Int. J. Chem. Sci.

: 10(1), 2012, 457-462

ISSN 0972-768X
www.sadgurupublications.com

IN VITRO ASSAY OF ALPHA AMYLASE INHIBITORY

ACTIVITY OF SOME INDIGENOUS PLANTS
M. A. BHUTKAR* and S. B. BHISE

Govt. College of Pharmacy, KARAD – 415124 (M.S.) INDIA

ABSTRACT
Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia and its type II
is the major form of diabetes, accounting for 90% of cases worldwide. The management of the blood
glucose level is a critical strategy in the control of diabetes complications. There are many and diverse
therapeutic strategies in the management of Type II diabetes. The inhibition of carbohydrate hydrolyzing
enzymes such as α-amylase can be an important strategy to lower postprandial blood glucose levels. Such
inhibitors which find application in the clinical practice for management of diabetes are known to be
associated with various gastrointestinal side effects. Therefore, it is the need of time to identify and
explore the amylase inhibitors from natural sources having fewer side effects. In the present study,
aqueous extracts from leaves, stems, seeds and roots of selected plants namely Tamarindus indica,
Catharanthus roseus and Caesalpinia bonducella which are used in the Ayurvedic traditional system of
medicine to treat diabetes were tested for their inhibitory effect on α-amylase. The results revealed that
aqueous extracts of leaves of T.indica 9 mg/mL, extracts from the stems and roots of C. roseus and C.
bonducella (9 mg/mL) exhibited significant (more than 70%) reduction in amylase activity. The highest
inhibition i.e. 87.26% was observed at a concentration of 9mg/mL with the aqueous extract of seeds of C.
bonducella.

Key words: Anti-diabetic, α-Amylase, Inhibitory effects.

INTRODUCTION

Diabetes mellitus is a major endocrine disorder affecting nearly 10% of the

population all over the World1. It is characterized by hyperglycemia and disturbances of
carbohydrate, protein and fat metabolisms, secondary to an absolute or relative lack of the
hormone insulin2. The number of people in the world with diabetes has increased
dramatically over recent years. It is also predicted that by 2030, India, China and the United
States will have the largest number of people with diabetes.3 Currently treatments of
diabetes, in addition to insulin supplement includes many oral hypoglycemic agents along
________________________________________
*
Author for correspondence; E-mail : [email protected]
458 M. A. Bhutkar and S. B. Bhise: In Vitro Assay of Alpha Amylase….

with appropriate diet and exercise. One therapeutic approach which may prove to be
beneficial for treatment of diabetes is to decrease the post-prandial hyperglycemia. This can
be achieved by retarding the absorption of glucose through the inhibition of the carbohydrate
hydrolyzing enzymes in the digestive tract. The ∝ glucosidase enzymes such as α-amylase
are responsible for the breakdown of oligo and/or disaccharide to monosaccharides.
Inhibitors of these enzymes delay carbohydrate digestion and prolong overall carbohydrate
digestion time causing a marked decrease in the rate of glucose absorption thereby blunting
the post prandial plasma glucose rise4. Examples of such inhibitors which find application in
the clinical practice for management of diabetes are acarbose, miglitol and voglibose.5
However, these drugs are known to be associated with various gastrointestinal side effects
such as abdominal pain, flatulence and diarrhea in the patients6,7. Therefore, it is the need of
time to identify and explore the amylase inhibitors from natural sources having fewer side
effects. The Indian traditional system of medicine practiced for over thousands of years have
reports of numerous anti- diabetic plants with no known side effects. Many plants and their
products have been widely prescribed and used for diabetic treatment all around the world
with less known mechanistic basis of their functioning. Thus, these natural products need to
be evaluated scientifically in order to verify for their anti-diabetic properties. The medicinal
plants selected for the study included Tamarindus indica (Family : Leguminosae),
Catharanthus roseus (Family Apocynaceae) and Caesalpinia bonducella L. belonging to
family fabaceae which are known to lower blood glucose levels and also used in Ayurvedic
medicines for treatment of number of ailments. The present investigation was undertaken to
make a comparative study for the ability of the selected plants to inhibit α-amylase activity.

EXPERIMENTAL
Material and methods
Reagents

All the chemicals used were of analytical grade obtained from S.D. Fine Chemicals
Pvt. Ltd., Mumbai, Sigma chemical company, USA and Loba chemicals, Mumbai.
Preparation of the plant extracts

The plant material was collected from Karad and surrounding areas. All the plant
materials were further identified and authenticated by the Department of Botany, Science
College, Karad. The plant parts were then separated and dried which were then powdered
using a grinder. For the aqueous extraction 50 gm of powdered plant material was stirred in
200 mL of distilled water. It was placed in a rotary shaker for 24 hr. Thereafter it was
subjected to centrifugation at 8000 rpm for 10 min. The resultant supernatant was filtered
Int. J. Chem. Sci.: 10(1), 2012 459

using Whatman No. 1 filter paper. The crude extract was subsequently oven dried at a
temperature of 35oC to form a powdery residue. The powdered dried crude extract was
dissolved in solvents for further studies.
Assay for α-amylase inhibition

The determination of α-amylase inhibition was carried out by quantifying the

reducing sugar (maltose equivalent) liberated under the assay conditions. The enzyme
inhibitory activity was expressed as a decrease in units of maltose liberated. A modified
dinitrosalicylic acid (DNS) method was adopted to estimate the maltose equivalent.8 1mL of
the aqueous extracts of the selected plant extracts were pre-incubated with α-amylase 1
U/mL for 30 min and thereafter 1 mL (1% w/v) starch solution was added. The mixture was
further incubated at 37°C for 10 min. Then the reaction was stopped by adding 1 mL DNS
reagent (12.0 g of sodium potassium tartrate tetrahydrate in 8 mL of 2 M NaOH and 96 mM
3, 5- dinitrosalicylic acid solution) and the contents were heated in a boiling water bath for 5
min. A blank was prepared without plant extracts and another without the amylase enzyme,
replaced by equal quantities of buffer (20 mM Sodium phosphate buffer with 6.7 mM
Sodium chloride, pH 6.9 at 20oC). The absorbance was measured at 540 nm. The reducing
sugar released from starch was estimated as maltose equivalent from a standard graph.
Acarbose was used as positive control. The aqueous plant extracts from different plant parts
were diluted in buffer to give a final concentration of 5mg/mL, 7mg/mL and 9mg/mL. The
anti-diabetic activity was determined through the inhibition of α-amylase which was
expressed as a percentage of inhibition and calculated by the following equations:
% reaction = (maltose) test / (maltose) control × 100
% inhibition = 100% reaction
Statistical analysis

All the analyses were carried out in triplicate and the results were expressed in mean
± SD.

RESULTS AND DISCUSSION

In the present study aqueous extracts of different parts of selected plants with known
antidiabetic activity were investigated for their potential to inhibit α-amylase activity. Three
different concentrations viz., 5, 7 and 9 mg/mL of aqueous extracts of leaves, stems, seeds
and roots of the selected plants were separately tested for the inhibition of α-amylase activity
(Table 1). Amongst the selected plants the aqueous extract of leaves of T. indica at 9mg/mL
concentration, had the highest amylase inhibition of 71.93% followed by C. roseus and C.
460 M. A. Bhutkar and S. B. Bhise: In Vitro Assay of Alpha Amylase….

bonducella with the inhibition of 68.02 and 55.19% respectively. The concentration of 9
mg/ml of C. roseus stem extract showed the highest inhibition of 74.88%, followed by C.
bonducella and T. indica which showed the inhibition of 70.22 and 63.54%, respectively for
their aqueous stem extracts. C. bonducella for its aqueous extracts of the seeds at the
concentration of 9 mg/mL exhibited the highest inhibition of 87.26% compared to the rest
of the other seed extracts of T. indica and C. roseus which showed inhibitory activity of
69.72 and 62.85%, respectively at the similar concentration. In case of aqueous extract of
the root samples from the selected plant species, C. bonducella at the concentration of 9
mg/mL exhibited maximum inhibitory activity with 85.93% inhibition followed by C. roseus
and T. indica which showed the inhibition of 70.44 and 59.17%, respectively. From the
results, it can be concluded that use of these plant extracts will be greatly beneficial to
reduce the rate of digestion and absorption of carbohydrates and thereby contribute for
effective management of diabetes by decreasing the post-prandial hyperglycemia. Future
studies will provide an insight for the molecular mechanisms by which these plant and their
active compounds regulate glucose homeostasis.
Table1: α-Amylase inhibitory activity of aqueous extracts from the leaves, stems, seeds
and roots of the selected plants

% Inhibition
Scientific name
5 mg/mL 7 mg/mL 9 mg/mL
Leaves
T. indica 09.07 ± 0.11 20.66 ± 0.12 55.19 ± 0.14
C. roseus 14.40 ± 0.01 32.73 ± 0.26 68.02 ± 0.22
C. bonducella 20.18 ± 0.14 35.16 ± 0.09 71.93 ± 0.21
Stems
T. indica 18.22 ± 0.16 31.79 ± 0.03 63.54 ± 0.19
C. roseus 15.19 ± 0.07 47.96 ± 0.20 70.22 ± 0.23
C. bonducella 23.10 ± 0.24 49.26 ± 0.17 74.88 ± 0.18
Seeds
T. indica 21.96 ± 0.13 38.11 ± 0.29 69.72 ± 0.18
C. roseus 19.50 ± 0.09 33.64 ± 0.15 62.85 ± 0.24
C. bonducella 29.98 ± 0.16 66.73 ± 0.17 87.26 ± 0.13
Cont…
Int. J. Chem. Sci.: 10(1), 2012 461

% Inhibition
Scientific name
5 mg/mL 7 mg/mL 9 mg/mL
Roots
T. indica 17.66 ± 0.11 29.54 ± 0.18 59.17 ± 0.25
C. roseus 23.80 ± 0.17 46.72 ± 0.21 70.44 ± 0.20
C. bonducella 25.52 ± 0.27 59.67 ± 0.19 85.93 ± 0.24
Data are mean ± SD of triplicates

100
% alpha-am ylase

80
inhibition

60
40
20
c
0
T. indica C.roseus C.bonduc
Plant extracts

Leaves Stems Seeds Roots

Fig. 1: Comparison of α -amylase inhibitory effect of aqueous extracts of different

plant parts from the selected plant species

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Accepted : 02.12.2011