European Journal of Biological Sciences 7 (1): 26-31, 2015

ISSN 2079-2085
© IDOSI Publications, 2015
DOI: 10.5829/idosi.ejbs.2015.7.01.91127

Alpha Amylase Inhibition Activity of Some Plants Extract of Teucrium Species

1
Zohre Mirzaalian Dastjerdi, 2Foroogh Namjoyan and Mohamad 3Ebrahim Azemi

Graduate of Pharmacy, Faculty of Pharmacy,

1

Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

2
Marine Pharmaceutical Research Center, Pharmacognosy Department,
Faculty of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
3
Department of Pharmacognosy, School of Pharmacy,
Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

Abstract: Diabetes Mellitus is a metabolic disorder characterized by high blood glucose level caused due to
deficiency of insulin secretion or insulin action. The inhibition of carbohydrate hydrolyzing enzymes such as
a-amylase can be an important strategy in the postprandial blood glucose level in patients with type II diabetes
Plants contains different chemical constituents with potential for inhibition of -amylase and hence maybe
used as therapeutic. three plants of Teucrium species of were tested for -amylase inhibition. Different
concentrations of extracts were incubated with enzyme substrate solution and the activity of enzyme was
measured. Also Acarbose was used as the standard inhibitor. Three plant extracts showed inhibition of
-amylase, The -amylase inhibitory activities of extracts are T. polium>T. oliverianum>T. Orientale. The IC50
value of Hydroalcoholic extract of T. polium was 3.63 mg/ml. The IC50 value of T. oliverianum and T. Orientale
were 3.86 and 13.93 mg/ml, respectively, when compared with acarbose (IC 50 value 0.037mg/mL). This study
supports that plants of Teucrium species exhibit considerable a-amylase inhibitory activities. all the fractions
(hydroalcoholic, dichloromethane and ethyl acetate) had potent inhibitory effects on the -amylase activity.
However, the lowest inhibitory potency was observed for the dichloromethane fraction. Determination of the
type of -amylase inhibition by these plant extracts could provide by successful use of plant chemicals as drug
targets.

Key words: T. polium T. oliverianum T. orientale -Amylase Inhibitory Effects Diabetes Mellitus

INTRODUCTION Available therapies for treating type II diabetes consist

Diabetes Mellitus (DM) is an extended metabolic
disease of several etiologies characterized by chronic
hyperglycemia with disorder of carbohydrate, fat and also
protein metabolism resulting from defects in insulin
secretion, insulin action or both of them [1]. The control
of hyperglycemia is very important in the treatment of all
forms of diabetes by reason that in the long term, acute
and chronic complications can happen when the blood
glucose concentration is not kept in the normal range
[2, 3]. The drugs widely used in clinic to manage or
handle diabetes are insulin, sulfonylureas, biguanide,
glycosidase inhibitors, aldosereductase inhibitor,
thiazolidinediones, carbamoylmethyl benzoic acid [4].
of stimulation of endogenous insulin secretion, increase
of activity of insulin at target tissues as well as inhibition
of alpha-amylase enzyme to reduce the degradation of
starch to decreasing glucose [5, 6].
One unique approach for decreasing postprandial
hyperglycemia is to reduce or slow down dietary
carbohydrate digestion. Inhibiting the enzymes involved,
such as the -amylase and -glucosidase enzymes, is a
strong the therapeutic goal of controlling the postprandial
glycemic reaction [7]. Also, -amylase inhibitors are one
of the anti-diabetic drug families, of which Acarbose
is the most well-known. These drugs have a very
strong advantage and are suitable for healing
noninsulin-dependent diabetes mellitus (type-2 diabetes)

Corresponding Author: Foroogh Namjoyan, Marine Pharmaceutical Research Center, Pharmacognosy Department,
School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
Tel: +986133361984, Fax: +9833361544, E-mail: namjoyan043@gmail.com.
26
 Europ. J. Biol. Sci., 7 (1): 26-31, 2014
[8, 9], but also induce gastrointestinal side effects that
reduce their use in a Preventive approach [10].
Accordingly, several researchers are investigating and
developing nutritional strategies to perfectly control
postprandial glycemia, without inducing negative
occasions in the digestive system, medicinal herbs due
to easy accessibility and also lower negative effects have
a special place in medicine to treat various diseases
[11- 13]. Active compounds derived from medicinal herbs
may also be a source of new -amylase inhibitors.
Furthermore, some plants include a great amount of
phenolic substances, therefore accordingly, they have
antioxidant activity [14].
Teucrium species plants, which are rich sources of
flavonoids, anthraquinone, steroid and phlobatannin,
have long been used to control hypertension,
inflammations, gastrointestinal disorders [15- 18] and
also leaves of Teucrium species have long been utilized
to management diabetes mellitus worldwide [19].
The hypoglycemic results of Teucrium species have been
supported by numerous researches, However, at the
least to our information, there are no previous studies on
the -amylase inhibitory effect on Teucrium species.
In this study, Teucrium species medicinal plants
(including T. oliverianum, T. Orientale and T. polium)
that are used to manage different diseases were screened
to discover possible new -amylase inhibition activity
using an in vitro -amylase inhibition assay.
MATERIALS AND METHODS
Chemical Materials: All chemical substances were
obtained from Sigma Aldrich ( USA ) and also Merck
(Germany) companies. The chemicals were of analytical
grade.
Plant Materials: Teucrium species young whole plants
were collected from Ahvaz Province, Iran in May 2013.
The plant was botanically identified and authenticated
by local Plant Biotechnologist, Department of Natural
Resources, khuzestan, Iran. The plants were dried at
ambient temperature (30-40°C) for 25-30 days Then they
were into fine powder.
Extraction and Fractionation Procedure: The air-dried
plants were carefully cleaned under running tap water in
order to remove any type of pollution and also air-dried in
shade, powdered in grinder and passed via sieve of mesh
size no-40. The extraction was handled aqueous ethanol
(30: 70) mixture by hot Soxhlet extraction technique and
27
the extract was condensed in a rotary evaporator under
decreased pressure, crude hydroalcoholic extract
(HAC). The dried crude extract was conserved in
airtight glass bottle at 4-8 . With serious model of
indomethacin-induced ulcer, HAC was discovered
extremely active also it was after that fractionated with
solvents of improving polarity. At first, HAC was
partition-fractionated with 1 :1 ( volume ratio ) of n-hexane
and ethanol ( 50% ) and the mixture was shaken intensely
and kept for about 30 min to make the two layers separate.
The upper layer comprising of n-hexane was removed and
concentrated in a rotary evaporator to obtain n-hexane
extract. The same procedure was repeated with the bottom
part layer by utilizing equivalent volume of additional
solvents such as, dichloromethane and ethyl acetate,
DCMC and EAC, respectively.
Assessment of -amylase Inhibition: The starch solution
(0.5% w/v) was obtained by stirring and boiling 0.25 g of
soluble potatostarch in 50 ml of deionized water for
15 min. The enzyme solution (0.5 unit/ml) was prepared
by mixing 0.001 g of -amylase (EC 3.2.1.1) in 100 ml of 20
mM sodium phosphate buffer(pH 6.9) containing 6.7 mM
sodium chloride. The extracts and/or fractions were
dissolved in DMSO to give suitable concentrations for
the assay. The color reagent was a solution containing
96 mM 3,5-dinitrosalicylic acid (20ml ), 5.31 M sodium
potassium tartrate in 2 M sodium hydroxide (8 ml) and
deionized water(12 ml ).One ml of each the extracts and/or
fractions and 1 ml of the enzyme solution were mixed in a
test tube and incubated at 25°C for 30 min. To 1ml of this
mixture was added 1 ml of the starch solution and the tube
was further incubated at 25°C for 3 min. Then, 1 ml of the
color reagent was added and the stoppered tube was
placed into an 85°C water bath. After 15 min, the reaction
mixture was removed from the water bath and cooled
thereafter, diluted with 9 ml distilled water and the
absorbance value determined at 540 nm using a Shimadzu
Multispect-1501spectrophotometer (Japan).
Individual blanks were prepared for correcting the
background absorbance. In this case, the color reagent
solution was added before the addition of starch
solution and then, the tube was placed into the water
bath. Then, the method was followed as described above.
Controls were conducted in an identical manner,
replacing extracts and/or fractions with 1 ml DMSO.
Acarbose solution (at the concentrations of 0.0094,
0.0184, 0.036, 0.07, 0.11, 0.21 ìg/ml) was used as positive
control. The inhibition percentage of -amylase was
assessed by the following formula:
 Europ. J. Biol. Sci., 7 (1): 26-31, 2014

I -amylase % =100×( AControl - ASample)/ AControl
AControl = ATest - ABlank
ASample = ATest - Ablank
The I -amylase % was plotted against sample
concentration and a logarithmic regression curve was
obtained in order to calculate the IC50 value which is
concentration of sample (mg/ml) necessary to decrease
the absorbance of -amylase solution by 50%.
Statistical Analysis: All of the statistical analyses
were performed using GraphPad Prism 3.02 statistical.
The data were expressed as mean ± SEM for here
experiments in each group. The IC50 values were estimated
by nonlinear curve-fitting and presented as their
respective 95% confidence limits.
RESULTS AND DISCUSSION
Diabetes mellitus is a chronic metabolic disorder
identified by hyperglycemia due to insulin insufficiency
and/or insulin resistance contributing to excess blood
glucose. It affected approximately 171 million people all
around the world in the year 2000 and the number is
projected to increase to around 366 million by 2030
[19]. Management of the blood glucose level is an
essential approach in the control of diabetes
complications. Inhibitors of carbohydrates hydrolysing
enzymes ( -amylase and -glucosidase) have been
helpful as oral hypoglycemic medicines for the control of
hyperglycemia exclusively in patients with type-2 diabetes
mellitus [5, 6, 20, 21]. Inhibition of these enzymes holds
of carbohydrate digestion and extend the total
carbohydrate digestion time, leading to a decrease in the
rate of glucose absorption and therefore reducing the
postprandial plasma glucose rise [22], Commonly used
synthetic inhibitory drugs such as acarbose and miglitol
18 possessed negative effects.
Traditionally, various parts of herbs were used
directly as a medication. Clinically effective substances
are now being obtained from plants, even those that have
not been categorized before as medicinal herbs.
Recently, traditional medicine (Phytotherapy) is often
used to treat several diseases, besides modern medicine.
A lot of natural extracts have been reported to have
antidiabetic activities and are utilized for the treatment of
diabetes. Herbal extracts have been used perfectly or
ultimately for the processing of numerous modern
medicines [10-12].
Teucrium species are the most widespread plant in
the world that is used for medical purposes. In traditional
medicine, this plant is used as a diuretic, treatment of
gout, rheumatism, hyperlipidemia, hyperglycemia,
hypertension,spleen and liver disease [14-17].
In this work, the inhibition activities of the
extracts obtained from T. polium, T. Orientale and
T. oliverianum were investigated on the -amylase
enzyme and IC50 values were calculated. Among the
plants studied, three species, demonstrated inhibitory
concentration dependent effects on the -amylase
activity. The strongest activity (at Concentration:
25mg/ml) was shown by the HAC extract of T. polium
(97.77%) T. Orientale DCMC extract revealed a weaker
activity. It is possibly because of the fact that at high
extract concentrations, there is certainly a confirmation
alter derived from authentic of substances to the enzyme
[23, 24]. The percentage inhibition and IC50 values
displayed by each extract is shown in Tables 1, 2 and 3,
The IC50 value of the positive control, acarbose, was
measured as 0.037 mg/mL.

Table 1: -Amylase inhibitory activities and IC50 values of different Concentrations of three extracts of T. Polium
Plant species Extracts Concentration (mg/mL) Inhibition (%) IC50 (mg/mL)
T. Polium DCMC 1.56 41.59 ± 0.64 3.01
3.12 50.45 ± 0.73
6.25 59.34 ± 1.01
12.5 68.20 ± 0.92
25 77.07 ± 0.49
EAC 1.56 49.32 ± 0.56 1.64
3.12 57.83 ± 1.11
6.25 66.37 ± 0.97
12.5 74.89 ± 1.21
25 83.41 ± 0.83
HAC 1.56 25.36 ± 0.12 3.63
3.12 43.45 ± 0.63
6.25 61.58 ± 0.76
12.5 79.67 ± 1.02
25 97.77 ± 1.21

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 Europ. J. Biol. Sci., 7 (1): 26-31, 2014

Table 2: -Amylase inhibitory activities and IC50 values of different Concentrations of three extracts of T. oliverianum
Plant species Extracts Concentration (mg/mL) Inhibition (%) IC50 (mg/mL)
T. Oliverianum DCMC 1.56 36.58 ± 0.23 4.73
3.12 45.24 ± 0.52
6.25 57.92 ± 0.38
12.5 68.57 ± 0.34
25 79.22 ± 1.04
EAC 1.56 32.83 ± 0.38 4.31
3.12 46.98 ± 0.44
6.25 61.16 ± 0.76
12.5 75.32 ± 0.62
25 89.47 ± 1.34
HAC 1.56 18.52 ± 0.29 3.86
3.12 35.13 ± 0.55
6.25 61.81 ± 1.17
12.5 82.42 ± 0.34
25 95.03 ± 1.22

Table 3: -Amylase inhibitory activities and IC50 values of different Concentrations of three extracts of T. Orientale
Plant species Extracts Concentration (mg/mL) Inhibition (%) IC50 (mg/mL)
T. Orientale DCMC 1.56 12.73 ± 0.07 22.59
3.12 22.39 ± 0.22
6.25 32.07 ± 0.76
12.5 41.73 ± 0.65
25 51.39 ± 0.76
EAC 1.56 21.77 ± 0.87 8.55
3.12 37.18 ± 023
6.25 43.62 ± 0.39
12.5 54.03 ± 0.41
25 61.44 ± 0.23
HAC 1.56 18.45 ± 0.08 13.93
3.12 28.44 ± 0.25
6.25 38.45 ± 0.23
12.5 48.44 ± 0.31
25 58.42 ± 0.28

The outcomes of this study show that the
administration of some of Teucrium species may
possibly control the postprandial blood glucose ranges
and confirm the use of these herbs suggested as a
treatment of diabetes in Traditional Medicine in Iran and
other areas.T.polium and T. oliverianum extract displayed
a good inhibitory activity on -amylase. Generally, our in
vitro studies indicated that Teucrium species, especially
T. polium and T. oliverianum, can function as organic
-amylase inhibitors and might possess beneficial
antidiabetic property in the type II diabetes mellitus.
This inhibitory activity of the Teucrium species
extracts may be as a result of the existence of various
phytochemicals like flavonoids, tannins, saponins,
anthraquinone, steroid, phlobatannin, terpenoid, in them
[25]. Previous research studies on -amylase inhibitors
identified from medicinal herbs recommend that a number
of capability inhibitors belong to flavonoid class that has
features of inhibiting -amylase activities [26]. In general,
the enzyme inhibitory activity of plant extracts not
just rely on the amount of especial phytochemicals
but additionally may depend on the quality of
especial phytochemicals. Additional researchers also
have reported that biological activities of phytochemicals
depend on the extent of hydroxylation and also
conjugation [27, 29]. Further, in vitro and in vivo research
are required to confirm the present observations,
findings on the isolation of active substances
contained in the extract and in vivo studies are necessary
to recognize a potential chemical substance entity for
clinical utilize in the therapy of diabetes and other
related disorders.
RESULTS
This study was financially supported by grant
U-88050 from the Ahvaz jundishapur university of
medical sciences, Ahvaz, IR Iran.

29
 Europ. J. Biol. Sci., 7 (1): 26-31, 2014
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