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Pharmacogn. Res. ORIGINAL ARTICLE

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In vitro Studies on the Inhibition of α‑Amylase and α‑Glucosidase

by Hydro‑ethanolic Extract of Pluchea lanceolata, Alhagi
pseudalhagi, Caesalpinia bonduc
Anupam Kumar Sachan, Ch. V. Rao1, Nikhil Kumar Sachan2
Department of Pharmaceutics, Dayanand Dinanath College, Institute of Pharmacy, Kanpur, 1Department of Pharmacology, Principal Scientist, National Botanical
Research Institute, Lucknow, Uttar Pradesh, 2Education Officer, University Grants Commission, New Delhi, India

ABSTRACT •  Among these three plants, Pluchea lanceolata showed potent enzyme
Background: Pluchea lanceolata (Rasna), Alhagi pseudalhagi (Jawasa), and inhibition as compared to other plant extracts.
Caesalpinia bonduc (Latakaranja) important medicinal plants widely used in
India as folk medicine. These plants have been used to control diabetes in
traditional medicinal systems. Objective: In the present study, 50% volume
per volume ethanolic extracts of P. lanceolata, A. pseudalhagi, and C. bonduc
subjected to in  vitro analysis of antidiabetic effect by alpha‑amylase and
alpha‑glucosidase inhibitory assay. Materials and Methods: Inhibitory
activity of the hydro‑ethanolic extract of the all three plants individually
against alpha‑amylase enzyme and alpha‑glucosidase enzyme were
examined in different concentrations  (3.90–500  µg/mL), where acarbose
used as a positive control. Results: The percentage inhibition of P. lanceolata
showed the highest alpha‑amylase and alpha‑glucosidase inhibitory activity.
Half‑maximal inhibitory concentration value P. lanceolata was found to
be 4.83  µg/ml and 11.94  µg/ml for alpha‑amylase and alpha‑glucosidase
inhibition. Conclusion: This study suggests that the hydro‑ethanolic extract
of all three plants have antidiabetic property, among these three plants
P. lanceolata showed potent enzyme inhibition as compared to other plant
extracts and standard acarbose.
Key words: Alpha‑amylase, alpha‑glucosidase, antidiabetic,
hydro‑ethanolic, in vitro

SUMMARY Abbreviations Used: PPA: Porcine pancreatic α‑amylase;

•  In the present study, 50% volume per volume ethanolic extracts of Pluchea
DNS: 3,5‑dinitrosalicylic acid, PBS: Potassium phosphate buffer solution,
lanceolata, Alhagi pseudalhagi, and Caesalpinia bonduc subjected to in vitro
analysis of antidiabetic effect by alpha‑amylase and alpha‑glucosidase pNPG: p‑Nitrophenyl‑α‑D‑glucopyranoside,
Access this article online
inhibitory assay IC50: Half‑maximal inhibitory concentration. Website: www.phcogres.com
•  Inhibitory activity of the hydro‑ethanolic extract of the all three plants Correspondence: Quick Response Code:
individually against alpha‑amylase enzyme and alpha‑glucosidase enzyme Dr. Anupam Kumar Sachan,
were examined in different concentrations  (3.90–500  µg/mL), where
Department of Pharmaceutics, Dayanand
acarbose used as a positive control
Dinanath College, Institute of Pharmacy,
•  The percentage inhibition of Pluchea lanceolata showed the highest
alpha‑amylase and alpha‑glucosidase inhibitory activity. Half‑maximal Ramaipur, Kanpur ‑ 209 214, Uttar Pradesh, India.
inhibitory concentration value Pluchea lanceolata was found to be 4.83 µg/ml E‑mail: [email protected]
and 11.94 µg/ml for alpha‑amylase and alpha‑glucosidase inhibition DOI: 10.4103/pr.pr_31_19

INTRODUCTION of glucose homeostasis, in which blood glucose level remains high after
consuming meal and plays an important role in the development of
Diabetes mellitus is a chronic multifactorial disorder and one of the type 2 diabetes and associated chronic complications, such as micro‑ and
non-communicable life‑threatening metabolic diseases involving huge macro‑vascular disorder.[5,6] Management of plasma glucose levels is
health‑care cost and high mortality rate. In 2015, it was found that it essential for delaying or preventing type‑2 diabetes. Insulin secretion
affecting 422 million adults globally. The majority of them were between
40 and 59  years and around 80% lived in middle‑  and low‑income
countries. It was found that more than 4.9 million deaths were caused This is an open access journal, and articles are distributed under the terms of the
Creative Commons Attribution‑NonCommercial‑ShareAlike 4.0 License, which
alone with diabetes and the number of diabetes patients will increase up allows others to remix, tweak, and build upon the work non‑commercially, as long as
to 55% by 2035, reaching 592 million aging between 20 and 79 years.[1,2] appropriate credit is given and the new creations are licensed under the identical terms.
These are non-infectious and non-transmissible. It is characterized by
chronic hyperglycemia with disturbance of carbohydrate, fat, and protein For reprints contact: [email protected]
metabolism due to the insufficient secretion of insulin by the pancreas
and by the resistance to the action on insulin in various issues, i.e., Cite this article as: Sachan AK, Rao CV, Sachan NK. In vitro studies on the
muscle, liver, and adipose, which results in impaired uptake of glucose.[3,4] inhibition of α-amylase and α-glucosidase by hydro-ethanolic extract of Pluchea
lanceolata, Alhagi pseudalhagi, Caesalpinia bonduc. Phcog Res 2019;11:310-4.
Postprandial hyperglycemia is one of the earliest observable abnormalities

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ANUPAM SACHAN, et al.: α-Amylase and α-Glucosidase Inhibition Activity of Three Plant Extract

through medication and/or dietary supervision, it is possible to reach this MATERIALS AND METHODS
goal.[7] Decreasing the postprandial glucose level is one of the therapeutic
approaches for treating type‑2 diabetes; for example slowing the glucose Chemicals and reagents
absorption through inhibition of the carbohydrates‑hydrolyzing Porcine pancreatic α‑amylase  (PPA), 3,5‑dinitrosalicylic acid  (DNS
enzymes present in the small intestinal brush border, α‑glucosidase, and color reagent), Potassium phosphate buffer solution  (PBS), p‑Nitrop
α‑amylase. These are responsible for the breakdown of oligosaccharides henyl‑α‑D‑glucopyranoside  (pNPG), α‑glucosidase, and ascorbose
and disaccharides into monosaccharides.[8‑10] Fruits and vegetables that were purchased from Sigma‑Aldrich  (St. Louis, USA). Soluble starch
are consumed worldwide have excellent sources of bioactive compounds potato, sodium potassium tartrate, sodium chloride, disodium
and having capacity reducing the risk of developing diabetes.[11,12] hydrogen phosphate, and sodium hydroxide were from Merck Chemical
Postprandial glucose levels can be regulated through α‑glucosidase Supplies (India). All the chemicals, including the solvents used in this
inhibition. Inhibition of these enzymes delay and in some cases halt study, were of analytical grade.
carbohydrate digestion, thus prolonging overall carbohydrate digestion
time, causing a reduction in the rate of glucose absorption and Collection and preparation of plant material
consequently reducing postprandial plasma glucose rise.[13] The plant material  (whole plant) of P. lanceolata  (Rasna),
Nowadays, α‑glycosidase inhibitors such as acarbose, miglitol, and A. pseudalhagi  (Jawasa), and C. bonduc  (Kant karaj) were collected
voglibose are oral blood glucose‑lowering drugs commonly used. from the wild area of Chambal Valley, District Etawah, Uttar Pradesh,
They decrease postprandial hyperglycemia without inducing insulin in the month of June and July during 2016. The plants were identified
secretion; these compounds do not induce hypoglycemia and have good and authenticated at source by Pharmacognosy and Ethnopharmacology
safety; although, the gastrointestinal adverse effect may limit long‑term Division Council of Scientific and Industrial Research‑National Botanical
compliance to therapy.[14] Research Institute  (NBRI), Lucknow. A  voucher specification  (No.:
NBRI‑standard operating procedures‑216) has been deposited in
Several medicinal plants species have been used to control diabetes in the
Institute repository. The plant materials were air dried and grounded
traditional medicinal systems of many cultures worldwide. The potential
into uniform powder with a grinder, passed through a sieve and stored
role of medicinal plants as inhibitors of α‑amylase and α‑glucosidase has
in airtight glass container for further use.[28]
been reviewed by several authors. A variety of plants has been reported
to show an enzymatic inhibitory activity, and so many are relevant to the The air‑dried powder of the plant was extracted by hot continuous
treatment of type‑2 diabetes.[8,15‑19] extraction with 500  ml of 50% volume per volume  (v/v) ethyl alcohol
as menstruum using Soxhlet extractor.[29,30] The hydro‑ethanolic extracts
The research for a new group of agents from natural resources,
so obtained were filtered through muslin cloth, and filtrates were
especially from traditional medicine becomes an attractive approach
evaporated under reduced pressure by using rotary evaporator and
for the treatment of postprandial hyperglycemia. It is revealed that
vacuum dried. The residue were then stored in desiccators. The extracts
there is a direct relationship between phenolic compounds, flavonoids,
derived are referred as hydro‑ethanolic extract of P. lanceolata (Rasna),
and tannins and the ability to inhibit α‑amylase and α‑glycosidase
hydro‑ethanolic extract of A. pseudalhagi (Jawasa), and hydro‑ethanolic
activities. These phenolic compounds have a positive effect on diabetes,
extract of C. bonduc (Latakaranja).
by inhibiting the two keys enzymes hydrolyzing carbohydrates in the
digestive tract.[8,20‑23]
In vitro methods employed in antidiabetic studies
The current study was undertaken to evaluate the hydro‑ethanolic α‑amylase inhibition activity
extract of whole plants of Pluchea lanceolata, Alhagi pseudalhagi, and
PPA  (enzyme commission 3.2.1.1) solution was dissolved in 20 mM
Caesalpinia bonduc for α‑amylase and α‑glycosidase inhibiting in vitro
phosphate buffer  (pH  6.9 with 6.7 mM sodium chloride) to give a
activities.
concentration of 1 U/ml. Starch solution  (1%, w/v) was obtained
In the present study, a survey was conducted in the remote villages by stirring 0.1  g of potato starch in 100  ml of 20 mM of phosphate
of Chambal Valley  (Etawah District‑Uttar Pradesh) with the help of buffer  (pH  6.9 with 6.7 mM sodium chloride) as a substrate. A  total
nongovernmental organization named Shri Jhabbulal Jan Jagrati Samiti, of 100 µl of plant extract solution and 100 µL of the enzyme were
Etawah to interact with the people living in small groups. Out of various preincubated at 37°C for 30 min. After preincubation 100 µl of a 1% starch
medicinal plants from Chambal Valley (Etawah District‑Uttar Pradesh), solution was added. The reaction mixtures were then incubated at 37°C
three plants were selected on the basis of their ethnobotanical information for 20 min. The reaction was stopped with 200 µL of DNS color reagent
used for the treatment of diabetes. Information was recorded, especially and placed in boiling water for 5 min and cooled to room temperature.
from native people and local traditional healers who were consulted Add 200 μl of reaction mixture into the 96‑well microplate after diluted
for their experiences for these plants for curing certain diseases and with 1.5 ml of distilled water. The α‑amylase activity was determined by
disorders. Data were collected through questionnaires. The aqueous measuring the absorbance of the mixture at 540 nm. Acarbose was used
solution of the outer shell of the seeds of C. bonduc is conventionally as positive control. Percentage inhibition was calculated by comparing
used by the tribal people of Andaman and Nicober Islands for the relief against control optical density with the test group.[22,31]
of the symptoms of diabetes mellitus. The aqueous and 50% ethanolic
extract of seeds produced antihyperglycemic and hypolipidaemic effect α‑glucosidase inhibitory activity
in normal and diabetic rats.[24] Hydroethanolic extract of the whole plant The α‑glucosidase inhibitory activity was performed with a set of
of P. lanceolata produced antidiabetic and wound healing activity in microwell. The enzyme solution containing 20 μl α‑glucosidase
normal and diabetic rats.[25] Whole plant of A. pseudalhagi useful in the (0.1 unit/ml) enzyme solutions were added in 96 microwell plate except
treatment of diabetes.[26] The hydro‑ethanolic extract of A. pseudalhagi blank well. A volume of 120 μl 0.1 M PBS solutions were added into the
obtained by hot continuous extraction was subjected to phytochemical well‑containing enzyme and 140 µl 0.1 M PBS in blank well and 160 µl
examination and pharmacological screening for antidiabetic activity PBS in extract blank well. Ten microliters of test samples (Acarbose or
in male Wistar rats after intraperitoneal administration using 18  h rat test samples) were added into the enzyme solution in microplate wells
fasted model, oral glucose tolerance test, and streptozotocin‑induced and then incubated for 15  min at 37°C. Twenty microliters of pNPG
diabetic rat model.[27] solutions were added to the microwell plate and incubated the plate for

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ANUPAM SACHAN, et al.: α-Amylase and α-Glucosidase Inhibition Activity of Three Plant Extract

15 min at 37°C. The reaction was terminated by adding 80 μl of 0.2 M as compared to acarbose  (IC50  ≈  29.33 μg/ml). A. pseudalhagi showed
sodium carbonate solution.[32] minimum α‑amylase inhibitory activity and IC50 value was found to be
• Test solution contains: 20 µl enzyme + 120 µl PBS + 10 µl of test 25.48 μg/ml. A comparison of α‑amylase inhibitory activity between the
samples + 20 µl pNPG + 80 µl stop reagent standard drug and extract of plants has been depicted in Figure 1.
• Control solution: All reaction mixture without test samples (20 µl
enzyme + 130 µl PBS + 20 µl pNPG + 80 µl stop reagent) α‑glucosidase inhibition activity
• Blank solution: All reaction mixture except α‑glucosidase In this study, the in  vitro α‑glucosidase inhibitory activities of the
enzyme (140 µl PBS + 10 µl of test samples + 20 µl pNPG + 80 µl hydro‑alcoholic extract of the whole plant of A. pseudalhagi, C. bonduc,
stop reagent) P. lanceolata was investigated. The results of experiment showed that there
• Extract blank solution: 10 µl extract + 160 µl PBS + 80 µl stop reagent. was a dose‑dependent increase in percentage inhibitory activity against
α‑glucosidase enzyme [Table 2]. Hydro‑alcohalic extracts of the whole
The absorbance of the wells was measured with a microplate reader plant of A. pseudalhagi, C. bonduc, P. lanceolata showed α‑glucosidase
at 405  nm, while the reaction system without plant extracts was used inhibitory potential. The half‑maximal inhibitory concentration values
as control. The system without α‑glucosidase was used as blank, and were determined using paranitrophenyl‑α‑D‑glucopyranoside as
acarbose was used as positive control. Each experiment was conducted substrate (in vitro) and tested sample concentration ranged from 9.30 to
in triplicate. The percentage enzyme inhibition and half‑maximal 500 μg/ml. P. lanceolata extract showed highest α‑glucosidase inhibitory
inhibitory concentration (IC50) was calculated. activity as compared to standard drug (acarbose). IC50 value P. lanceolata
was found to be 11.94 μg/ml. IC50 value of A. pseudalhagi extract was
Calculation of half‑maximal inhibitory concentration
found to be 70.26 μg/ml. It was also showed potent enzyme inhibition
The concentration of plant extracts required to scavenge 50% of the as compared to acarbose  (IC50  ≈  202.03 μg/ml). C. bonduc showed
radicals  (IC50) was calculated by using the percentage scavenging minimum α‑glucosidase inhibitory activity and IC50 value was found
activities at five different concentrations of the extracts. to be 480.25 μg/ml. A comparison of α‑glucosidase inhibitory activity
Percentage inhibition (I%) was calculated by between the standard drug and extract of plants is depicted in Figure 2.
I% = (Ac–As)/Ac × 100 Inhibition of α‑amylase and α‑glucosidase enzymes involved in
Where Ac is the absorbance of the control and As is the absorbance of the digestion of carbohydrates, which can significantly decrease the
the sample. postprandial increase of blood glucose after a mixed carbohydrate
diet and therefore can be play an important role in the management
RESULTS AND DISCUSSION of postprandial blood glucose level in type  2 diabetic patients and
Anti‑diabetic plants have a major role in inhibiting the glucose level thus borderline patients.[34,35] According to numerous in  vitro studies,
providing protection to human against hyperglycemia. Realizing the inhibition of α‑amylase and α‑glucosidase is believed to be one of the
facts his research was carried out to evaluate the anti‑diabetic activity of most effective approaches for diabetes care.[32,36]
hydro‑ethanolic extract of the selected plants. The in vitro antidiabetic
activity of these plants extract was detected by measurement of glucose
uptake in L6 cell lines.[33]

α–Amylase inhibition activity

In this study, the in  vitro α‑amylase inhibitory activities of the
hydro‑ethanolic extract of the whole plant of A. pseudalhagi, C. bonduc,
and P. lanceolata was investigated. The results of the experiment showed
that there was a dose‑dependent increase in percentage inhibitory
activity against α‑amylase enzyme [Table 1].
The IC50 values were determined using potato starch  (1%, w/v) in 20
mM phosphate buffer  (pH  6.9 containing 6.7 mM sodium chloride)
is used as substrate  (in  vitro) and tested sample concentration ranged
from 3.90 to 500 μg/ml. P. lanceolata extract showed highest α‑amylase
Figure  1: Half‑maximal inhibitory concentration value  (µg/ml) of test
inhibitory activity as compared to the standard drug (acarbose). IC50 value
samples showed α‑amylase inhibition potential. Note: Lower half‑maximal
P. lanceolata was found to be 4.83 μg/ml. IC50 value of C. bonduc extract
inhibitory concentration value means higher efficacy
was found to be 9.81 μg/ml. It was also showed potent enzyme inhibition

Table 1: α‑Amylase inhibition data at different concentration of test samples

Concentration Percentage of inhibition

(µg/ml) Alhagi pseudalhagi Caesalpinia bonduc Pluchea lanceolata Standard (acarbose)
3.90 28.22 32.39 45.80 12.49
7.81 39.12 48.56 51.36 34.96
15.63 45.56 61.53 57.51 45.51
31.25 57.25 74.12 65.23 51.03
62.50 65.12 84.37 74.25 59.62
125.00 70.21 97.11 81.23 75.92
250.00 88.14 100 92.97 84.20
500.00 98.35 100 100.00 97.52

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ANUPAM SACHAN, et al.: α-Amylase and α-Glucosidase Inhibition Activity of Three Plant Extract

Table 2: α‑Glucosidase inhibition data at different concentration of test samples

Concentration Percentage of inhibition

(µg/ml) Alhagi pseudalhagi Caesalpinia bonduc Pluchea lanceolata Standard (acarbose)
3.90 22.02 0.36 39.43 4.63
7.81 30.25 5.31 45.12 10.64
15.63 38.90 8.65 57.23 13.62
31.25 44.31 12.36 69.25 37.75
62.50 51.10 16.96 80.49 39.83
125.00 60.85 24.88 89.21 47.18
250.00 72.04 31.12 93.08 56.20
500.00 97.98 50.63 99.82 66.52

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