Federation (2019), the number of patients is
projected to increase to 642 million by 2040 [2].
An important problem of pharmacovigilance is
that existing pharmacotherapy can effectively
reduce hyperglycemia, but it is not always able to
stabilize fluctuations in glycemic values during the
day and maintain it at an optimal level [3, 4, 5, 6]. A
sudden rise in blood glucose levels, causing
hyperglycemia in type 2 diabetes patients happens
due to hydrolysis of starch by pancreatic α-amylase
and uptake of glucose by intestinal α-glucosidases
[7, 8, 9, 10]. The inhibition of enzymes involved in
the breakdown of starch (α-amylase) and uptake of
glucose (α–glucosidase) has been suggested to be a
useful approach to the management and prevention
of type 2 diabetes and dietary phytochemicals, have
promising potential [11, 12, 13, 14]. Amylase inhibitors
are also known as starch blockers because they
contain substances that prevent dietary starch from
being absorbed by the body. Starches are complex
carbohydrates that cannot be absorbed unless they
are first broken down by the digestive enzyme
amylase and other secondary enzymes [15, 16, 17,
18].
Therefore, the optimization of pharmacotherapy,
search and study of new drugs with antidiabetic
activity for the prevention and treatment of this
disease and its dangerous complications is a topical
issue of pharmacy and medicine.
One such area is phytotherapy, as it has a number
of advantages over traditional therapy with using
oral synthetic agents, namely, it is low-toxic, has a
mild pharmacological effect and can be used for
long periods without significant side effects, is well
combined with synthetic drugs, has a complex
activity through a number of biologically active
compounds [19, 20, 21, 22]. Particular attention
deserves the combinations of different medicinal
plants because such herbal mixtures will have more
biologically active substances that will influence on
all links of the pathogenetic mechanism of
development of diabetes mellitus and its
complications [23, 24, 25, 26].
In addition, acarbose is a medication clinically
used to inhibit α-glucosidase and α-amylase.
Unfortunately, its long-term administration resulted
in side effects including abdominal distention and
diarrhea. Alternative plant-derived products with
better safety potential may also be used for the
management of diabetes mellitus [27, 28, 29, 30].
Thus, the aim of our research was to study an
inhibitory α-amylase activity of the herbal mixtures,
which are used in folk medicine for the prevention
and treatment of diabetes mellitus type 2 and with
established hypoglycemic, hypolipidemic,
antioxidant, hepatoprotective, pancreatoprotective
activity in pharmacological study in vivo [23, 24, 25,
26] and the defined phytochemical composition that
determines such pharmacodynamics [16, 17, 18, 19,
20, 21].
Methods
Plant materials: The herbal raw materials
harvested in June to August 2019 in Ternopil region
(Ukraine) were used. After harvesting, the raw
materials were dried, crushed and brought back to
standard according to the general GACP
requirements [31]. The plants were identified by
Department of Pharmacognosy with Medical
Botany, I.Horbachevsky Ternopil National Medical
University, Ternopil, Ukraine. The voucher
specimens of the herbal raw materials have been
deposited in Departmental Herbarium for future
record.
For the study were used the five different herbal
mixtures, composition of which is given in Table 1.
Chemicals and standards: chemical reference
substance (CRS) of acarbose were of primary
reference standard grade (≥ 95 % purity HPLC) and
were purchased from Sigma-Aldrich Chemical
Company (Germany), as well as α-amylase. Water
used in the studies was produced by MilliQ Gradient
water deionizaton system (USA).
Extraction procedure: the samples of herbal raw
materials (10 g) were placed into a 100 mL conical
flask with120 mL of distilled water. The extractions
were carried out in a water bath for 30 min. The
resulting extracts were filtered using Whatmann

PhOL Savych, et al. 1445 (pag 1443-1449)
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filter paper No1. Then the filtrates were
evaporated by rotary evaporator and were
lyophilized to dryness. The lyophilized powders of
each herbal mixture were stored at 4 °C for further
use.
Inhibition of α-amylase enzyme: the method is
based on enzyme inhibition, so the transformation
of starch to reducing oligosaccharides that react
with 3,5-dinitrosalicylic acid is blocked. A total of 500
µL of samples of the studied extracts with a range
of concentrations100-1000 µg/mL were added to
500 µL of 0.20 mM phosphate buffer (pH 6.9 with
0.006 M sodium chloride) containing α-amylase
solution (0.5mg/mL) and were incubated at 25°C for
10 min. Thereafter, it was added 500 µL of (1% w/v)
starch solution in 0.02 M sodium phosphate buffer
(pH 6.9 with 0.006 M sodium chloride) to each tube
and was incubated at 25°C for 10 min. The reaction
was stopped with 1.0 mL of 3,5-dinitrosalicylic acid
colour reagent (12.0 g of sodium
potassium tartrate tetrahydrate in 8 mL of 2 M
NaOH and 96 mM 3,5- dinitrosalicylic acid solution).
Then the tubes were incubated in the boiling water
bath for 5 min and cooled to room temperature. The
reaction mixture was diluted by adding 10 mL of
distilled water and absorbance was measured at 540
nm using the spectrophotometer Shimadzu 1800-UV
(Japan). Experiment was performed in triplicate.
Acarbose was used as a positive control [5].
Calculation of 50% Inhibitory Concentration
(IC50): the inhibitory concentration of the water
extracts of the herbal mixtures required to inhibit
the activity of the enzyme by 50%, IC50 was
calculated by regression analysis using the
percentage scavenging activities at five different
concentrations of the extracts. Inhibition (I %) was
calculated by:
       
  
Results and Discussion
Management of the blood glucose level is a
critical strategy in the control of diabetes
complications. Inhibitors of saccharide hydrolysing
enzymes (α-amylase) have been useful as oral
hypoglycemic drugs for the control of
hyperglycemia especially in patients with type-2
diabetes mellitus [32, 33, 34]. Inhibition of this
enzyme delay carbohydrate digestion and prolong
overall carbohydrate digestion time, causing a
reduction in the rate of glucose absorption and
consequently reducing the postprandial plasma
glucose rise [35, 36, 37].
The experimental studies of antidiabetic activity
in vitro of investigated herbal mixtures with a range
of concentrations100-1000 µg/mL were performed
by inhibition of α-amylase activity compared with
acarbose.
The relationship between the increase in the
inhibitory activity of α-amylase and the
concentration of aqueous extracts of herbal
mixtures was revealed. During the study of
inhibition of α-amylase enzyme it was established
that the IC50 of the water extracts of the sample 1
was 699.49 µg/mL; the sample 2 – 758.15 µg/mL; the
sample 3 – 781.76µg/mL; the sample 4 – 700.17
µg/mL; the sample 5 – 646.52 µg/mL (Table 2). The
IC50 value of standard drug acarbose against α-
amylase was 246.22 µg/mL.
Inhibition of intestinal pancreatic α-amylase
activities results in delayed carbohydrate digestion
of absorbable monosaccharides leading to a drop in
postprandial hyperglycemia [38]. The search for a
new α-amylase inhibitor from herbal mixtures is a
striking method for the management of
postprandial hyperglycemia. Secondary metabolites
such as tannins, phenolic acids, and flavonoids are
the main phytoconstituents that possess α-amylase
inhibitory activity [39].
Conclusions
For the first time, it was conducted the study an
inhibitiry α-amylase activity of the water extracts of
the herbal mixtures, which are used in folk medicine
for the prevention and treatment of diabetes
mellitus type 2 and with established hypoglycemic,
hypolipidemic, antioxidant, hepatoprotective,
pancreatoprotective activity in pharmacological
study in vivo and the defined phytochemical
composition that determines such
pharmacodynamics. The present study showed a
high inhibitory activity of herbal mixtures to
pancreatic α-amylase, which is one of the
mechanisms of prevention and treatment of type 2
diabetes.

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ISSN: 1827-8620
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Table 1. Composition of the herbal mixtures
Herbal mixtures
Herbal drug component
Portion in the mixture, %
Relative ratio
Sample 1
Urtica dioica leaf
Cichorium intybus roots
Rosa majalis fruits
Elymus repens rhizome
Taraxacum officinale roots
26.32
26.32
21.05
15.79
10.52
5
5
4
3
2
Sample 2
Arctium lappa roots
Elymus repens rhizome
Zea mays columns with stigmas
Helichrysum arenarium flowers
Rosa majalis fruits
26.32
26.32
21.05
15.79
10.52
5
5
4
3
2
Sample 3
Inula helenium rhizome with roots
Helichrysi arenarium flowers
Zea mays columns with stigmas
Origanum vulgari herb
Rosa majalis fruits
Taraxacum officinale roots
10.0
20.0
20.0
20.0
20.0
10.0
1
2
2
2
2
1
Sample 4
Cichorium intybus roots
Elymus repens rhizome
Helichrysum arenarium flowers
Rosa majalis fruits
Zea mays columns with stigmas
26.32
26.32
21.05
15.79
10.52
5
5
4
3
2
Sample 5
Urtica dioica leaf
Taraxacum officinale roots
Vaccinium myrtillus leaf
Rosa majalis fruits
Mentha piperita herb
20.0
20.0
20.0
20.0
20.0
1
1
1
1
1

PhOL Savych, et al. 1449 (pag 1443-1449)
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the samples of the herbal mixtures
Herbal mixtures
Concetration, µg/mL
Inhibition, %
IC50, µg/mL
Sample 1
100
22.17± 3.65
699.49
200
30.97± 2.98
400
41.15± 3.51
800
52.97± 4.28
1000
61.64± 3.49
Sample 2
100
20.47± 3.31
758.15
200
28.58± 4.42
400
39.13± 5.28
800
51.27± 3.63
1000
59.34± 3.75
Sample 3
100
21.58± 3.53
781.76
200
32.11± 3.94
400
38.28± 2.37
800
50.56± 3.63
1000
60.18± 3.74
Sample 4
100
20.65± 3.62
700.17
200
31.07± 2.86
400
40.95± 3.73
800
53.01± 3.85
1000
58.75± 3.92
Sample 5
100
23.04± 3.76
646.52
200
31.65± 4.93
400
42.82± 2.71
800
54.47± 3.83
1000
63.18± 3.17
Acarbose (standart)
100
33.98± 1.92
246.22
200
47.37± 2.13
400
58.75±2.46
800
69.58± 2.06
1000
75.94± 1.99
Note: Values are expressed as mean ± SD (n=3).