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http://informahealthcare.com/phbISSN 1388-0209 print/ISSN 1744-5116 online
Editor-in-Chief: John M. PezzutoPharm Biol, 2014; 52(2): 199–207
! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2013.823551
ORIGINAL ARTICLE
Evaluation of in silico, in vitro a-amylase inhibition potential andantidiabetic activity of Pterospermum acerifolium bark
Paramaguru Rathinavelusamy, Papiya Mitra Mazumder, Dinakar Sasmal, and Venkatesan Jayaprakash
Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi-835215, Jharkhand, India
Abstract
Context: Pterospermum acerifolium (L.) Willd (Sterculiaceae) has been traditionally used in thetreatment of diabetes mellitus but no scientific data has been published supporting theclaimed ethnomedical use.Objective: The present study was designed to estimate the in silico, in vitro a-amylase inhibitionpotential and anti-diabetic activity of Pterospermum acerifolium bark.Materials and methods: In silico studies were performed between human pancreatic a-amylase(HPA) and b-sitosterol by using autodock 4.2 software. In vitro a-amylase inhibition study wascarried out with 50% ethanol extract of the bark (PABEE) and its various fractions. The activeethyl acetate fraction (PABEF) was sub-fractionated into three fractions (PABE1, PABE2 andPABE3). Two doses (15 and 30 mg/kg) based on acute toxicity studies, of the above fractionswere subjected to antidiabetic screening in vivo by STZ-nicotinamide induced type II diabeticrats.Results: In silico studies showed the potent inhibition of b-sitosterol on human pancreaticamylase (HPA) with an estimated inhibition constant (Ki) of 269.35 nmol and two hydrogenbond interactions. PABEF showed marked a-amylase inhibition (69.94%) compared to otherfractions. Diabetic rats treated with PABE3 (30 mg/kg) reduced the levels of fasting bloodglucose, HbA1c, ALT, AST, ALP, triglycerides, total cholesterol, TBARS significantly (p50.01) andincreased the levels of HDL-C, catalase, GSH, SOD significantly (p50.01) as compared to that ofdiabetic control animals. Histological studies on PABE3 treated group showed remarkablepositive changes in b-cells.Conclusion: The present study confirmed the antihyperglycemic activity along with its statuson hepatic biomarkers, antihyperlipidemic and antioxidant properties of Pterospermumacerifolium bark.
Keywords
3OLE, autodock, oxidative stress,streptozotocin-nicotinamide,type II diabetes
History
Received 13 March 2013Revised 28 May 2013Accepted 5 July 2013Published online 25 September 2013
Introduction
Diabetes mellitus (DM) is characterized by chronic hyper-
glycemia resulting from defects in insulin secretion or action,
or both, with impaired carbohydrate, lipid and protein
metabolism. It is divided into insulin dependent (Type I)
and non-insulin dependent (Type II) diabetes mellitus (Aylin
et al., 2007). Its frequency worldwide is expected to continue
to grow by 6% per annum and it is predicted that by 2025
India, China and the United States will have the largest
number of people with diabetes and more importantly, type II
diabetes that accounts for 90–95% of all diabetes (King et al.,
1998; Muller, 2001). The pathogenesis of type 2 diabetes is
complex involving progressive development of insulin resist-
ance in liver and peripheral tissues accompanied by a
defective insulin secretion from pancreatic b-cells leading to
overt hyperglycemia (Cheng, 2005). Currently available
therapeutic strategies for treating type II diabetes is limited
with generally inadequate efficacy and a number of serious
adverse effects (Srinivasan & Ramarao, 2007). Many trad-
itional plant treatments for diabetes mellitus are used
throughout the world, both in developing and developed
countries, but few of the medicinal plant treatments for
diabetes have received scientific scrutiny, for which World
Health Organization (WHO) has also recommended attention
(WHO, 1980).
Pterospermum acerifolium (L.) Willd (Sterculiaceae) is a
large tree up to 24 m in height and 2.5 m in girth. It is widely
distributed in the regions of sub-Himalayan tract and outer
valleys from Yamuna eastwards to West Bengal, and in
Assam and Manipur, up to an altitude of 1200 m (Mazumder
et al., 2011). The plant is commonly known as Kanakchampa,
Karnikara, Muchukunda and Matsakanda and has tradition-
ally been used as a haemostatic, anti-inflammatory, anthel-
mintic, laxative and antihyperglycemic, as well as a treatment
for blood troubles, small pox, leucorrhoea, leprosy, ulcer,
tumors, ear pain and stomachache (Chopra et al., 1956;
Kirtikar & Basu, 1935). A significant number of
Correspondence: Dr. (Mrs) Papiya Mitra Mazumder, Departmentof Pharmaceutical Sciences, Birla Institute of Technology, Mesra,Ranchi, Jharkhand 835215, India. Tel: 0651 2275290. E-mail:[email protected]
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phytoconstituents have been isolated from all parts of the
plant (Dixit et al., 2011). However, the antidiabetic potential
of Pterospermum acerifolium bark has not yet been exten-
sively studied. Hence, this work is designed to evaluate the in
silico, in vitro a-amylase inhibition potential and antidiabetic
activity of Pterospermum acerifolium bark.
Materials and methods
Plant material
Pterospermum acerifolium bark collected from the campus of
BIT Mesra, Ranchi during August of 2011. The plant material
was identified and authenticated by Dr. K. Karthikeyan,
taxonomy department of Botanical Survey of India (BSI),
Kolkata. A voucher specimen (CNH/48/2012/Tech II/805)
was deposited in the herbarium of Department of
Pharmaceutical Sciences, BIT Mesra, Ranchi for future
reference.
Extraction and fractionation
Powdered bark (1 kg) was extracted with 50% ethanol for 72 h
at room temperature and concentrated to yield 74.61 g of
PABEE. The extract (25 g) was dissolved in distilled water
and extracted successively with hexane (PABHF), chloroform
(PABCF) and ethyl acetate (PABEF), and concentrated on
rotavapour (Buchi Labortechnik AG, Schweiz) under reduced
pressure at 40 �C to yield PABHF (3.46 g), PABCF (5.71 g)
and PABEF (14.28 g) fractions.
Molecular docking studies
Amongst the various phytoconstituents reported in the bark of
Pterospermum acerifolium, b-sitosterol is the common sec-
ondary metabolite which had shown various activities
including type II diabetes (Gupta et al., 2011). Therefore,
b-sitosterol was selected as the ligand for in silico studies.
The autodock 4.2 docking software was used to perform
molecular docking simulation between human pancreatic
amylase and b-sitosterol. The crystal structure of HPA
complexed with acarviostatin (PDB CODE: 3OLE) at
1.55 A resolution was downloaded from the RCSB Protein
Data Bank (http://www.rcsb.org/pdb/home/home.do).
MGLTools-1.4.6 was used to prepare protein (protein.pdbqt)
and to write grid parameter file (protein.gpf) and docking
parameter file (ligand.dpf). Protein preparation includes: (i)
removal of water and ions and extraction of co-crystallized
ligand; (ii) addition of polar hydrogens; (iii) assignment of
AD4 atom type; and finally (iv) assignment of Gasteiger
charges. The grid maps representing the native ligand in the
actual docking target site were calculated with autogrid4 with
box dimension of 60� 60� 60 A and spacing of 0.375 A by
taking the centre of the ligand as the centre of the grid.
Docking of the ligand was done with default parameters
except keeping 50 runs.
a-Amylase inhibition assay
A modified form of the Sigma-Aldrich (St. Louis, MO)
method was used (Ali et al., 2006). Briefly, a starch solution
(0.5% w/v) was obtained by stirring 0.125 g of potato starch in
25 ml of 20 mM sodium phosphate buffer with 6.7 mM
sodium chloride, pH 6.9 at 65 �C for 15 min. Enzyme (2
units/ml) solution was prepared by dissolving a-amylase in
ice cold distilled water. In screw cap plastic tubes, 40 ml of
plant extracts (1 mg/ml in dimethyl sulfoxide), 160 ml of
distilled water and 400 ml of starch solution (0.5% w/v) were
mixed. The reaction was started by the addition of 200 ml of
enzyme solution. The tubes were incubated at 25 �C for 3 min.
The above mixture (200ml) was removed and added on to a
separate tube containing 100 ml of dinitrosalicylic acid (DNS)
color reagent solution (96 mM 3,5-dinitrosalicyclic acid,
5.31 M sodium potassium tartarate in 2 M sodium hydroxide)
and kept in a water bath at 85 �C for 15 min, cooled and
quantified at 540 nm. The a-amylase inhibition was expressed
as 100 – % reaction, where the % reaction¼ (maltose in test/
maltose in control)� 100.
Subfractionation of ethyl acetate fraction
The ethyl acetate fraction was further fractionated by column
chromatography. Crude fraction (7 g) was mixed with silica
gel (mesh 60–120) and slurry was prepared to make the
fractions adsorbed with silica gel for uniform mixing. Using
dichloromethane as solvent column was packed with silica gel
(mesh 100–200). The column was eluted with dichloro-
methane, followed by methanol: dichloromethane in the
regular increased proportion of methanol. Each fraction was
tested on TLC plate for its homogeneity and were combined
into three fractions and concentrated on a rotavapour (Buchi
Labortechnik AG, Schweiz) under reduced pressure at 40 �Cto yield the subfractions PABE1 (1.53 g), PABE2 (2.14 g) and
PABE3 (2.87 g).
Animals
Healthy male albino rats (Wistar strain 180–200 g) were
procured from the animal house of Birla Institute of
Technology. They were housed in clean polypropylene
cages with free access to standard laboratory pellet diet and
water at room temperature with an inverted 12:12 h light–dark
cycle and relative humidity of 60%. All the animals were
acclimatized for 7 days prior to the experiments. The
experimental protocols were approved by the Institutional
Animal Ethics Committee (BIT/PH/IAEC/34/2011dt
10.12.2011).
Acute oral toxicity studies and dose fixation
Toxicity studies conducted on female mice based on OECD
guidelines 423 for acute oral toxicity-acute toxic class
method. Three animals were used in each step. The dose
level to be used as the starting dose was selected from one of
the four fixed levels, 5, 50, 300 and 2000 mg/kg body weight.
For the ethyl acetate fraction (PABEF), 2000 mg/kg was fixed
as the initial dose and for the subfractions (PABE1, PABE2
and PABE3) 300 mg/kg was fixed as the initial dose and the
test substance was administered in a single dose by using an
intragastric tube. Animals were observed individually after
dosing at least once during the first 30 min, periodically
during the first 24 h, with special attention given during the
first 4 h, and daily thereafter, for a total of 14 days.
200 P. Rathinavelusamy et al. Pharm Biol, 2014; 52(2): 199–207
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Induction of diabetes
Streptozotocin (STZ) was dissolved in citrate buffer (pH 4.5)
and nicotinamide was dissolved in normal physiological
saline. Non-insulin dependent diabetes mellitus was induced
in overnight fasted rats by a single intraperitoneal injection of
STZ (60 mg/kg b.w.): 15 min later, the rats were given the
intraperitoneal administration of nicotinamide (120 mg/kg
b.w.). Hyperglycemia was confirmed by the elevated glucose
levels determined at 72 h. Animals with blood glucose
concentration more than 250 mg/dL were included in the
study (Pellegrino et al., 1998).
Experimental design
Animals were divided in to 11 groups and each group
consisted of 6 animals.
Group I: Normal control (2% Tween 80; 1 ml/kg b.w.)
Group II: Diabetic control rats receiving sterile water
Group III: PABEF1; Diabetic animals treated with PABEF
200 mg/kg b.w.
Group IV: PABEF2; Diabetic animals treated with PABEF
400 mg/kg b.w.
Group V: PABE1-1; Diabetic animals treated with PABE1
15 mg/kg b.w.
Group VI: PABE1-2; Diabetic animals treated with PABE1
30 mg/kg b.w.
Group VII: PABE2-1; Diabetic animals treated with PABE2
15 mg/kg b.w.
Group VIII: PABE2-2; Diabetic animals treated with PABE2
30 mg/kg b.w.
Group IX: PABE3-1; Diabetic animals treated with PABE3
15 mg/kg b.w.
Group X: PABE3-2; Diabetic animals treated with PABE3
30 mg/kg b.w.
Group XI: STD drug; Diabetic animals treated with
600 mg/kg b.w. (Ananda Prabu et al., 2012) of
glibenclamide.
Plant extracts, glibenclamide and vehicle were adminis-
tered via an intragastric tube for 30 days.
Oral glucose tolerance test
Oral glucose tolerance test (OGTT) was performed in overnight
fasted normal and drug treated rats on the 15th day of treatment.
A glucose load (2 g/kg) was given to each rat orally with a
feeding syringe exactly after 30 min post administration of
extract, standard drug or vehicle. The blood glucose profile of
each rat was determined at time 0 min (prior to the glucose
load) and 30, 60 and 120 min of post-glucose administration.
Food was withheld 18 h prior to administration of glucose
during the course of experimentation (Gandhi et al., 2012).
Biochemical analysis
Fasting blood glucose was measured on days 0, 14, 21 and 28
by using a glucometer (Bayer’s blood glucose measuring kit
with counter strips). On the 30th day, blood was collected in
EDTA coated tubes for measuring glycosylated hemoglobin
(HbA1C) by using a commercial kit (Coral Clinical Systems,
Goa, India) and blood was collected in tubes without
anticoagulant for serum preparations. Serum was analyzed
for ALT (alanine transaminase), AST (aspartate transaminase),
ALP (alkaline phosphatase) by cogent Serum-ALT, AST, ALP
estimation kits (Span Diagnostics Limited, Surat, India);
triglycerides, total cholesterol (TC), HDL-C was estimated
by their respective kits (Span Diagnostics Limited, Surat,
India). For antioxidant enzyme assays, a portion of the liver
tissue was dissected out, washed with ice-cold saline imme-
diately, and dried on tissue paper. The tissue homogenate was
prepared in ice cold phosphate buffer saline (PBS) (0.1 M, pH
7.4) and centrifuged at 10 000 g at 4 �C for 20 min. The
supernatant was quantified for the antioxidant enzyme param-
eters. TBARS (Slater & Sawyer, 1971), catalase (Aebi, 1994),
superoxide dismutase (Misra & Fridovich, 1972) and reduced
glutathione (Moron et al., 1979) were estimated.
Histopathology of pancreas
The pancreas from normal control, diabetic control and
maximum drug dose treated animals were blotted free of
mucus. The tissues were washed in normal saline, cut in to the
desired size and fixed in 10% formalin for 24 h. After fixation,
tissues were dehydrated and embedded in paraffin. Sections
of tissue were made in microtome of 5 mm in thickness and
mounted on slides. The mounted slides were stained with
hematoxylin and eosin for photographic observations.
Statistical analysis
The results were expressed as mean� standard error of the
mean (SEM). Statistical analysis of all the data was evaluated
according to one-way analysis of variance (ANOVA) using
statistical software Graphpad prism version 6. The signifi-
cance of difference was evaluated using one way ANOVA
followed by Dunnett’s multiple comparison test. Probability
values of aaaap50.0001, aaap50.001, aap50.01, ap50.05
were compared with normal control. Probability values ofbbbbp50.0001, bbbp50.001, bbp50.01, bp50.05 were com-
pared with disease control.
Results
Molecular modelling studies
To investigate the interaction of b-sitosterol, molecular
docking simulations of the binding of b-sitosterol along
with acarbose at the HPA active site was carried out using
Autodock 4.2. From this study we found that acarbose forms
three H-bond interactions with the active site residues Tyr2,
Thr6 and Asn5 (Figure 3), while the hydroxyl oxygen of
b-sitosterol has one hydrogen bond with hydroxyl hydrogen
of Thr6 and hydroxyl hydrogen of b-sitosterol has a hydrogen
bond with backbone carbonyl oxygen of Thr6 (Figures 1 and
2). The estimated free energy binding of b-sitosterol was
found to be �8.39 kcal mol�1 with an estimated inhibition
constant (Ki) of 269.35 nmol whereas the estimated free
energy binding of acarbose was �6.07 kcal mol�1 with an
estimated inhibition constant (Ki) of 28.52 mmol which infers
clearly that b-sitosterol is more potent than acarbose.
In vitro a-amylase inhibition study
The results of a-amylase inhibition studies revealed that the
ethyl acetate fraction (PABEF) possessed a higher enzyme
DOI: 10.3109/13880209.2013.823551 Antidiabetic potential of Pterospermum acerifolium 201
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inhibition potential (69.94%) compared to all other fractions,
followed by the 50% ethanolic extract (PABEE), which
showed marked enzyme inhibition potential (26.51%).
PABHF (�69.02%) and PABCF (�12.75%) did not show
positive percentage of inhibition. Hence, for further studies,
the ethyl acetate fraction (PABEF) was selected.
Acute oral toxicity
Acute oral toxicity studies revealed the nontoxic nature of
bark of P. acerifolium at the dose levels tested. No lethality or
toxic reactions were observed throughout the study. Mortality
was not recorded during 14 days on drug treated animals.
Hence, the doses were fixed as 200 and 400 mg/kg for
PABEF; 15 and 30 mg/kg for subfractions (PABE1, PABE2
and PABE3).
Effect of P. acerifolium on OGTT
In the oral glucose tolerance test, the blood glucose levels of
glucose treated experimental animals were increased mark-
edly at 30 min. PABE3 (15 and 30 mg/kg) inhibited the
increasing blood sugar level significantly (p50.05) at the
60th and 120th min when compared with disease control
(Table 1).
Effect of P. acerifolium on fasting blood glucose levels,body weight and HbA1c
Fasting blood glucose levels were measured in all experimental
rats at time zero and on the 14th, 21st and 28th day. PABE3
(30 mg/kg) showed a significant reduction (p50.01) in blood
glucose levels on the 14th, 21st and 28th day as compared to the
disease control group (Table 2). The body weight of the
diabetic control animals was reduced markedly compared to
that of normal animals, PABE3 (30 mg/kg) showed a notice-
able increase in body weight compared to diabetic control
animals (Table 3). The levels of glycosylated hemoglobin
(HbA1c) of diabetic control animals were increased signifi-
cantly (p50.0001) compared to that of normal animals,
PABE3 (30 mg/kg) decreased the level of HbA1c significantly
(p50.01) compared to diabetic control (Table 3).
Effect of P. acerifolium on levels of serum liverenzymes and lipid parameters
The levels of ALT, AST and ALP were found to be increased
significantly in diabetic control animals (p50.0001) as
compared to that of normal animals. Diabetic animals treated
with PABE3 (30 mg/kg) showed the significant reduction
Figure 2. Hydrogen bond interactions between b-sitosterol and aminoacid residues in the active site pocket of human pancreatic a-amylase.
Figure 1. Comparison of binding mode of b-sitosterol in the active sitepocket of human pancreatic a-amylase.
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(p50.01) on levels of ALT, AST and ALP compared to the
diabetic control (Table 4). In lipid parameters, the levels of
triglycerides and total cholesterol of diabetic control animals
were increased significantly (p50.0001) compared to that of
normal vehicle treated animals and the level of HDL-C
(p50.0001) on the same animals were decreased significantly
compared to that of normal vehicle treated animals. Treatment
with PABE3 (30 mg/kg) decreased the level of triglycerides
(p50.001) and total cholesterol (p50.01) and increased the
level of HDL-C (p50.01) significantly when compared to
that of normal control (Table 5).
Effect of P. acerifolium on in vivo antioxidant enzymesstatus
The level of thiobarbituric acid reactive substances (TBARS)
were found to increase significantly (p50.0001) in diabetic
control animals compared to that of normal vehicle treated
animals and the levels of antioxidant enzymes such as
catalase, SOD and GSH were found to decrease significantly
(p50.0001) in diabetic control animals compared to that of
the normal group. Diabetic animals treated with PABE3
(30 mg/kg) showed a significant reduction (p50.01) of the
Figure 3. Hydrogen bond interactions between acarbose and aminoacid residues in the active site pocket of human pancreatic a-amylase.
Table 1. Effect of ethyl acetate fraction and subfractions of Pterospermum acerifolium bark on changes in blood glucoselevel during OGTT.
Blood glucose level (mg/dl)
Treatment 0 min 30 min 60 min 120 min
Normal control 75.77� 0.48 120.20� 1.24 114.80� 2.10 81.83� 2.242Diabetic control 345.80� 6.79aaaa 452.00� 3.20aaaa 444.30� 6.38aaaa 401.80� 5.15aaaa
PABEF-1 239.20� 13.85 297.70� 17.48 282.80� 17.60 255.80� 6.86PABEF-2 221.00� 7.79 277.80� 8.55 259.80� 7.15 248.20� 6.23PABE1-1 238.70� 13.29 288.50� 10.14 272.50� 10.34 260.70� 13.66PABE1-2 228.00� 11.12 282.50� 14.43 262.50� 15.22 244.70� 11.82PABE2-1 282.80� 8.92aa 343.80� 7.60aa 338.70� 5.68aaa 332.50� 7.58aaa
PABE2-2 287.70� 9.40aaa 341.50� 9.17aa 337.20� 10.14aaa 330.50� 11.78aaa
PABE3-1 216.80� 16.51 267.00� 11.74b 245.00� 10.19b 227.00� 11.62b
PABE3-2 211.20� 10.77b 265.70� 9.68b 237.00� 9.28b 222.70� 11.61b
STD drug 191.00� 11.62bb 240.50� 12.83bb 209.20� 12.27bbb 195.20� 11.52bbb
The results are expressed as mean� standard error of the mean (SEM). p Values of aaaap50.0001, aaap50.001, aap50.01were compared with normal control. p Values of bbbp50.001, bbp50.01, bp50.05 were compared with disease control.
DOI: 10.3109/13880209.2013.823551 Antidiabetic potential of Pterospermum acerifolium 203
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levels of TBARS compared to the diabetic control (Table 6)
and the same increased the levels of catalase (p50.05), SOD
(p50.01) and GSH (p50.01) significantly compared to the
disease control (Table 6).
Histopathological studies
In diabetic control rats, the microscopic section of pancreas
showed the features of insulitis and marked evidence of
degeneration of b-cells revealing cellular swelling and
cytolysis with picnotic and fragmented nuclei. As compared
to the diabetic control, animals treated with the ethyl acetate
fraction and subfractions of Pterospermum acerifolium
showed lesser features of insulitis, amplified islet cells
granulation and proper islet cells arrangement along with its
density (Figure 4).
Discussion
Diabetic patients, particularly those with type II diabetes, are
at a considerable risk of excessive morbidity and mortality
from cardiovascular, cerebrovascular and peripheral vascular
diseases leading to myocardial infarction, strokes and ampu-
tations (Watkins, 2003). Pterospermum acerifolium bark was
traditionally used for the treatment of diabetes mellitus, but
there was no in-depth proper scientific justification. Hence,
the in silico antidiabetic activity of b-sitosterol was
determined which was isolated and reported as a major
phytoconstituent earlier from the bark of Pterospermum
Table 2. Effect of ethyl acetate fraction and subfractions of Pterospermum acerifolium bark on changes in blood glucose level.
Blood glucose (mg/dl)
Treatment Day 0 Day 14 Day 21 Day 28
Normal control 68.00� 1.07 75.17� 0.48 74.17� 0.40 77.00� 0.37Diabetic control 286.50� 9.52 342.50� 5.81aaaa 373.30� 7.84aaaa 424.50� 5.15aaaa
PABEF-1 296.30� 11.57 241.30� 13.75 240.50� 17.86a 188.50� 11.60PABEF-2 285.70� 9.39 222.50� 8.09 198.20� 13.90 177.50� 8.07PABE1-1 301.50� 11.14 240.30� 13.43 212.70� 11.46 177.70� 8.72PABE1-2 298.30� 10.40 230.00� 13.43 196.70� 11.96b 175.80� 13.51PABE2-1 297.30� 9.52 285.00� 8.93aa 290.80� 8.10aaa 282.80� 9.70aaa
PABE2-2 303.00� 10.44 289.00� 9.56aaa 277.80� 11.50aaa 276.30� 9.69aaa
PABE3-1 295.80� 10.53 217.50� 16.53b 181.70� 15.96b 161.70� 14.59b
PABE3-2 298.70� 9.35 213.70� 10.73bb 174.70� 12.58bb 151.50� 11.95bb
STD drug 297.20� 10.42 193.00� 11.44bbb 145.20� 12.23bbb 117.80� 12.03bbb
The results are expressed as mean� standard error of the mean (SEM). p Values of aaaap50.0001, aaap50.001, aap50.01,ap50.05 were compared with normal control. p Values of bbbp50.001, bbp50.01, bp50.05 were compared with diseasecontrol.
Table 3. Effect of ethyl acetate fraction and subfractions ofPterospermum acerifolium bark on body weight changes and glycosy-lated Hb.
Body weight (g)
Treatment Initial Final Glycosylated Hb (%)
Normal control 196.80� 3.64 238.10� 4.04 2.74� 0.26Diabetic control 195.70� 4.96 150.40� 4.91 7.93� 0.72aaaa
PABEF-1 198.20� 5.36 202.60� 5.02 6.19� 0.03PABEF-2 195.30� 4.49 200.30� 5.02 6.09� 0.16PABE1-1 197.90� 3.75 201.40� 3.73 5.77� 0.05PABE1-2 201.70� 2.16 208.90� 2.41 5.47� 0.03PABE2-1 200.90� 4.57 174.70� 4.56 7.01� 0.08aaa
PABE2-2 200.10� 2.39 187.00� 1.77 6.75� 0.08aa
PABE3-1 200.40� 3.29 205.60� 3.05 5.35� 0.04b
PABE3-2 197.20� 3.50 222.30� 4.11 4.97� 0.05bb
STD drug 199.00� 4.37 227.00� 3.88 3.32� 0.08bbb
The results are expressed as mean� standard error of the mean (SEM).p Values of aaaap50.0001, aaap50.001, aap50.01 were compared withnormal control. p Values of bbbp50.001, bbp50.01, bp50.05 werecompared with disease control.
Table 5. Effect of ethyl acetate fraction and subfractions ofPterospermum acerifolium bark on levels of lipid parameters.
TreatmentTriglycerides
(mg/dl)Totalcholesterol
(mg/dl)HDL-C(mg/dl)
Normal control 91.64� 0.52 57.35� 0.40 59.37� 0.40Diabetic control 215.60� 1.47aaaa 163.40� 3.66aaaa 14.35� 0.36aaaa
PABEF-1 144.60� 1.02 106.00� 0.51 33.78� 0.48PABEF-2 139.90� 1.06 103.80� 0.56 35.13� 0.66PABE1-1 136.30� 0.89 102.80� 0.46 35.38� 0.34PABE1-2 131.10� 0.85b 98.77� 0.4b 37.45� 0.43b
PABE2-1 197.90� 1.50aaaa 148.70� 0.47aaaa 21.35� 0.37aaaa
PABE2-2 192.20� 1.60aaa 144.40� 0.42aaa 24.39� 0.35aaa
PABE3-1 115.90� 1.14bb 82.13� 6.65b 46.80� 0.53b
PABE3-2 106.20� 1.20bbb 77.83� 0.53bb 48.90� 0.49bb
STD drug 127.50� 1.08b 65.79� 0.57bbb 52.45� 0.34bbb
The results are expressed as mean� standard error of the mean (SEM).p Values of aaaap50.0001, aaap50.001 were compared with normalcontrol. p Values of bbbp50.001, bbp50.01, bp50.05 were comparedwith disease control.
Table 4. Effect of ethyl acetate fraction and subfractions ofPterospermum acerifolium bark on levels of serum liver enzymes.
Treatment ALT (IU/L) AST (IU/L) ALP (IU/L)
Normal control 25.83� 0.27 31.92� 0.22 11.97� 0.22Diabetic control 54.74� 0.50aaaa 72.30� 0.40aaaa 27.27� 0.09aaaa
PABEF-1 46.53� 0.03 61.34� 0.33 24.41� 0.07PABEF-2 43.62� 0.48 59.29� 0.34 22.21� 0.05PABE1-1 38.82� 0.22 58.64� 0.46 21.51� 0.09PABE1-2 38.04� 0.24b 56.43� 0.34b 20.28� 0.08PABE2-1 50.33� 0.38aaaa 71.17� 0.48aaaa 27.53� 0.05aaaa
PABE2-2 51.41� 0.38aaa 68.91� 0.27aaa 26.44� 0.04aa
PABE3-1 37.52� 0.41 46.85� 0.24b 18.21� 0.05PABE3-2 34.80� 0.23bb 44.43� 0.37bb 16.61� 0.07bb
STD drug 28.93� 0.23bbb 36.95� 0.21bbb 14.18� 0.03bbb
The results are expressed as mean� standard error of the mean (SEM).p Values of aaaap50.0001, aaap50.001, aap50.01 were compared withnormal control. p Values of bbbp50.001, bbp50.01, bp50.05 werecompared with disease control.
204 P. Rathinavelusamy et al. Pharm Biol, 2014; 52(2): 199–207
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acerifolium (Muhit et al., 2010). The molecular modeling
results undoubtedly revealed b-sitosterol as a potential
inhibitor of HPA in comparison with acarbose. One of the
most important therapeutic approaches to treat diabetes is to
target the carbohydrate hydrolyzing enzymes, which are
responsible for postprandial hyperglycemia (PPHG).
a-Amylase is one of those most important enzymes which
is responsible for breakdown of carbohydrates and inhibitors
of these enzyme delay carbohydrate digestion and prolong
overall carbohydrate digestion time, causing a reduction in the
rate of glucose absorption and consequently blunting the
postprandial plasma glucose rise (Ali et al., 2006). In the
present study, 50% ethanol extract (PABEE) and fractions
(PABHF, PABCF and PABEF) were studied for its a-amylase
inhibition potential and found PABEF (ethyl acetate fraction)
as active against a-amylase inhibition. Hence, PABEF was
selected for further subfractionation and in vivo studies.
Postprandial hyperglycemia contributes to microvascular
and macrovascular complications during diabetic conditions
and effective control of postprandial hyperglycemia plays a
vital role in early intervention and prevention of diabetic
complications (Gandhi et al., 2012). In the present study,
PABE3 (30 mg/kg) treated diabetic rats significantly reduced
the blood glucose levels at 60th and 120th min when
compared to that of diabetic control and it implies that
there is further scope of the drug to be studied in detail on
animal models of diabetic complications.
Administration of STZ leads to necrosis of the b-cells
through the formation of alkylating free radicals. The rate of
insulin synthesis was diminished and the administration of
nicotinamide, a poly-ADP-ribose synthetase inhibitor, pro-
tected the function of the islets by preventing the decrease in
the levels of NAD and proinsulin. This phenomenon partially
reversed the inhibition of insulin secretion (Srinivasan & Pari,
2012). Persistent hyperglycemia, the common characteristic
of diabetes, can cause most diabetic complications and in all
patients. Treatment should therefore aim to lower blood
glucose to near-normal levels (Shirwaikar et al., 2005). In the
present study, PABE3 (15 and 30 mg/kg) reduced the blood
glucose level at 14th, 21st and 28th days significantly and it
showed clearly its antihyperglycemic effect which may be due
to its increased release of insulin from existing b-cells.
STZ-nicotinamide induced diabetes is characterized by the
severe loss of body weight due to the destruction of structural
proteins, which are known to contribute to body weight
(Ananda Prabu et al., 2012). Treatment of diabetic animals
with PABE3 (30 mg/kg) prohibited the loss of body weight
when compared to that of diabetic control animals and
showed its ability to reduce hyperglycaemia. HbA1C is a
nonenzymatic glycated product of the hemoglobin b-chain
and it is normally present at low levels in circulating red cells
because of the glycosylation reaction between Hb and
circulating glucose, but in the presence of excess plasma
glucose this glycation is increased, thus making the HbA1C a
useful index of glycemic control (Yavari, 2011). Diabetic
animals treated with PABE3 (15 and 30 mg/kg) reduced the
HbA1c levels significantly compared to that of diabetic
control group and it could also be due to its anti-
hyperglycemic property.
AST, ALT and ALP are the biomarker enzymes needed to
evaluate hepatic disorders. Increase in the activities of these
enzymes in diabetic rats validated hepatic damage (Navarro
et al., 1993). Significant reductions in the activities of these
enzymes in PABE3 (30 mg/kg) treated diabetic animals
indicated the hepatoprotective role in preventing diabetic
complications. The elevation of serum triglycerides, total
cholesterol and the decline in the level of HDL-C was well
documented in diabetic animals (Shirwaikar et al., 2005).
Significant reduction in the level of triglycerides, total
cholesterol and rise in the level of HDL-C was observed in
PABE3 (30 mg/kg) treated diabetic animals and it may be
directly attributed to improvements in insulin levels upon the
treatment or indirectly to its influence on various lipid
regulation systems (Shirwaikar et al., 2005).
A marked increase in the level of TBARS and decrease in
the levels of antioxidant enzymes such as catalase, GSH and
SOD were observed in diabetic animals. The increased levels
of TBARS in diabetic animals were due to activation of the
lipid peroxidation system in tissues and may lead to cellular
infiltration and cell damage (Srinivasan & Pari, 2012).
Superoxide dismutase is an important defense enzyme,
which catalyzes the dismutation of superoxide radicals
(McCord et al., 1976), and catalase is a hemoprotein, which
catalyzes the reduction of hydrogen peroxides and protects
Table 6. Effect of ethyl acetate fraction and subfractions of Pterospermum acerifolium bark on levels of antioxidant status.
Treatment TBARS Catalase Red.Glutathione SOD
Normal control 0.98� 0.19 74.82� 3.67 49.16� 1.96 7.86� 0.89Diabetic control 1.74� 0.78aaaa 30.78� 3.16aaaa 24.17� 2.07aaaa 3.7� 0.35aaaa
PABEF-1 1.45� 0.36 47.32� 2.71a 34.81� 1.87a 4.32� 0.16a
PABEF-2 1.39� 0.12 51.33� 1.73 36.06� 2.11 4.67� 0.32PABE1-1 1.39� 0.81 50.16� 3.34 35.41� 1.32 4.53� 0.16PABE1-2 1.35� 0.87 55.31� 1.77 36.11� 1.81 4.86� 0.31PABE2-1 1.69� 0.32aaa 30.74� 2.17aaaa 27.33� 1.82aaa 3.8� 0.16aaa
PABE2-2 1.67� 0.18aa 33.21� 1.61aaa 29.11� 2.11aa 3.93� 0.22aa
PABE3-1 1.18� 0.32b 56.81� 1.32 38.11� 1.09b 5.86� 0.21b
PABE3-2 1.14� 0.03bb 59.31� 1.72b 40.81� 1.12bb 6.01� 0.11bb
Std Drug 1.05� 0.17bbb 68.87� 2.16bbb 44.86� 2.07bbb 6.41� 0.09bbb
Thio barbituricacid reactive substances (TBARS) expressed as nM of MDA/mg protein; Catalase expressed as nmoles ofH2O2 consumed/min/mg protein; Superoxide dismutase (SOD) expressed as U/mg protein; Glutathione expressed as mgof GSH/mg protein. The results are expressed as mean� standard error of the mean (SEM). p Values of aaaap50.0001,aaap50.001, aap50.01, ap50.05 were compared with normal control. p Values of bbbp50.001, bbp50.01, bp50.05 werecompared with disease control.
DOI: 10.3109/13880209.2013.823551 Antidiabetic potential of Pterospermum acerifolium 205
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tissues from highly reactive hydroxyl radicals (Chance et al.,
1952). GSH is one of the essential compounds for maintaining
cell integrity against ROS, as it can scavenge free radicals and
reduce hydrogen peroxide and the exhaustion of GSH
promotes oxidative stress with a cascade of effects on the
functional and structural integrity of cells and organelle
membranes (Masella et al., 2004). The liver plays a major role
in glutathione homeostasis and is the main export organ for
glutathione (Townsend et al., 2003). Treatment with PABE3
(30 mg/kg) reduced the TBARS levels and increased the
levels of antioxidant enzymes compared to that of diabetic
control and it clearly implied its role against oxidative stress.
(A) (B)
(C) (D)
(E) (F)
(G)
Figure 4. Histopathological observation of experimental rats pancreas after 30 days of treatment (A) normal control; (B) diabetic control; (C) diabeticanimals treated with PABEF (400 mg/kg); (D) diabetic animals treated with PABE1 (30 mg/kg); (E) diabetic animals treated with PABE2 (30 mg/kg);(F) diabetic animals treated with PABE3 (30 mg/kg); (G) diabetic animals treated with glibenclamide (600 mg/kg).
206 P. Rathinavelusamy et al. Pharm Biol, 2014; 52(2): 199–207
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Histopathological changes showed the prominent changes in
islet cell arrangement and density in Pterospermum acer-
ifolium treated animals, which also evidenced its diabetic
activity. The active subfraction PABE3 was subjected to
chromatographic studies in the reported chromatographic
conditions and solvent system to examine the presence of
b-sitosterol (Misar et al., 2010). We have found two major
components, but their Rf values did not match with the
reported b-sitosterol Rf value. Hence, there was no correlation
between b-sitosterol and in vivo antidiabetic activity shown
by PABE3, but by our inference we confirm that some
antidiabetic phytoconstituents other than b-sitosterol were
present in that active fraction which was responsible for the
activity.
Conclusion
From the present study it can be concluded that PABEF
showed maximum a-amylase inhibition potential and its
subfraction PABE3 showed maximum efficacy in reducing
hyperglycemia. The present study demonstrated for the first
time the alpha-amylase inhibitory potential and elaborated
antidiabetic activity of the bark of Pterospermum acerifolium.
Further studies to isolate the bioactive constituents respon-
sible for the antidiabetic activity and detailed cellular assays
to estimate the mechanism of action are in progress on the
active subfraction PABE3. The above study also reveals
the scope of the bark of Pterospermum acerifolium in the
screening of diabetic complications.
Acknowledgements
The authors would like to acknowledge the Department of
Pharmaceutical Sciences Birla Institute of Technology,
Mesra, Ranchi, India for providing the necessary facilities
to carry out the study and the University Grants Commission
(UGC) for providing financial assistance. The authors are
thankful to Dr. K.K. Singh, Chairman-Veterinary Pathology
division, Birsa Agricultural University, Ranchi for assistance
in histopathology observations.
Declaration of interest
The authors report no declarations of interest. The authors
alone are responsible for the content and writing of the paper.
This work was financially supported by University Grant
Commission (UGC) under the scheme of major research
project (MRP), New Delhi, India. (Grant No: F.39-157/2010
(SR))
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