This study investigated the potential protective effects of Bombax ceiba extract fractions against palmitate-induced pancreatic beta-cell death. Cells responded to palmitate in a dose-dependent manner with decreased viability at higher concentrations, consistent with previous research. Optimal non-toxic concentrations of 50 μg/ml for B. ceiba butanol fraction and 125 μM for palmitate were determined. When co-incubated, B. ceiba fractions modestly contributed to beta-cell death under lipotoxic conditions compared to palmitate alone, though not significantly. The study suggests future research use lower B. ceiba concentrations to ensure no cytotoxic effects under lipotoxic conditions.

1. Potential protective effect of Bombax ceiba extract fractions
against palmitate-induced pancreatic β-cell death
Introduction
Method
Results Discussion
References
Student number: 13013802
Supervisor: Dr. David Watson
In a Type 2 diabetic state, relative insulin deficiency
and peripheral insulin resistance leads to increased
plasma concentration of free fatty acids (FFAs), which
exert toxic effects on peripheral tissue - ‘lipotoxicity’.
Non-esterified saturated FFAs such as palmitate
(C16:0) cause β-cell apoptosis through de novo
ceramide synthesis, JNK activation and reactive
oxygen species (ROS)-induced ER stress [1].
Oxidative and nitrosative stress have an established
role in the pathogenesis of both Type 2 Diabetes
Mellitus (T2DM). It is conceivable, therefore, that
antioxidant therapy should be beneficial. In fact,
inhibition of ROS has been shown to protect against
palmitate-induce β-cell death in vitro [2]. Plant
extracts with anti-oxidant properties exert
cytoprotective effects through free radical
scavenging.
B. ceiba possesses anti-oxidant properties [3] and
has been reported to exert anti-diabetic effects;
lowering blood glucose, cholesterol and triglyceride
levels in a streptozotocin-induced diabetic rat model
[4]. This study used an in vitro rat pancreatic β-cell
line (BRIN-BD11) to focus specifically on palmitate, a
known physiologically relevant inducer of β-cell death,
to model cell death in T2DM.
The aim of this study is to investigate the potential
protective effect of butanol and ethyl acetate fractions
of B. ceiba extract against palmitate-induced β-cell
death.
[1] Igoillo-Esteve, M., Marselli, L., Cunha, D.A., Ladrière, L., Ortis, F., Grieco, F.A., Dotta, F., Weir, G.C.,
Marchetti, P., Eizirik, D.L. and Cnop, M., (2010). Palmitate induces a pro-inflammatory response in human
pancreatic islets that mimics CCL2 expression by beta cells in type 2 diabetes. Diabetologia 53, 1395-1405.
[2] Lin, N., Chen, H., Zhang, H., Wan, X. and Su, Q. (2012). Mitochondrial reactive oxygen species (ROS)
inhibition ameliorates palmitate-induced INS-1 beta cell death. Endocrine 42, 107-117.
[3] Gandhare, B., Soni, N. and Dhongade, H.J., (2010). In vitro antioxidant activity of Bombax ceiba.
International Journal of Biomedical Research 1, 31-36.
[4] Bhavsar, C. and Talele, G.S., (2013). Potential anti-diabetic activity of Bombax ceiba. Bangladesh Journal
of Pharmacology 8, 102-106.
[5] Welters, H.J., Smith, S.A., Tadayyon, M., Scarpello, J.H. and Morgan, N.G., (2004). Evidence that protein
kinase Cdelta is not required for palmitate-induced cytotoxicity in BRIN-BD11 beta-cells. Journal of molecular
endocrinology 32, 227-235.
[6] Welters, H.J., Diakogiannaki, E., Mordue, J.M., Tadayyon, M., Smith, S.A. and Morgan, N.G., (2006).
Differential protective effects of palmitoleic acid and cAMP on caspase activation and cell viability in pancreatic
β-cells exposed to palmitate. Apoptosis 11, 1231-38.
• BRIN-BD11 cells were cultured in RPMI-1640
growth media supplemented with 2mM L-
Glutamine, 10% FCS (Foetal Calf Serum),
100U/ml Penicillin and 100µg/ml Streptomycin, at
37ºC and 5% CO2.
• Palmitate was conjugated to Bovine Serum
Albumin (BSA) at a molar ratio of 3.3:1.
• Cells were treated with 125µM palmitate alone or
in combination with 50µM of B. ceiba (butanol or
ethyl acetate fractional extracts).
• % cell viability was determined by MTT assay.
0 .2 5 % B S A 1 2 5  M P a lm ita te
0
5 0
1 0 0
1 5 0
T re a tm e n t
Cellviability(%)
C o n tro l
B . c e ib a
(b u ta n o l)
B . c e ib a
(e th y l a c e ta te )*
* *
The optimum palmitate and B. ceiba concentrations to use for the co-
incubation study were determined by performing dose response assays
(Figure 1 and 2).
C
o
n
tro
l
3
1
.2
5
6
2
.5
1
2
5
2
5
0
5
0
0
1
0
0
0
0
2 0
4 0
6 0
8 0
1 0 0
[P a lm ita te ] ( M )
Cellviability(%)
*
*
*
*
*
*
Figure 1 Palmitate dose response. BRIN-BD11
cells were treated with 0-1000µM palmitate and
incubated at 37ºC and 5% CO2 for 24 hours before
cell viability was determined by MTT assay with
absorbance measured at 540nm. Results expressed
as mean % cell viability +/- SEM and significance
calculated by one-way ANOVA and Tukey’s test.
*P<0.05 vs. control, n=4.
Figure 1 and 2 suggested that the optimum concentrations of palmitate and B
ceiba to use for the co-incubation study were 125µM and 50µM, respectively.
The subsequent co-incubation study (Figure 3), showed that B. ceiba extracts
displayed no protective effect against β-cell death under lipotoxic conditions.
Figure 3 Palmitate + B. ceiba co-incubation. BRIN-BD11 cells were treated with 125µM palmitate alone or in
combination with 50µg/ml B. ceiba (butanol or ethyl acetate) extract and incubated at 37ºC and 5% CO2 for 24
hours before cell viability was determined by MTT assay with absorbance measured at 540nm. Results
expressed as mean % cell viability +/- SEM and level of significance calculated by two-way ANOVA and Tukey’s
test. *P<0.05 vs. control, n=3.
The aim of this study was to investigate the potential
protective effect of butanol and ethyl acetate fractions
of B. ceiba extract against palmitate-induced β-cell
death.
Cells responded to palmitate treatment as expected,
with increasing concentrations of palmitate (0-
1000µM) decreasing cell viability in a dose-dependent
manner (Figure 1). This result is consistent with other
in vitro studies of palmitate-induced β-cell death using
BRIN-BD11 cells [5, 6].
Figure 2 suggested that 50µg/ml B. ceiba (butanol
fraction) extract was the most appropriate non-toxic
concentration to use for the co-incubation study since
this concentration did not affect cell viability
(100.54%, P>0.05 vs. control). Insufficient B. ceiba
(ethyl acetate fraction) extract was available to
complete a full dose response, therefore the
optimised concentration of B. ceiba (butanol fraction)
extract was used.
Figure 3 showed that, as expected, both B. ceiba
extract fractions (butanol and ethyl acetate) alone are
not toxic (P>0.05). However, in co-incubation with
palmitate, B. ceiba modestly contributes to β-cell
death under lipotoxic conditions, albeit not significant
to the control (P>0.05). Cell viability is decreased
further from 51.61% (palmitate alone) to 37.83%
(butanol fraction) and 36.66% (ethyl acetate fraction).
In light of this result, future studies investigating the
potential cytoprotective effect of B.ceiba against
palmitate toxicity may use lower concentrations (i.e.
25µg/ml) to ensure that there is no cytotoxic effect of
B. ceiba under lipotoxic conditions.
C
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l
6
.2
5
1
2
.5
2
5
5
0
1
0
0
2
0
0
0
5 0
1 0 0
1 5 0
[B .c e ib a (b u ta n o l)] ( g /m l)
Cellviability(%)
*
Figure 2 B. ceiba (butanol) extract dose
response. BRIN-BD11 cells were treated with 0-
200µg/ml B. ceiba (butanol) and incubated at 37ºC
and 5% CO2 for 24 hours before cell viability was
determined by MTT assay with absorbance
measured at 540nm. Results expressed as mean %
cell viability +/- SEM and significance calculated by
one-way ANOVA and Tukey’s test. *P<0.05 vs.
control, n=3.