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International Journal of Biological & Pharmaceutical Research SCREENING OF

LICHEN EXTRACTS FOR IN VITRO ANTIDIABETIC ACTIVITY USING ALPHA
AMYLASE INHIBITORY ASSAY

Article  in  International Journal of Biological & Pharmaceutical Research · January 2015

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Rashmi Shivanna. et al. / International Journal of Biological & Pharmaceutical Research. 2015; 6(5): 364-367.

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IJBPR
International Journal of Biological
&
Pharmaceutical Research
Journal homepage: www.ijbpr.com

SCREENING OF LICHEN EXTRACTS FOR IN VITRO

ANTIDIABETIC ACTIVITY USING ALPHA AMYLASE INHIBITORY
ASSAY
Rashmi Shivanna, Hengameh Parizadeh and Rajkumar H. Garampalli*
Department of Botany, Manasagangotri, University of Mysore, Mysore 570 006, Karnataka, India.

ABSTRACT
Diabetes mellitus is a clinical condition characterized by hyperglycemia in which an elevated amount of glucose
circulates in the blood plasma. Alpha amylase inhibitors are used to achieve greater control over hyperglycemia in type 2
diabetes mellitus. The present study intends to screen novel alpha amylase inhibitors from natural sources like lichens in order
to minimize the toxicity and side effects of the inhibitors currently used to control hyperglycemia. The aim of this work was to
evaluate the in vitro antidiabetic activity of methanol and ethyl acetate extracts of lichens at various concentrations. Two
lichens were collected from Mysore district, Karnataka and identified as Flavoparmelia caperata (L.) Ach. and Physcia aipolia
(Ehrh. ex Humb.) Furnr. The alpha amylase inhibition assay showed that the methanolic extract of F. caperata had highest
inhibition of 49% at 15mg/ml, followed by P. aipolia with 46% inhibition. The results of the work, therefore clearly indicate
the potential of these extracts to manage hyperglycemia. Hence lichens could be exploited in the development of active agents
in regulating the postprandial glucose level to control diabetes.

Key Words: Flavoparmelia caperata, Physcia aipolia, Lichens, Antidiabetic.

INTRODUCTION
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 (Bhutkar and Bhise, 2013). 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 (Wild et al., 2004).
The management of the blood glucose level is a
critical strategy in the control of diabetes complications.
One therapeutic approach to prevent postprandial
hyperglycemia is to retard the digestion and absorption of
carbohydrates in the gastrointestinal tract through
Corresponding Author
Rajkumar H. Garampalli
Email: dr.hgrajkumar@botany.uni-mysore.ac.in
inhibition of enzymes such as α-amylase (Prabhakar et al.,
2013). The α-amylase is the one of the main enzymes in
human that catalyses the hydrolysis of 1,4-glucosidic
linkage of complex carbohydrates like starch into simple
sugars namely, maltose. Inhibition of the α - amylase
activity is one of the possible mechanisms that can be
potentially used for controlling diabetes. Controlling the
glucose production from complex carbohydrates is
considered to be effective in controlling diabetes
(Jumepaeng et al., 2013).
Examples of some inhibitors, which find
application in the clinical practice for the management of
diabetes are Acarbose, miglitol and voglibose (Bailey,
2003). Although the inhibitory drug may inhibit α-amylase
in controlling glucose level in type 2 diabetic patients, have
undesirable side effects, especially flatulence, abdominal
pain and diarrhea (Chakrabarti and Rajagopalan, 2002).
Therefore, it is the need of time to identify and explore the

Rashmi Shivanna. et al. / International Journal of Biological & Pharmaceutical Research. 2015; 6(5): 364-367.
amylase inhibitors from natural products having fewer side
effects. Natural products involve retarding the absorption
of glucose by inhibiting the carbohydrate hydrolyzing
enzyme, such as amylase. The inhibition of this enzyme
delay carbohydrate digestion and protract overall
carbohydrate digestion time, resulting in the reduction in
glucose absorption rate and consequently dulling the
postprandial plasma glucose rise (Tamil et al., 2010).
Common advantages of herbal drugs are effectiveness,
safety and acceptability (Valiathan, 1998).
The aim of this present study is to evaluate alpha
amylase inhibitory activity of lichens. Lichens constitute
an intimate symbiotic association of fungi with a group of
cyanobacteria or green algae or both (Ahmadjian, 1993).
Lichens are inherently resistant to microbial infection due
to the production of large numbers of unique secondary
metabolites (Huneck, 1999). Lichen substances exhibit a
great diversity of biological effects, including
antimicrobial, antiinflammatory, analgesic, antipyretic,
antiproliferative and cytotoxic activities, and there has
been a growing interest in the pharmaceutical properties of
compounds derived from lichens (Boustie and Grube,
2005). Hence an attempt has been made to standardize a
natural drug to cure diabetes.
MATERIALS AND METHODS
Collection and identification of lichens
Lichen samples Flavoparmelia caperata (L.) Ach.
and Physcia aipolia (Ehrh. ex Humb.) Furnr were collected
from Manasagangotri campus, University of Mysore,
Mysore. The lichens were identified based on
morphological, anatomical and color tests (Awasthi, 1988).
Preparation of extracts
Collected lichens were washed with distilled
water and kept to dry at room temperature. The dried
lichen materials were ground to fine powder and extracted
by soxhlet apparatus using methanol and ethyl acetate as
solvents. The extracts were filtered using Whatman filter
paper no. 1 Filtered extracts were concentrated by air-
drying for 4 –5 days or until the extracts crystallized, and
Fig 1. Inhibition (%) of amylase enzyme by different
concentrations of lichen Flavoparmelia caperata extracts
365
the weight/yield of the crude extracts were determined and
preserved at 5℃ in airtight bottles until further use.
Amylase inhibitory assay
The Alpha amylase inhibitory activity of lichens
with different concentrations was determined against
fungal Diastase by following the method of Karthik et al.
(2011). The enzyme (0.5%) was prepared in phosphate
buffer (pH 6.8). Briefly, 500μl of different concentrations
of lichen extracts and 500μl of 0.1M phosphate buffer (pH
6.8) containing amylase were incubated at 25°C for 10
min. After preincubation, 500μl of a 1% starch solution in
0.1M phosphate buffer (pH 6.8) was added to each tube
and further incubated at 25°C for 10 min. The reaction was
stopped by addition of 1ml of dinitrosalicylic acid reagent.
The same was performed for control where extract was
replaced with buffer. The test tubes were placed in a
boiling water bath for 10 min and cooled. To each tube,
10ml of distilled water was added and the absorbance (A)
was measured at 540nm. The percentage (%) inhibition
was calculated using the formula:
% Inhibition = [A540Control - A540 Extract /
A540Control] x100
RESULTS
In the present study methanol and ethyl acetate
extracts of selected lichens were investigated for their
antidiabetic activity to inhibit α-amylase activity. The
amylase inhibitory activity of the lichen extracts was
determined against fungal Diastase (amylase). The extract
caused a dose dependent inhibition of amylase activity.
Three different concentrations viz., 5, 10 and 15 mg/ml of
methanol and ethyl acetate extracts of lichens
Flavoparmelia caperata and Physcia aipolia both of which
belongs to the family Parmeliaceae and physciaceae
respectively were separately tested for the inhibition of α-
amylase activity. Amongst the selected lichens the
methanolic extract of F. caperata showed highest
inhibition of 49% at 15mg/ml (Fig 1), followed by with
46% P. aipolia (Fig 2). Whereas ethyl acetate extract
showed 43% in F. caperata and 32% in P. aipolia at
concentration of 15mg/ml.
Fig 2. Inhibition (%) of amylase enzyme by different
concentrations of lichen Physcia aipolia extracts
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Rashmi Shivanna. et al. / International Journal of Biological & Pharmaceutical Research. 2015; 6(5): 364-367.

DISCUSSION proteinases in the gut of insects and inhibit their normal

Literature on amylase inhibitory activity of
lichens is very scanty, particularly from Indian lichens.
Ramalina conduplicans caused higher inhibition of enzyme
activity followed by Ramalina hossei, Parmotrema
tinctorum, Usnea sinensis, Everniastrum cirrhatum and
Parmotrema pseudotinctorum. Pseudotinctorum (Vinayaka
et al., 2013) evaluated the amylase inhibitory activity of
methanol extract of a macrolichen Heterodermia
leucomela and showed about 38% of inhibition of amylase
enzyme activity at 25mg/ml concentration of the extract.
Phytochemical analysis of F. caperata had revealed the
presence of Tannins, Flavonoids, Proteins, Triterpenes
Carbohydrates and steroids. P. aipolia showed only
Proteins, Triterpenes, Carbohydrates and steroids. Lichenic
acids like Atranorin, Usnic acid, Protocetraric acid were
present in F. caperata and Atranorin, Zeorin in P. aipolia
(Rashmi and Rajkumar, 2014). Some of the enzyme
inhibitors present in natural products act by directly
blocking the active centre of the enzyme at various local
sites (Sama et al., 2012). Hence diabetics tend to have low
alpha amylase levels in order to keep their glucose levels
under control. Lichens also use alpha amylase inhibitors as
a defence mechanism as a protection from insects. These
inhibitors alter the digestive action of alpha amylases and
feeding behaviour. Therefore, alpha amylase inhibitors
have potential roles in controlling blood sugar levels and
crop protection (Kumaran et al., 2010). Thus, the data
presented here indicate that the methanolic extract of
Flavoparmelia caperata possesses significant in vitro
antidiabetic activity.
CONCLUSION
Alpha amylase is the enzyme responsible for
hyperglycemia by inhibiting its activity. From the results, it
can be concluded that the above mentioned lichen extracts
are having anti diabetic properties will be greatly beneficial
to reduce the rate of digestion and absorption of
carbohydrates and thereby contribute to effective
management of type 2 diabetes by decreasing the post-
prandial hyperglycemia. Future studies will provide an
insight for the molecular mechanisms by which these
lichens and their active compounds regulate glucose
homeostasis.
ACKNOWLEDGEMENT
One of the authors (Rashmi Shivanna) are
thankful to the University of Mysore for awarding UGC
NON-NET fellowship to carry out this work.

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