Inhibitory Effects of Ethanolic
Extract of Physalis minima on Amylase, Lipase and Alpha
Glucosidase
SN Raju, D Sathis
Kumar*, Otilia Banji, David
Banji, P Yogeswaran, D Narender Prasad and A Harani
Nalanda
College of Pharmacy, Nalgonda, Andhra Pradesh-508
001, India.
ABSTRACT:
The aim of the present
study was to assess the effects of ethanolic extract of Physalis minima on the
amylase, lipase and alpha glucosidase activity in
vitro. Powder of whole plant of Physalis minima was extracted in ethanol and
the extract was assayed for the measurement of inhibitory effects on activities
of enzymes. The extracts rich in bioactive phytochemicals
showed inhibitory activity on the amylase, lipase and alpha glucosidase,
thus suggesting that extract might be useful in the treatment to limit dietary
fat and glucose absorption and the accumulation of fat in adipose tissue. The
extracts of Physalis minima may be safe, natural and cost effective for
reducing fat and glucose absorption.
KEYWORDS: Physalis minima, amylase, lipase, alpha glucosidase.
INTRODUCTION:
Herbal medicine is a
major component in all indigenous peoples' traditional medicine and a common
element in Ayurvedic, homeopathic, naturopathic, traditional oriental, and
Native American Indian medicine. WHO notes that out of 119 plant-derived
pharmaceutical medicines, about 74 percent are used in modern medicine in many
ways that correlated directly with their traditional uses as plant medicines by
native culture. Medicinal plants have the advantage of
having little or no side effects. Some of them are being used in traditional
systems of medicine from hundreds of years in many countries of the world.
NIDDM (non insulin dependent Diabetes Melitus) has
also been associated with an increased risk for premature arteriosclerosis due
to vascular disease. An ideal treatment for diabetes would be a drug that not
only controls the glycemic level but also prevents
the development of arteriosclerosis and other complications of diabetes.[1] Long before till the use of
insulin became common, indigenous remedies were used for the treatment of
diabetes mellitus and hyperlipidemia. There has been
an increasing demand from patients for the use of natural products with anti
diabetic and anti hyperlipidemic activity. This is
largely because insulin cannot be used orally and insulin injections are
associated with the risk of hepatic and other body functions. The undesirable
side effects and contra indications of synthetic drugs, and the fact that they
are not suitable for use during pregnancy, have made scientists look towards
hypoglycemic agents of plant origin [2]. Many herbs and plant
products have been shown to have antihyperglycemic
and hyperlipidimic action [3-4]. It had
been reported that digestive enzymes such as lipase, amylase and alpha glucosidase is responsible for the digestibility of protein
starch & lipid.[5] The membrane-bound intestinal alpha-glucosidases hydrolyze oligosaccharides, trisaccharides, and disaccharides to glucose and other monosaccharides in the small intestine. Alpha-glucosidase inhibitors are saccharides that act as competitive inhibitors of enzymes needed
to digest carbohydrates (specifically alpha-glucosidase enzymes in the brush border of the small
intestines). Pancreatic alpha-amylase hydrolyzes complex starches to
oligosaccharides in the lumen of the small intestine. Alpha-glucosidase
inhibitors also blocks pancreatic alpha-amylase in addition to inhibiting
membrane-bound alpha-glucosidases.
A
high post prandial blood glucose response is
associated with micro and macro cellular complications in diabetes, and is more
strongly associated with the risk for cardiovascular disease than are fasting
glucose levels potent glucosidase inhibitors such as acarbose and voglibose have
already been clinically used for diabetic and obese patients6. α-Amylase is an enzyme of low molecular mass (45000 to
50000). According to the site of action, it is a digestive juice enzyme. α-Amylase catalyzes hydrolysis of the α-1,
4-glycoside linkage in the polysaccharide molecule, forming maltose and glucose
from amylose. Beside maltose and glucose, dextrins are also formed from amylopectins
and glycogen, because the enzyme does not act on the 1, 6-glycoside linkages.
Amylase is mostly synthesized in pancreatic acini, and partly in
salivary glands. Lipases (triacylglycerol lipases EC
3.1.1.3) are enzymes which have been classically employed to carry out
hydrolysis of triglycerides with concomitant production of free fatty acids.
Lipase may also be elevated in chronic pancreatitis but, if severe destruction
of the acinar tissue has occurred, serum levels may
be below those normally detected. Obstruction of the pancreatic duct caused by
a calculus or carcinoma may also result in increased serum lipase levels.
Increased serum lipase may also be observed in chronic or acute renal disease,
after endoscopic retrograde pancreatography or
treatment with opiates. Merina and etal explained that in diabetic rats, varying degrees of
cellular damage was observed in the pancreatic islets. There was a decrease in
population of cells with nuclear pyknosis, karyarerrhsis and karyolysis with
degranulation and cytolysis. By regenerating
pancreas, it can control the lipase elevation [7]. Physalis minima belong to Solanaceae family, distributed in South Asian countries. Physalis minima is commonly found on the
bunds of the fields, waster lands, around the houses, on roadsides, etc., where
the soil is porous and rich in organic matter. It is an annual herbaceous plant
having a very delicate stem and leaves. The
whole plant is
bitter, appetizing, tonic, diuretic, laxative, and useful in inflammations,
enlargement of the spleen and abdominal troubles8. The present invitro study tested the hypothesis that ethanolic extract
of Physalis minimacan
inhibit the enzymatic activity of amylase, lipase and alpha glucosidase in vitro.
MATERIALS AND METHODS:
Preparation
of ethanolic Plant extracts:
Plant material of
Physalis minima was collected from local areas of Nalgonda,
Andhra Pradesh and plant was authentified by Mr. Madhavachetty, Botanist, S.V.University,
Thirupati, Andhra Pradesh.
Plant was dried in the shade and ground into uniform powder using milling
machine.
The extraction procedures were carried out
for about 18 hrs using soxhlet apparatus with 70%
ethanol as a solvent. Initially the shade dried plant of Physalis minima was
taken in a grinder mixture to obtain a coarse powder and then passed through a
40 mesh.9 The powder (500gms) of plant was
defatted with hexane and later extraction procedure was carried out using
ethanol. Then residue was collected and used for experiment. The extract of
Physalis minima was concentrated to dryness under reduced pressure.
Preliminary
Phyto Chemical Screening10:
The ethanolic fractions of plant were
subjected to qualitative chemical investigation for identification of Phytoconstituents.
In-
vitro Amylase inhibitory activity:
Ethanolic extract of Physalis minima was used
in various concentrations (2000 µl, 1500 µl, 1000 µl, 500 µl). 1ml of substrate
was taken in all the test tubes and was kept for incubation at 370C
for 5 min. After incubation, 100 µl of Amylase solution was added in to all the
tubes then 100 µl of extract of different dilutions was added and it was incubated
at 370C for 15 minutes. To the resulting solution, 2,500 µl of
working coloring reagent was added then made up to 10ml with acetate buffer of
pH 4.8. The absorbance was measured at 660 nm. The reading was noted. The
data’s were presented in Table No: 1
Calculation:
Amylase activity =
(Absorbance per minute of sample/ Absorbance
per minute of Calibrator) x Calibrator value
Absorbance /min of calibrator = 0.040
Value of calibrator = 100 unit/ltr.
Amylase inhibition activity = [100 – ((100 x
Amylase activity)/Value of Calibrator))]
In-vitro
Lipase inhibitory activity:
Lipase working reagent and Lipase calibrator
(Lipase 450U/L) were obtained from reckon diagnostics private limited. To each
1ml of working reagent taken in 4 individual test tubes, 0.05ml of Physalis
minima extract of 500, 1000, 1500, 2000 µl concentration and blank solution (buffer) was added. All
test tubes were incubated at room temperature or 370C for 5min.,
then 0.05ml of calibrator was added to all test tubes and again incubated at
room temperature or 370C for 5min.
The rate of decrease in turbidity measured at 340nm is proportional to
the lipase activity. The data was presented in Table No: 2
Lipase activity =
(Absorbance per minute of sample/ Absorbance
per minute of Calibrator) x Calibrator value
Absorbance /min of calibrator = 0.030
Value of calibrator = 450 unit/ltr.
Lipase inhibition activity = [100 – (100 x
Lipase activity)/Value of Calibrator)]
In-vitro
alpha glucosidase inhibitory activity:
Physalis minima ethanolic
extract was used to investigate the in-vitro inhibitory effect of alpha glucosidase enzymes. After fasting, small intestine of goat
between duodenum and cecum (Upper Part) was cut,
rinsed with ice-cold saline and homogenated with maleate buffer (pH 6). Small intestine homogenate was used
as an enzyme source. The 500 µl of enzyme & 100 µl of extract of different
concentration and Acarbose (1000mcg/ml) were taken in
to different test tubes and pre incubated for 15min, at 370C. Then
500 µl of 100 mM maltose(2%) as a substrate was added
to all the test tubes and incubated for 15min at room temperature and
centrifuged. 0.6ml of supernatant liquid was collected from all the test tubes
separately and it was mixed with 0.8ml of alkaline CuSO4 individually The solution
was heated in water bath for 8min and cooled. After cooling, phosphomolybdic acid was added to the mixture and made to
10ml with distilled water. Glucose concentration was measured using glucose
kit. In case of maltase inhibitory test, maltose was used as a substrate. The
data was presented in Table No: 3
Table
no: 1 Amylase activity and inhibitory effects of Physalis minima.
Concentration
(mcg/ml) |
Amylase
activity (units/L) |
Amylase
inhibitory activity |
||
Physalis
minima |
Control |
Physalis
minima |
Control |
|
500 |
81.14±2.459** |
|
18.86 |
- |
1000 |
79.50±9.937** |
|
20.5 |
- |
1500 |
77.86±1.419** |
|
22.14 |
- |
2000 |
79.50±3.756** |
|
20.5 |
- |
0 |
- |
100±3.756** |
- |
0 |
The values are mean±SD
of 6 values. Means with superscripts (**) within a column are significantly
different from each other at p<0.01 as determined by Dennett’s Multiple
comparison test. F value for PM is
30.2, df (5, 12). |
Table
no: 2 Lipase activity and inhibitory effects of Physalis minima.
Concentration
(mcg/ml) |
Lipase
activity (units/L) |
Lipase
inhibitory activity |
||
Physalis
minima |
Control |
Physalis
minima |
Control |
|
500 |
372.58±8.380** |
|
17.20 |
- |
1000 |
416.12±8.380** |
|
7.52 |
- |
1500 |
396.77±8.380** |
|
11.82 |
- |
2000 |
382.25±8.380** |
|
15.05 |
- |
0 |
- |
450±14.51** |
- |
0 |
The values are mean±SD
of 6 values. Means with superscripts (**) within a column are significantly
different from each other at p<0.01 as determined by Dennett’s Multiple
comparison test. F value for PM is
90.41, df (5, 12). |
Statistical
analysis:
All the data was subjected to analysis of
variance (ANOVA). The data (mean± standard deviation) shown are mean value and
the significance differences was compared by using Dennett’s Multiple
comparison test at the p<0.05 probability level. ANOVA was carried out by
using GRACHPADPRISM version 4.2 software.
RESULTS:
The ethanolic extract of Physalis minima was
concentrated on water bath to a dry residue and kept in a desiccator. The
percentage yield was 5.8%w/w for ethanolic extract of Physalis minima. The Phytochemical screening and quantitative estimation of the
percentage crude yields of extracts studied had shown that the whole plants of Physalis minima was rich in alkaloids,
carbohydrates, phenolic compounds, tannins.
The experiment was performed to analyze
amylase inhibition activity of ethanolic extract of our plant from 500mcg/ml to
2000mcg/ml. Analysis of data confirms that amylase inhibition activity was
maximum at 1500mcg/ml of Physalis minima compared to 100U/L of standard. In
case of Physalis minima, inhibition activity was increased gradually up to
1500mcg/ml, and then its inhibition activity was decreased slightly in
2000mcg/ml. According to the experimental results, it was certainly confirmed
that the ethanolic extract of plant inhibits the activity of amylase enzymes.
Table 3: In vitro effect of
alpha glucosidase inhibitory activity
Concentration (mcg/ml) |
% inhibition activity |
|
Physalis minima |
Standard |
|
500 |
34.31±1.259** |
|
1000 |
50.99±1.984** |
51.78±2.615** |
1500 |
48.08±0.525** |
|
2000 |
48.06±1.959** |
|
The
values are mean±SD of 6 values. Means with
superscripts (**) within a column are significantly different from each other
at p<0.01as determined by Dennett’s Multiple comparison test. F value is
90.41, df (5, 12). |
The experiment was performed to analyze
lipase inhibition activity of ethanolic extract of our plant by reckon
diagnostics from 500mcg/ml to 2000mcg/ml. Analysis of data confirms that lipase
inhibition activity was maximum at 500mcg/ml of Physalis minima compared to
450U/L of standard. In case of Physalis minima, inhibition activity was
decreased initially up to 1000mcg/ml, and then its inhibition activity was
increased slightly up to 2000mcg/ml. According to the experimental results, it
was certainly confirmed that the ethanolic extract of plant inhibits the activity of lipase
enzymes.
The experiment was performed to analyze alpha
glucosidase inhibition activity of ethanolic extract
of Physalis minima on goat intestine homogenate from 500mcg/ml to 2000mcg/ml.
Analysis of data confirms that alpha glucosidase
inhibition activity was maximum at 1000mcg/ml of Physalis minima(p<0.01)
compared to 1000mcg/ml of standard. In case of Physalis minima, inhibition
activity was increased up to 1000mcg/ml, and then its inhibition activity was stablished up to 2000mcg/ml. According to the experimental
results, it was certainly confirmed that the ethanolic extract of plant
inhibits the activity of alpha– glucosidase enzymes
such as maltase. Graph I represented the enzyme inhibitory activity of ethanolic
extract of Physalis minima.
DISCUSSION:
We tested our plant extract for inhibitory
action against in vitro pancreatic lipase & confirmed that extract contain
lipase inhibitors that act in a dose dependent manner. Starch which is the
predominant ingredient of human food is rapidly degraded in the
gastrointestinal tract by salivary and pancreatic α amylase to maltose
which is further hydrolyzed by maltase localized in the brush border of small
intestine to glucose. Glucose is immediately absorbed leading to hyperglycemia
and consequently to hyperinsulinemia. Both phenomena
are undesirable in diabetics and in obese patients. Inhibition of the digestion
of starch leads to a decrease and a retardation of glucose absorption. In
nature, α amylase inhibitors are found in wheat and other grains[11]. Several inhibitors of
amylase and α glucosidase have been developed[12]. Animal experiments with
high doses of absorbable alpha glucosidase inhibitors
indicate that lysosomal storage of glycogen may occur[13]. The result strongly
suggests that our plant extract inhibited the glucose level by inhibiting glucosidase activity and alpha amylase activity. NIDDM has also been associated with an
increased risk for premature arteriosclerosis due to increase in triglycerides
and LDL levels. An ideal treatment for diabetes would be a drug that not only
controls the glycemic level but also prevents the
development of arteriosclerosis & other complication of diabetes. The
higher lipid levels in diabetic patient are due to increased mobilization of
free fatty acids from peripheral despots & also due to lipolysis
caused by hormones. According to the above result our plant leads to inhibition
of lipid peroxidation and control of lipolytic hormone like lipase. It had been reported that digestive
enzymes such as lipase, amylase and alpha glucosidase
were inhibited by tannins in young chicks which decrease the digestibility of
protein starch & lipid [14,15]. The
mechanism of inhibition on maltase intestinal enzyme by Ethanolic extracts of both
plants could be due to polyphenol content. For
example, tea polyphenol such as catechin
have been found to inhibit glucosidase activity and
glucose transport [16]. The tannin (polyphenol)
has specific property such as ability to precipitate some proteins. This
precipitation is presumed to occur by the formation of hydrogen bond between
hydroxyl groups of tannins and the peptide linkage of the protein. As per our
study, tannins present in Physalis minima extracts might have significantly
precipitated the enzymes such as maltase. The extracts rich in bioactive phytochemicals showed inhibitory activity on the amylase,
lipase and alpha glucosidase, thus suggesting that
extract might be useful as a treatment in limiting dietary fat and glucose
absorption and the accumulation of fat in adipose tissue.
By our study we can conclude that Physalis minima has significant inhibitory activity against
amylase, lipase and alpha glucosidease which might be
helpful in preventing of suppressing the progress of various disorders. The
extracts of Physalis minima may provide a safe, natural and cost effective for
reducing fat and glucose absorption.
ACKNOWLEDGEMENT:
We are thankful to
The Management, Nalanda College of Pharmacy, Nalgonda, and Mr.Pradeep,
Scientist, Suven Lab,
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Received on 07.10.2009
Accepted on 02.12.2009
© A&V Publication
all right reserved
Research Journal of Pharmacognosy and Phytochemistry. 2(2): March -April 2010, 159-162