Pharmaceutical Biology

ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: https://www.tandfonline.com/loi/iphb20

Anti-inflammatory action of Tamarind seeds

reduces hyperglycemic excursion by repressing
pancreatic β-cell damage and normalizing
SREBP-1c concentration

Sushant S. Sole, B. P. Srinivasan & Atul S. Akarte

To cite this article: Sushant S. Sole, B. P. Srinivasan & Atul S. Akarte (2013) Anti-inflammatory
action of Tamarind seeds reduces hyperglycemic excursion by repressing pancreatic β-cell
damage and normalizing SREBP-1c concentration, Pharmaceutical Biology, 51:3, 350-360, DOI:
10.3109/13880209.2012.729067

To link to this article: https://doi.org/10.3109/13880209.2012.729067

Published online: 15 Nov 2012.

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Pharmaceutical Biology, 2013; 51(3): 350–360
© 2013 Informa Healthcare USA, Inc.
ISSN 1388-0209 print/ISSN 1744-5116 online
DOI: 10.3109/13880209.2012.729067

RESEARCH ARTICLE

 nti-inflammatory action of Tamarind seeds reduces

A
hyperglycemic excursion by repressing pancreatic β-cell
damage and normalizing SREBP-1c concentration
Sushant S. Sole, B. P. Srinivasan, and Atul S. Akarte

Department of Pharmacology, Delhi Institute of Pharmaceutical Sciences and Research, University of Delhi,
New Delhi, India

Abstract
Context: Tamarindus indica L. (Leguminosae) is widely used as a traditional medicine for the management of diabetes
mellitus (DM) in India, in addition to its anti-inflammatory activity. The present study has been designed to understand
the correlation involved between antidiabetic and anti-inflammatory action of aqueous seed extract of T. indica (TSE)
in diabetic rats.
Objective: In view of the fact that fatty acid synthesis and insulin release from islets of pancreas are regulated by sterol
regulatory element-binding proteins (SREBP-1c) and cytosolic calcium, respectively, the objectives of present study
were to determine the influence of TSE on SREBP-1c mRNA and to investigate the intracellular islets calcium [Ca2+]I
involvement and β-cell mass preservation in insulin secretagogue action of TSE.
Materials and methods: The effect of 4 weeks oral treatment (120 and 240 mg/kg) of high-performance liquid
chromatography (HPLC) standardized TSE was studied in streptozotocin (STZ)-induced diabetic male Wistar rats.
Reverse transcription-PCR (RT-PCR) and a spectrofluorometer were used for mRNA concentration and islets [Ca2+]I
determination, respectively. The TUNEL assay was followed to study the pancreatic apoptosis.
Results: TSE (120 and 240 mg/kg) showed positive correlation with [Ca2+]I and insulin release. The anti-inflammatory
action of TSE was significant on nitric oxide (NO) and tumor necrosis factor-α (TNF-α) in addition to a favorable effect
on β-cell neogenesis and improved mRNA concentration of SREBP-1c.
Discussion and conclusion: The results suggest that anti-inflammatory action of Tamarind seeds on β-cell cells of islets
and cytokines contribute toward its antidiabetic activity by way of complex mechanisms of [Ca2+]I handling and
through SREBP-1c gene in liver.
Keywords:  Diabetes mellitus, cytosolic calcium, oxidative stress, β-cell neogenesis, tumor necrosis factor α, sterol
regulatory element binding protein-1c, serum nitric oxide, lipid profile, islets apoptosis

Introduction activities due to a continuum of tissue insults leading

Diabetes mellitus (DM) is a disease that results in
chronic inflammation and apoptosis in pancreatic
islets in either type 1 or type 2 DM patients and is
characterized by abnormal insulin secretion or insulin
receptor or post receptor events affecting metabolism
in addition to damaging liver, kidney, and β-cells of
pancreas (Baynes, 1991). In DM, chronic damage is
associated with elevated oxidative or inflammatory
to more severe diabetic complications (Ndisang, 2010).
However, the protection of pancreatic islets β-cells
from selective destruction can be one of the targets for
treatment of DM. Thus, it is necessary to look for new
and more efficacious drugs and to make use of the
vast reserves of phytotherapy for medicinal purposes.
Tamarindus indica L. [(Leguminosae (Caesalpiniaceae)]
commonly known as Tamarind, occurs in the tropical

Address for Correspondence: Department of Pharmacology, Delhi Institute of Pharmaceutical Sciences and Research, Pushp Vihar, Sector-3,
MB Road, New Delhi 110017, India. Tel.: +91-11-29554327; Fax: +91-11-29554503. E-mail: sush.sole@gmail.com
(Received 26 June 2012; accepted 07 September 2012)

350

regions of the world, and can be found in more than 50
countries. Tamarind seeds have been reported to contain
polyphenolic compounds like epicatechin, procyanidin
polymers and are used in traditional medicine for the
management of DM (Iyer & Iyer, 1995). Furthermore, the
aqueous extract of Tamarind seeds was found to have
potent antidiabetic and antihyperlipidemic activities in
streptozotocin (STZ) induced diabetic male rat (Maiti
et al., 2004, 2005).
Oxidative stress is one of the key mechanisms in the
pathogenesis of diabetes-related vascular dysfunction.
STZ-induced β-cell damage involves many complicated
mechanisms, one of which is production of reactive
oxygen species, particularly NO either from STZ (Herold
et al., 1997) or neighboring macrophages (de Groot et al.,
2003). Peroxynitrite and proinflammatory cytokines such
as tumor necrosis factor-α (TNF-α), have been found to
cause DNA damage and apoptosis in human and rat
islets (Hadjivassiliou et al., 1998). Furthermore, it has
been reported that SREBP-1c expression and its nuclear
abundance is low in liver of STZ-induced diabetic rats
and increases markedly with insulin treatment. This fact
presents a direct relation between insulin and fatty acid
metabolism (Shimomura et al., 1999).
A rise in the [Ca2+]I, owing to influx through voltage-
gated l-type Ca2+ channels in the plasma membrane,
leads to insulin secretion from the pancreatic β-cell
(Prentki & Matschinsky, 1987). Thus, it is imperative to
consider the action of Tamarind seeds on [Ca2+]I, to ascer-
tain whether it has insulin secretagogue effect or not.
Therefore, by putting these views together, the aim of
study was to investigate the effect of TSE on: (1) insulin
secretion and blood glucose, (2) [Ca2+]I in isolated rat
pancreatic islets, (3) β-cell proliferation, apoptosis, and
neogenesis, (4) cytokine (TNF-α), lipid profile specifi-
cally high-density lipoprotein (HDL), low-density lipo-
proteins (LDL), cholesterol, and NO and, (5) SREBP-1c
mRNA concentration in liver in STZ-induced diabetic rat
model.
Materials and methods
Preparation of aqueous extract of seeds of T. indica
Seeds of T. indica were collected from Kharibauli, New
Delhi in the month of May 2011 and authentication
was done by Dr Roshini Nayar, Scientist at the National
Bureau of Plant Genetic Resources, New Delhi, India
(voucher No. NHCP/NBPGR/2010–52). An aqueous
extract of the seeds of T. indica was prepared using the
method mentioned by National Institute of Health and
Family Welfare, India (Khillare, 2000). Briefly, after incu-
bation for 2 days at 40°C, the seeds of T. indica were pow-
dered in a grinder. Powder (100 g) suspended in 500 mL
redistilled water and the extraction was performed
in Soxhlet apparatus for 18 h. A deep brown aqueous
extract was obtained which was filtered using a coarse
sieve filter paper (Whatman paper grade 591:7–12 µm).
The filtrate was then dried under reduced pressure and
© 2013 Informa Healthcare USA, Inc.
Insulin mimetic Tamarind seeds and β-cell protection  351
finally lyophilized. Phytochemical screening and stan-
dardization of the extract was done before commence-
ment of the in vivo study. The aqueous extract yielded
2.8 g lyophilized powder (0.28%) from 1 kg of Tamarind
seeds.
Analytical high-performance liquid chromatography
(HPLC)
Freeze-dried material (TSE 5 g obtained from 17.8 kg of
Tamarind seeds) was extracted with petroleum ether in
a Soxhlet apparatus (3 h) to remove lipid content. After
drying, the solids were extracted with methanol (3 × 3 h)
(Owen et al., 2003). Reversed-phase analytical HPLC
was conducted on a Hewlett-Packard (HP) 1090 liquid
chromatogram fitted with a C-18 (250 × 4, 6.5 µ). For the
separation of individual compounds, the mobile phase
consisted of phosphoric acid in doubly distilled water
(A) and acetonitrile (B) utilizing the following gradient:
95% A for 0.01 min, 70% A for 35 min, 70% A for 36 min,
and 95% A for 40 min. The flow rate of the mobile phase in
both cases (A and B) was 1.0 mL/min. The column tem-
perature and injection volume was maintained at 30°C
and 10 µL, respectively. The chromatogram was scanned
up to 20 min, which was detected at 280 nm, followed by
washing and reconditioning of the column. The analysis
was done in triplicate.
Animals
Male Wistar rats weighing between 150–200 g used
for the study were obtained from the Animal House
of the Delhi Institute of Pharmaceutical Sciences and
Research. Rats were housed in colony cages (four rats
per cage), at an ambient temperature of 25°C with 12 h
light:12 h dark cycle. Rats had free access to standard
food and water ad libitum. The Principles of Laboratory
Animal Care (NIH, 1985) were followed throughout
the duration of experiment. All the experimental pro-
cedures were conducted according to the Institutional
Animal Ethical Committee (Protocol No.2/DIPSAR/
IAEC/2010) and Committee for the Purpose of Control
and Supervision on Experiments on Animals (CPCSEA)
guidelines.
Drugs and chemicals
The biochemical kits used in the experiment were
obtained from, serum NO (Biovision, U.S.A., Milpitas,
CA, USA, Cat#K262-200), Apo-BrdU-IHCTM In Situ DNA
Fragmentation Assay Kit (Biovision, U.S.A, Cat#K403-
50), TNF-α (Raybiotech, U.S.A. Inc., Norcross, GA, USA,
cat#ELR-TNF-α-001), RPMI 1640 (Sera Laboratories
International Ltd., West Sussex, UK), STZ (Sigma
Chemicals, St Louis, MO, USA), metformin (Ranbaxy Ltd.,
Gudgaon, India), glimepiride (Batch no. P010743397)
Manufacturer: Hetero Labs Ltd. (Hyderabad, India).
This was provided by: Panacea Biotech Ltd. Malpur,
Baddi, Solan (India), Taq DNA polymerase (Bioline Ltd.,
London, UK; Cat-A5209, 0200), benzyl penicillin, and
streptomycin (Sigma-Aldrich, Munich, Germany).
352  S. S. Sole et al.
Experimental design/animal group
Induction of type 2 diabetes
STZ was injected at dose level of 90 mg/kg intraperito-
neal (prepared freshly in 0.1 M citrate buffer pH 4.5) to
2-day-old neonatal rat. Controls were injected with an
equivalent volume of citrate buffer. After 6 weeks of injec-
tion, animals were evaluated for fasting blood glucose
level. The fasting glucose level of 140 mg/dL was the cri-
teria for selection of diabetic rats (Weir et al., 1981) A total
of 40 male rats were used and divided into five groups
(n = 8): group 1, normal untreated rats; group 2, dia-
betic control rats; group 3, diabetic rats treated with TSE
extract (120 mg/kg); group 4, diabetic rats treated with
TSE (240 mg/kg); and group 5, diabetic rats treated with
metformin (100 mg/kg). The basis for selection of doses in
present study was previous reports of antidiabetic action
of Tamarind seeds (Maiti et al., 2004). The treatment
period was for 4 weeks. In the morning, after adminis-
tration of last dose, blood samples were collected under
fasting conditions and body weight was measured. The
pancreas was then isolated and was immersed and fixed
in 10% phosphate-buffer formalin solution, to prepare a
paraffin section.
Determination of biochemical metabolic parameters
The fasting blood glucose and body weights were mea-
sured on day 0, 14, 21, and 28 (data for 14th and 21th
day not represented). Plasma insulin was determined
by enzyme linked immunosorbent assay (Mercodia,
Uppsala, Sweden), on day 28. Four weeks after the
administration of drugs (TSE, metformin), under the
ether anaesthesia blood was collected from the hearts
of animals by cardiac puncture prior to killing, placed in
EDTA vacutainer tubes and centrifuged at 4000g, at 4°C
for 15 min, and stored at –80°C until analysis. The ali-
quots of plasma samples were used for the quantification
of TNF-α and insulin level. Serum was separated by sub-
jecting the coagulated blood to centrifugation at 4000g,
for 15 min and further used for the NO, HDL, LDL, and
cholesterol determination using ELISA, on day 28.
Isolation and culture of islets from normal rats
Animals were sacrificed by decapitation and pancreatic
islets of Langerhans were isolated (Lacy & Kostianovsky,
1967), using digestion with collagenase obtained from
Clostridium histolyticum (Sigma-Aldrich). Digestion and
sedimentation of islets were carried out in Hanks’ solution
containing 5.5 mM glucose. Islets were then handpicked
under a stereomicroscope and transferred to Petri dishes
containing culture medium RPMI 1640 supplemented
with 100 U/mL benzyl penicillin, 0.1 mg/mL streptomycin,
2 mM l-glutamine, and 10% (vol/vol) heat-inactivated fetal
calf serum. Islets were cultured free floating at 37°C, in 5%
CO2/95% O2 atmosphere and incubation was done for 48 h.
Measurement of cytosolic Ca2+ concentrations
Pancreatic islet cells were obtained from adult rats and
cultured overnight in RPMI 1640 culture medium. The
 Pharmaceutical Biology
cells were loaded with Ca2+ indicator by incubation
with 2 µM fura-2/AM for 45 min in the culture medium.
They were then washed in medium containing 140 mM
NaCl, 5.9 mM KCl, 1.28 mM CaCl2, 1.2 mM MgCl2, 25 mM
HEPES, pH 7.4, and 1 mg/mL bovine serum albumin. The
cells (~4 × 106) were suspended in 2.5 mL of fresh washing
medium in a stirred cuvette placed in a spectrofluorome-
ter (Perkin Elmer 50 B; Perkin Elmer, Waltham, MA, USA)
for monitoring [Ca2+]I changes using 340 or 340/380 nm
excitation ratio and emitted fluorescence of 510 nm at
37°C. Intracellular Ca2+ levels could not be calibrated
directly, and thus the [Ca2+]I changes presented are
only relative. After a stable baseline was reached, islets
were supplemented with 0 or 100 µg/mL TSE, or 0 or
50 μM glimepiride in the presence or absence of 20 mM
d-glucose. Time that the cells spent in the absence of
glucose, that is, the time needed for washing, suspending
and establishment of baseline, was 10–15 min. For each
experiment a separate preparation of islets was used
(Thomas et al., 1991).
Immunocytochemistry
Whole pancreas from rats was removed under anesthesia
and fixed in 10% buffered formalin for 24 h. Tissues were
dehydrated in graded series of alcohol, embedded in paraf-
fin, sectioned at 5 µ thickness and used for immunostain-
ing. The tissue sections were stained with hematoxylin and
eosin while the remaining serial sections were used for
immunostaining. Serial sections of the rat pancreas were
immunostained by streptavidin-biotin peroxidase method
using prediluted polyclonal antibodies. All sections were
deparaffinized in xylene bath to remove the excess wax.
The slides were placed in two changes of absolute alcohol
for 3 min each. The same procedure was repeated with 90%
alcohol. The slides were placed in blocking reagent in order
to block the endogenous peroxidase activity for 5 min,
which was prediluted with 5 volumes of 100% ethanol. The
slides were placed in two changes of 70% alcohol for 3 min
each. The excess alcohol around the sections was removed
and the slides were quickly immersed in Tris buffer, pH 7.6
for 5 min. Two drops of tissue conditioner was added and
the sections were incubated for 5 min and then rinsed in
buffer solution. Prediluted primary polyclonal anti-guinea
pig antibody to insulin (1:1,000) (Genetex, Irvin, CA, USA)
raised against human insulin was added to the sections
and incubated for 1 h. The secondary antibody for insulin
was anti-rabbit polyclonal antibodies. After incubation for
half an hour, the sections were rinsed with Tris buffer, per-
oxidase solution was added, incubated for 30 min and later
rinsed with the buffer. AEC (3-amino, 9-ethyl carbazole)
chromogen substrate was added to the sections and was
incubated for 15 min and rinsed with distilled water. The
sections were counter-stained with Harris haematoxylin
for 45 s to facilitate nuclear identification (Hsu et al., 1981).
DNA fragmentation assay
For detection and localization of apoptosis in the pan-
creas, we used the technique of TUNEL. Briefly, sections

were deparaffinized, hydrated, and digested with pro-
teinase K (20 μg/mL), and then added biotinylated dUTP
to the 3′ end of DNA fragments by incubating sections in
0.05 mol/l Tris–HCl buffer (pH 7.6) with 0.03 U/μL TdT
and 0.04 nmol/μL biotin-11-dUTP at 37°C for 1 h. The
sections were rinsed in PBS. Endogenous peroxidase
was blocked with 0.3% H2O2 in distilled H2O. The sections
were rinsed with PBS and covers with 2% blocking solu-
tion in 0.1 mol/L sodium maleate to reduce background
staining. The sections were then incubated with avidin-
peroxidase complexes in phosphate-buffered saline
(PBS) (1:50) for 30 min and rinsed with PBS (3 × 5 min).
Peroxidase activity was visualized with 3,3-diaminoben-
zidine until the brown product was clearly visible. The
sections were then counter-stained with methyl green.
The positive apoptotic cells were the cells with brown
nucleus (Matsuno et al., 1997).
Insulin mimetic Tamarind seeds and β-cell protection  353
data obtained in present study in addition to previ-
ous reports of polyphenols content in Tamarind seeds
(Siddhuraju, 2007; Razali et al., 2012) led us to highlight
our investigation with perspective of catechin and epi-
catechin. Analytical reversed-phase HPLC of the extract
of Tamarind seeds as depicted in Figure 1, revealed the
presence of flavonoids, catechin, and epicatechin. The
peak for catechin and epicatechin was observed at 16.35
and 20.58 in test sample (Figure 1C), which can be cor-
roborated with the peaks of standard at 16.33 (Figure 1A)
and 20.48 (Figure 1B), respectively.

RNA isolation and real-time PCR analysis

Real-time PCR amplifications for SREBP1C (Genbank
ID-78968) was conducted using Light-Cycler® 480 SYBR
Green I Master (Roche, Basel, Switzerland) according
to the manufacturer’s instructions. Reaction conditions
were 10× PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl,
and 2.5 mM MgCl2), 20 pM oligonucleotide, 300 μM
dNTPs, 1:1000 SYBR Green I nucleic acid stain, 0.5 U
Taq DNA polymerase (recombinant), and 50 ng tem-
plates cDNA in a 25 ml reaction volume. The forward
primer was 5′- GGA GCC ATG GAT TGC ACA TT- 3′; the
reverse primer was 5′-AGG AAG GCT TCC AGA GAG
GA-3′. Thermal cycling conditions were 95°C for a 3 min
denaturation step, followed by 40 PCR cycles (94°C for
30 s, 56°C for 30 s, and 72°C for 1 min) and reactions were
performed in a Light Cycler 480 (Roche) instrument.
Fluorescence was detected at the end of the 56°C seg-
ment in the PCR step. PCR products of β-actin-F 5′-TCA
CCC ACA CTG TGC CCC ATC TAC GA-3′ and β-actin-R
5′ CAG CGG AAC CGC TCA TTG CCA ATGG-3′ primers
gene, were used as internal standards. All assays were
carried out in triplicate. Real-time PCR analysis and sub-
sequent calculations were performed on Light- Cycler®
480 software (Roche, version LCS480 1.2.0.169).

Statistical analysis
All values are means ± SEM. Data analysis was done with
one way analysis of variance followed by Dunnet’s mul-
tiple test where diabetic group was considered as positive
control (Sigma Plot11, San Jose, CA, USA).

Results
Analysis of catechin and epicatechin in Tamarind seeds
(HPLC)
Keeping in view of the ethno-pharmacological impor-
tance of T. indica seeds, preliminary studies were Figure 1.  Reversed-phase high-performance liquid chromatogram
(RP-HPLC) of (A) catechin (B) epicatechin, and (C) TSE. TSE,
undertaken for standardization. The preliminary phy- tamarind seed extract. The peaks for catechin and epicatechin, at
tochemical study revealed that TSE was positive for retention time 16.35 and 20.58, were observed in the chromatogram
alkaloid, tannin, and flavonoid contents. The primary of the extract along with other components.

© 2013 Informa Healthcare USA, Inc.

354  S. S. Sole et al.

Body weight, blood glucose, and plasma insulin
Before the supplementation of TSE and metformin, there
were no significant differences of baseline body weight
of the rats (Figure 2). The TSE (120 and 240 mg/kg) and
metformin (100 mg/kg) treated rats showed significant
increase in body weight as compared with diabetic groups
after 4 weeks of study. Before treatment, the fasting glu-
cose level was significantly higher (p < 0.05) in all groups
when compared with the normal group. After 4 weeks,
groups treated with TSE showed dose-dependent reduc-
tion of fasting glucose versus diabetic group (p < 0.05).
The insulin level was significantly decreased in diabetic
rats in respect to nondiabetic control. After 28 day of TSE
supplementation to the diabetic rats, there was a significant
elevation in insulin level in respect to diabetic control group,
in dose-dependent manner (p < 0.05, Figure 2). Of the two
doses of TSE tested, the 240 mg/kg dose was found to be the
most effective in reducing fasting blood glucose levels.
Serum NO and TNF-α concentrations
Vehicle-treated diabetic rats led to significant increase in NO
and TNF-α concentration compared with the correspond-
ing vehicle-treated normal rats. The daily administration of
TSE at both the dose levels (120 and 240 mg/kg) showed a
dose-dependent decrease in TNF-α concentration when
compared with the corresponding diabetic control (p < 0.05,
Table 1). In the TSE supplemented groups, the levels of NO
were decreased by 22 and 43% at 120 and 240 mg/kg dose
levels, respectively. This suggests that TSE could be a poten-
tial NO scavenger along with its anti-inflammatory activity
which was represented by reduction in TNF-α level.
Cytosolic Ca2+ concentrations
Stimulation of isolated islets with glucose produced
the expected increase of intracellular Ca2+, especially at
the lowest glucose concentration (Figure 3). It has been
shown before that Ca2+ oscillations of individual β-cells
within the mouse and rats islet are synchronized, so that
oscillations are observed besides at the level of the whole
islet (Valdeolmillos et al., 1993). In the absence of glu-
cose, both TSE (100 µg/mL) and glimepiride (50 µM) sig-
nificantly increased the islet cytosolic Ca2+ (TSE 22.22%;
*p < 0.05, glimepiride 40%; **p < 0.01, n = 8) contents.
The addition of 20 mM d-glucose alone increased the
islet cytosolic Ca2+ contents. In the presence of 20 mM
d-glucose, both TSE and glimepiride significantly
increased islet cytosolic Ca2+ contents (TSE 42%; #p < 0.05,
glimepiride 60%; ###p < 0.001, n = 8) when compared with
d-glucose alone treated islets (*p < 0.05 and **p < 0.01 for
difference from 0 mM glucose, #p < 0.05 and ###p < 0.001 for
difference from 20 mM glucose; Figure 3).
Serum lipid (HDL, LDL, and cholesterol) profile and
SREBP-1c mRNA in rat liver
The aqueous extract of Tamarind seeds at 120 and 240 mg/
kg dose, decreased the elevated levels of serum LDL

Figure 2.  Influence of Tamarind seed extract on body weight (g), blood glucose, and insulin in diabetic rats. The values are the means ± SEM
from eight animals in each group. *p < 0.05 vs. diabetic group. TSE, Tamarind seed extract. Bar graph with dark shade represents body weight
before treatment whereas, with light shade after treatment (TSE-120 and 240 mg/kg, metformin-100 mg/kg, saline-for normal rats, orally for
4 weeks).

 Pharmaceutical Biology

and cholesterol, significantly (p < 0.05). The significant
improvement was noted in serum HDL level. Although
unable to bring down the normal level of serum lipid and
inflammatory cytokine (TNF-α), the TSE had marked
effect on the elevated concentration of these parameters
(Figure 4).
It has been well established that acetyl CoA carboxyl-
ase and cytosolic HMG-CoA synthase, the key enzymes
of fatty acid and cholesterol synthesis, respectively, are
regulated by SREBP-1c during the transcriptional step
(Lopez et al., 1996). Accordingly, we investigated the
influence of TSE on SREBP-1c mRNA in liver. As shown in
Figure 4, amplification efficiency (E) for gene was deter-
mined by linear regression analysis of the fluorescent
data from the exponential phase of PCR (Peirson et al.,
Table 1.  Effect of Tamarind seed extract on TNF-α and serum NO.
Nitric oxide
TNF-α (pg/mL) (nmol/µL)
Normal 11.25 ± 0.59* 1.85 ± 0.27*
Diabetic 24.50 ± 0.75 10.02 ± 0.50
TSE-120 mg/kg 17.87 ± 0.61* 7.74 ± 0.23*
TSE-240 mg/kg 14.37 ± 0.73* 5.64 ± 0.13*
Metformin 12.75 ± 0.36* 5.46 ± 0.18*
#The values are the means ± SEM. *p < 0.05 vs. diabetic group
(n = 8). Data analysis was done with one way analysis of variance
(ANOVA) followed by Dunnet’s multiple test. The diabetic group
without drug treatment was compared with the normal, TSE
treated (120, 240 mg/kg), and Metformin (standard) groups. The
route of drug (saline in case of normal group) administration for
all the groups was oral. NO-serum, TNF-α-plasma. Duration of
treatment was 4 weeks after diabetes induction.NO, nitric oxide;
TNF-α, tumor necrosis factor-α; TSE, Tamarind seed extract.
Figure 3.  Effect of Tamarind seed extract on the pattern of cytosolic calcium concentration in islets of pancreas. The values are the means ±
SEM from eight animals in each group. *p < 0.05; **p < 0.01; ***p < 0.001 vs. diabetic group. TSE, Tamarind seed extract, Glime, glimeperide,
Glu, glucose. Data analysis was done with one way analysis of variance (ANOVA) followed by Dunnet’s multiple test.
© 2013 Informa Healthcare USA, Inc.
Insulin mimetic Tamarind seeds and β-cell protection  355
2003). Quantitative RT-PCR results showed increase in
liver SREBP-1c mRNA concentrations after 4 weeks treat-
ment of TSE as compared with corresponding vehicle-
treated diabetic rats.
Influence on histopathology changes and cell
apoptosis in the pancreas of diabetic rats
Histopathology evaluation of the pancreas of diabetic
rats revealed a high frequency of degenerative changes,
such as moderate decrease in the number of insulin-
positive granules and atrophy, pyknosis, degeneration,
and necrosis in the islets (Figure 5E, H). Whereas, degen-
erative changes occurred at a low frequency, in the TSE
and metformin groups, with only a slight decrease in the
number of insulin-positive granules and no marked islet
atrophy, degeneration, or necrosis (Figure 5F, I).
TSE and metformin reduced pancreatic cell apoptosis
in diabetic treated rats. By contrast, morphological fea-
tures of apoptosis, including pyknotic nuclei, were read-
ily detectable in pancreatic sections from diabetic rats
(Figure 5B, C).
Discussion
Herbal extract is a mixture of many compounds and
produce complex chromatogram. However, under the
experimental conditions used in this work, no interfer-
ence from these constituents was observed. Figure 1A
and B shows the HPLC chromatograms of catechin and
epicatechin standard, while Figure 1C shows the HPLC
chromatogram of TSE. The presence of catechin, epicat-
echin along with other chemical constituents has been
356  S. S. Sole et al.

Figure 4.  Effects of Tamarind seed extract on serum low-density lipoprotein (LDL), high-density lipoprotein (HDL), Cholesterol and sterol
regulatory element-binding protein-1c (SREBP-1c) concentration (liver). The values are the means ± SEM from eight animals in each group.
*p < 0.05 vs. diabetic group. TSE, Tamarind seed extract. Amplification efficiency (E) for gene was determined by linear regression analysis of
the fluorescent data from the exponential phase of PCR.

reported in Tamarind seed pericarp (Sudjaroen et al.,
2007) which is further validated in the present findings
(Figure 1). In general, the antihyperglycemic nature of
TSE is supported by the fact that the polyphenols consid-
ered for their insulin mimetic action (Daisy et al., 2010)
present in the extract, exhibit antioxidant activity and
radical scavenging ability.
Thus, speculation can be made to justify the protective
effect of TSE on pancreatic islets might be due to syner-
gistic action of catechin and epicatechin.
In the present study, TSE was tested after chronic dos-
ing (once daily) in a preclinical rat model of STZ-induced
type 2 diabetes. Body weight of diabetic rats was found
to be less during the course of development which might
be due to the accelerated lipolysis, whereas, significant
weight gain was observed in the rats treated for 4 weeks
with TSE and metformin. The TSE-treated group showed
significant antihyperglycemic effect, associated with
increased plasma insulin activity. An enhanced insulin
level caused by TSE is the primary factor for its glucose
and lipid lowering activity (Sachdewa & Khemani, 2003).
Neonatal STZ Wistar rat model is a well-characterized
model for type 2 diabetes. STZ rats develop persistent dia-
betes rapidly after 6 weeks of age and shows diabetes like
symptoms such as lack of insulin release in response to
glucose, glucose intolerance, and depletion of pancreatic
insulin store (Weir et al., 1981; Porte, 1991; Masiello et al.,
1998). Since, TSE has been reported to have antidiabetic

 Pharmaceutical Biology
 Insulin mimetic Tamarind seeds and β-cell protection  357

Figure 5.  Effect after 4 weeks daily dosing of Tamarind seed extract on apoptosis [deoxynucleotidyltransferase-nick-end-labelling (TUNEL)
assay] changes in the pancreata of diabetic rats; Hematoxylin and eosin staining and anti-insulin antibody immunostaining from (A, D,
G) normal (B, E, H) vehicle-treated diabetic rats, and (C, F, I) TSE-treated diabetic rats, respectively. Original magnification ×400. H&E,
hematoxylin and eosin.

action (Maiti et al., 2004), it was of interest to analyze
whether it affects islet [Ca2+]I and insulin release or not.
In the pancreatic β-cells the oscillations in [Ca2+]I is of
particular physiological importance. An elevated con-
centration of glucose within the β-cell ultimately leads to
membrane depolarization and an influx of extracellular
calcium. The resulting increase in the [Ca2+]I is thought to
be one of the primary triggers for exocytosis of insulin-
containing secretary granules (Weigle, 1987; Chou & Ipp,
1990). The present results indicate that TSE increased
islet [Ca2+]I significantly in the presence and absence of
20 mM d-glucose in cultured cells (Figure 3). The data are
consistent with the fact reported by Gilon and Henquin
(1992) wherein ATP and ADP are considered to be the
connecting linkage between increased glucose metabo-
lism and the ionic events that lead to release of insulin.
Furthermore, Bormann and Melzig (2000) reported that
the flavonoids might be responsible for inhibition of
metallopeptidases which are involved in the degradation
of neuropeptides. It certainly seems possible that the
stimulatory flavonoids in TSE (catechin, epicatechin) acts
on islet function, at least in part, via alterations in [Ca2+]I
flux (Sudjaroen et al., 2005; Siddhuraju, 2007). TSE was
able to mobilize [Ca2+]I in pancreatic islet both in absence
and presence of glucose might be due to presence of fla-
vonoids, suggesting inhibition of neuropeptides degra-
dation in the cells of the islets of Langerhans and in the
basolateral surfaces of pancreatic acinar cells which are
thought to be involved in increase of [Ca2+]I (Adeghate &
Donáth, 1990; Larson, 1979). Nevertheless, possibility of
other insulin secretary mechanisms such as interaction
with calmodulin (Nishino et al., 1984) and protein kinase
C (Gschwendt et al., 1983) cannot be eliminated.
SREBP-1c regulates the transcription of genes
involved in cholesterol and fatty acid metabolism (Gang
et al., 2007). The effect of insulin on SREBP-1c have been
corroborated by in vivo studies showing that SREBP-1c
expression and nuclear abundance were low in the liver
of STZ-induced diabetic rats, and markedly increased
after insulin treatment (Valerio et al., 2006). The stimu-
lating action of TSE on SREBP-1c mRNA expression
might be due its insulin secretagogue effect. Therefore,
the inflammation which is thought to play a key role in
the pathophysiology of DM (Haffner, 2006) might involve

© 2013 Informa Healthcare USA, Inc.

358  S. S. Sole et al.
Figure 6.  The proposed mechanism for anti-inflammatory action of Tamarind seeds contribute to antidiabetic activity. SREBP-1c, sterol
regulatory element-binding protein-1c; STZ, streptozotocin.
in hypolipedmic action of TSE. The mechanisms that
trigger the activation of TNF-α in type 2 diabetes are not
fully understood. It is likely that local hypoxia, induced
by capillary occlusion, and high levels of advanced
glycosylation end-products, associated with the devel-
opment of diabetic complications induces TNF-α activa-
tion (Beisswenger et al., 1995; Pankewycz et al., 1995).
Adipocytes synthesize and secrete biologically active
molecules, known as “adipocytokines” which affect
insulin mediated actions on a cellular level (Boden,
1997). These chemical messengers including TNF-α
(Ruan & Lodish, 2003) play a key modulator linking high
free fatty acids and inflammation. Although there is a
significant correlation between type 2 DM and insulin
resistance as well as obesity, the pathophysiology of
these relationships is not well understood. In the present
study, TSE exhibited a significant hypolipidemic activ-
ity, decreasing total cholesterol and LDL cholesterol in
serum and caused significant reduction in plasma TNF-α
(Figure 4). A decrease in serum cholesterol might be
associated with the epicatechins content of TSE (Chan
et al., 1997). The results obtained in this study are in
 Pharmaceutical Biology
accordance with the antioxidant action of T. indica in
hypercholestelomic hamsters wherein, it resulted in
hypolipidemic action (Martinello, 2006). These effects
caused an improvement of glucose disposal and utiliza-
tion and improved insulin sensitivity in the TSE-treated
rats.
It is known that STZ generated oxygen free radicals
cause cytotoxicity which leads to β-cell apoptosis and
death (Oberley, 1988). The destruction of β-cells is medi-
ated by changes in the expression of antiapoptotic or
proapoptotic proteins. STZ could induce NO formation
and which further leads to mitochondrial membrane
potential changes and hence the release of cytochrome
C, which triggers apoptosis (Hirst et al., 2010). In the pres-
ent study, the diabetic rats showed significant increase in
NO and decrease in insulin secretions and vice-versa in
normal rats. It is reported that increased serum levels of
NO indirectly reflect the presence of either endothelial
dysfunction or vascular injury, including microvascular
complications (Matata & Galiñanes, 2001). TSE treat-
ment to diabetic rats had shown a decrease in serum NO
level, but it significantly increased the plasma insulin

level and these findings can be correlated with number
of polyphenolic phytochemicals, such as resveratrol,
quercetin (Kawada et al., 1998), and catechins (Pannala
et al., 1997) which have been reported to inhibit the effect
of reactive nitrogen species. Chan et al. (1997) found that
epigallocatechin gallate could inhibit inducible nitric
oxide synthase activity and its mRNA expression in
lipopolysaccharide-activated macrophage. In the pres-
ent study, histopathological evaluation of the pancreas,
revealed a high frequency of degenerative changes and
necrosis in the islets of diabetic rats. Pons and Aoki (1995)
reported that the number of glucagon producing α- and
somatostatin-producing delta cells was increased in dia-
betes and it appears that somatostatin-producing cells
rise in number to compensate for the relative reduction
in insulin-secreting cells. This up-regulation of α-cells
of pancreas control the glucagon release to maintain
glucose homeostasis by way of feedback mechanism of
somatostatin. Furthermore, DNA fragmentation study
(TUNEL assay) in present investigation revealed that TSE
treatment has significantly reduced the apoptosis in pan-
creatic islets which further stimulates β-cell neogenesis
as in Figure 5B, C it is evident that insulin-positive cell
mass is increased compared to diabetic insulin-positive
cells. Correlations could also be found in our study
between the total phenolic content and NO scavenging
ability of the extract. The results are in agreement with
those reported by Komutarin et al. (2004), where seed
coat extract of Tamarind seeds suppressed NO in murine
macrophage cell line and freshly isolated peritoneal
macrophages.
Conclusions
The diagrammatic representation of the proposed mecha-
nism of TSE for anti-inflammatory action contributing to
hypoglycemia in type 2 diabetes is presented in Figure 6.
Thus, we conclude that TSE is efficacious in improving glu-
cose homeostasis and possess anti-inflammatory action
in a rat model of DM with defects in insulin sensitivity and
secretion. The beneficial effect of the TSE on glycemia con-
trol is clearly associated with significantly increased β-cell
mass and flavonoids content in extract. Furthermore, the
stimulating action of TSE on SREBP-1c mRNA level may
possibly be due to its insulin secretagogue effect. Action of
TSE could extend beyond glycemic regulation via modula-
tion of insulin secretion, to include a potential reduction
in β-cell failure which is commonly observed in diabetic
patients. Therefore, TSE can be considered for further
development as a therapeutic agent for impaired blood
glucose and DM.
Acknowledgments
The author acknowledges Panacea Biotech and Ranbaxy,
India for providing glimiperide and metformin,
respectively.
© 2013 Informa Healthcare USA, Inc.
Insulin mimetic Tamarind seeds and β-cell protection  359
Declaration of interest
The study was supported by Government of NCT Delhi
[MH-2203-O.E.1.1(1)(1)(1)(4)], India. The authors report
no declaration of interest.
References
Adeghate E, Donáth T. (1990). Distribution of neuropeptide Y and
vasoactive intestinal polypeptide immunoreactive nerves in
normal and transplanted pancreatic tissue. Peptides, 11, 1087–1092.
Baynes JW. (1991). Role of oxidative stress in development of
complications in diabetes. Diabetes, 40, 405–412.
Beisswenger PJ, Makita Z, Curphey TJ, Moore LL, Jean S, Brinck-Johnsen
T, Bucala R, Vlassara H. (1995). Formation of immunochemical
advanced glycosylation end products precedes and correlates
with early manifestations of renal and retinal disease in diabetes.
Diabetes, 44, 824–829.
Boden G. (1997). Role of fatty acids in the pathogenesis of insulin
resistance and NIDDM. Diabetes, 46, 3–10.
Bormann H, Melzig MF. (2000). Inhibition of metallopeptidases by
flavonoids and related compounds. Pharmazie, 55, 129–132.
Chan MM, Fong D, Ho CT, Huang HI. (1997). Inhibition of inducible
nitric oxide synthase gene expression and enzyme activity by
epigallocatechin gallate, a natural product from green tea. Biochem
Pharmacol, 54, 1281–1286.
Chou HF, Ipp E. (1990). Pulsatile insulin secretion in isolated rat islets.
Diabetes, 39, 112–117.
Daisy P, Balasubramanian K, Rajalakshmi M, Eliza J, Selvaraj J.
(2010). Insulin mimetic impact of Catechin isolated from Cassia
fistula on the glucose oxidation and molecular mechanisms of
glucose uptake on Streptozotocin-induced diabetic Wistar rats.
Phytomedicine, 17, 28–36.
de Groot M, Schuurs TA, Leuvenink HG, van Schilfgaarde R. (2003).
Macrophage overgrowth affects neighboring nonovergrown
encapsulated islets. J Surg Res, 115, 235–241.
Gang X, Hideaki KD, Ross L, Valerie F, Duvivier K, Nitin T. (2007). Down
regulation of GLP-1 and GIP receptor expression by hyperglycemia
possible contribution to impaired incretin effects in diabetes.
Diabetes, 56, 1551–1558.
Gilon P, Henquin JC. (1992). Influence of membrane potential changes
on cytoplasmic Ca2+ concentration in an electrically excitable
cell, the insulin-secreting pancreatic B-cell. J Biol Chem, 267,
20713–20720.
Gschwendt M, Horn F, Kittstein W, Marks F. (1983). Inhibition of the
calcium- and phospholipid-dependent protein kinase activity
from mouse brain cytosol by quercetin. Biochem Biophys Res
Commun, 117, 444–447.
Hadjivassiliou V, Green MH, James RF, Swift SM, Clayton HA, Green IC.
(1998). Insulin secretion, DNA damage, and apoptosis in human
and rat islets of Langerhans following exposure to nitric oxide,
peroxynitrite, and cytokines. Nitric Oxide, 2, 429–441.
Haffner SM. (2006). The metabolic syndrome: inflammation, diabetes
mellitus, and cardiovascular disease. Am J Cardiol, 97, 3A–11A.
Herold KC, Baumann E, Vezys V, Buckingham F. (1997). Expression
and immune response to islet antigens following treatment with
low doses of streptozotocin in H-2d mice. J Autoimmun, 10,
17–25.
Hirst DG, Robson T. (2010). Nitrosative stress as a mediator of apoptosis:
implications for cancer therapy. Curr Pharm Des, 16, 45–55.
Hsu SM, Raine L, Fanger H. (1981). Use of avidin-biotin-peroxidase
complex (ABC) in immunoperoxidase techniques: a comparison
between ABC and unlabeled antibody (PAP) procedures. J
Histochem Cytochem, 29, 577–580.
Iyer SR, Iyer RR. (1995). Anti leishmanial therapy–the changing scene. J
Assoc Physicians India, 43, 234.
Kawada N, Seki S, Inoue M, Kuroki T. (1998). Effect of antioxidants,
resveratrol, quercetin, and N-acetylcysteine, on the functions of
360  S. S. Sole et al.
cultured rat hepatic stellate cells and Kupffer cells. Hepatology, 27,
1265–1274.
Khillare, B. (2000). Orientation Training Course on Research
Methodology in Reproductive Biomedicine. New Delhi, India:
Department of Reproductive Biomedicine, National Institute of
Health and Family Welfare, Government of India.
Komutarin T, Azadi S, Butterworth L, Keil D, Chitsomboon B, Suttajit
M, Meade BJ. (2004). Extract of the seed coat of Tamarindus indica
inhibits nitric oxide production by murine macrophages in vitro
and in vivo. Food Chem Toxicol, 42, 649–658.
Lacy PE, Kostianovsky M. (1967). Method for the isolation of intact
islets of Langerhans from the rat pancreas. Diabetes, 16, 35–39.
Larson LI. (1979). Innervation of the pancreas by substance P,
encephalin, vasoactive intestinal polypeptide, and gastrin/CCK
immunoreactive nerves. J Histochem Cytochem, 27, 1283–1284.
Lopez JM, Bennett MK, Sanchez HB, Rosenfeld JM, Osborne TF. (1996).
Sterol regulation of acetyl coenzyme A carboxylase: a mechanism
for coordinate control of cellular lipid. Proc Natl Acad Sci USA, 93,
1049–1053.
Maiti R, Das UK, Ghosh D. (2005). Attenuation of hyperglycemia
and hyperlipidemia in streptozotocin-induced diabetic rats by
aqueous extract of seed of Tamarindus indica. Biol Pharm Bull, 28,
1172–1176.
Maiti R, Jana D, Das UK, Ghosh D. (2004). Antidiabetic effect of aqueous
extract of seed of Tamarindus indica in streptozotocin-induced
diabetic rats. J Ethnopharmacol, 92, 85–91.
Martinello FSM, Soares JJ, Franco AC, Santos A, Sugohara SB, Garcia
C, Curti SA, Uyemura. (2006). Hypolipemic and antioxidant
activities from Tamarindus indica L. pulp fruit extract in
hypercholesterolemic hamsters. Food Chem Toxicol, 44, 810–818.
Masiello P, Broca C, Gross R, Roye M, Manteghetti M, Hillaire-Buys D,
Novelli M, Ribes G. (1998). Experimental NIDDM: development
of a new model in adult rats administered streptozotocin and
nicotinamide. Diabetes, 47, 224–229.
Matata BM, Galiñanes M. (2001). Effect of diabetes on nitric oxide
metabolism during cardiac surgery. Diabetes, 50, 2603–2610.
Matsuno T, Sasaki H, Nakagawa K, Ishido N, Matsuda H, Sadamori H,
Inagaki M, Saito S, Yagi T, Haisa M, Orita K, Tanaka N. (1997). Fas
antigen expression and apoptosis in kidney allografts. Transplant
Proc, 29, 177–178.
Ndisang JF. (2010). Role of heme oxygenase in inflammation insulin
signaling diabetes and obesity. Medit Inflam, 359, 732–738.
Nishino H, Naitoh E, Iwashima A, Umezawa K. (1984). Quercetin
interacts with calmodulin, a calcium regulatory protein.
Experientia, 40, 184–185.
Oberley LW. (1988). Free radicals and diabetes. Free Radic Biol Med, 5,
113–124.
Owen RW, Haubner R, Mier W, Giacosa A, Hull WE, Spiegelhalder B,
Bartsch H. (2003). Isolation, structure elucidation and antioxidant
potential of the major phenolic and flavonoid compounds inbrined
olive drupes. Food Chem Toxicol, 41, 703–717.
Pankewycz OG, Guan JX, Benedict JF. (1995). Cytokines as mediators
of autoimmune diabetes and diabetic complications. Endocr Rev,
16, 164–176.
 Pharmaceutical Biology
Pannala AS, Rice-Evans CA, Halliwell B, Singh S. (1997). Inhibition of
peroxynitrite-mediated tyrosine nitration by catechin polyphenols.
Biochem Biophys Res Commun, 232, 164–168.
Peirson SN, Butler JN, Foster RG. (2003). Experimental validation of
novel and conventional approaches to quantitative real-time PCR
data analysis. Nucleic Acids Res, 31, e73.
Pons P, Aoki A. (1995). Differential proliferation of somatostatin and
glucagon cells in rat pancreatic islets submitted to various stimuli.
Ann Anat, 177, 221–227.
Porte JR., D. (1991). β-Cell in type 2 diabetes mellitus. Diabetes, 40,
166–180.
Prentki M, Matschinsky FM. (1987). Ca2+, cAMP, and phospholipid-
derived messengers in coupling mechanisms of insulin secretion.
Physiol Rev, 67, 1185–1248.
Razali N, Sarni MJ, Muthalib AF, Subramaniam S, Aziz AA. (2012). Effects
of various solvents on the extraction of antioxidant phenolics from
the leaves, seeds, veins and skins of Tamarindus indica L. Food
Chem, 131, 441–448.
Ruan H, Lodish HF. (2003). Insulin resistance in adipose tissue: direct
and indirect effects of tumor necrosis factor-alpha. Cytokine
Growth Factor Rev, 14, 447–455.
Sachdewa A, Khemani LD. (2003). Effect of Hibiscus rosa sinensis
Linn. ethanol flower extract on blood glucose and lipid profile in
streptozotocin induced diabetes in rats. J Ethnopharmacol, 89,
61–66.
Shimomura I, Bashmakov Y, Ikemoto S, Horton JD, Brown MS,
Goldstein JL. (1999). Insulin selectively increases SREBP-1c mRNA
in the livers of rats with streptozotocin-induced diabetes. Proc Natl
Acad Sci USA, 96, 13656–13661.
Siddhuraju P. (2007). Antioxidant activity of polyphenolic compounds
extracted from defatted raw and dry-heated Tamarindus indica L.
seed coat. J Food Sci Technol, 40, 982–990.
Sudjaroen Y, Haubner R, Wurtele G, Hull WE, Erben G, Spiegelhalder B,
Tahraoui A, El-Hilaly J, Israili ZH. (2007). Ethnopharmacological
survey of plants used in the traditional treatment of hypertension
and diabetes in south-eastern Morocco (Errachidia province). J
Ethnopharmacol, 110, 105–117.
Thomas PJ, Gaspers LD, Pharr C, Thomas JA. (1991). Continuous
measurement of mitochondrial pH gradients in isolated
hepatocytes by difference ratio spectroscopy. Arch Biochem
Biophys, 288, 250–260.
Valdeolmillos M, Nadal A, Soria B, García-Sancho J. (1993). Fluorescence
digital image analysis of glucose-induced [Ca2+]i oscillations in
mouse pancreatic islets of Langerhans. Diabetes, 42, 1210–1214.
Valerio A, Cardile A, Cozzi V, Bracale R, Tedesco L, Pisconti A, Palomba
L, Cantoni O, Clementi E, Moncada S, Carruba MO, Nisoli E. (2006).
TNF-alpha downregulates eNOS expression and mitochondrial
biogenesis in fat and muscle of obese rodents. J Clin Invest, 116,
2791–2798.
Weigle DS. (1987). Pulsatile secretion of fuel-regulatory hormones.
Diabetes, 36, 764–775.
Weir GC, Clore ET, Zmachinski CJ, Bonner-Weir S. (1981). Islet secretion
in a new experimental model for non-insulin-dependent diabetes.
Diabetes, 30, 590–595.