This document describes an evaluation of the cytotoxic effects of curcumin-loaded nanoemulsion (CRM-NE) using a Sulforhodamine B (SRB) assay on glioblastoma cell lines. The SRB assay measures cell viability by staining cellular proteins. Testing found that CRM-NE showed higher cytotoxicity against glioblastoma cells than curcumin alone, reducing cellular viability in a dose-dependent manner. CRM-NE was more effective than the cancer drug adrenomycin, indicating it may be useful for treating brain tumors. The SRB assay is a widely-used, accurate and reproducible method for evaluating drug cytotoxicity, even suitable for large-scale screening.

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Research Article
Sulforhodamine B (SRB) Assay Of Curcumin Loaded Nanoemulsion By Using Glioblastoma Cell
Line
Sagar Kishor Savale*
Department of Pharmaceutics, R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, MS,
India
Received: 25 May 2017 Revised: 5 June 2017 Accepted: 10 June 2017
Abstract
Objective: The Sulforhodamine B (SRB) assay was measure drug-induced cytotoxicity and cell
proliferation for large-scale drug-screening applications. Its principle is based on the ability of the
protein dye sulforhodamine B to bind electrostatically and pH dependent on protein basic amino acid
residues of trichloroacetic acid-fixed cells. Under mild acidic conditions it binds to and under mild basic
conditions it can be extracted from cells and solubilized for measurement.
Method: Various reports were taken from research articles published in journals, data from various
books and other online available literature.
Conclusion: It has been widely used in cytotoxicity and cell growth studies. Curcumin loaded
nanoemulsion (CRM-NE) shows maximum cytotoxic effect against glioblastoma cell line.
Keywords: Sulforhodamine B (SRB), Curcumin, Curcumin-Nanoemulsion, cytotoxicity, cellular
viability.
Introduction
Sulforhodamine B (SRB) cell cytotoxicity assay
is one of the most widely methods used to detect
cell viability or drug cytotoxicity. This assay
relies on the ability of SRB to bind cellular
protein components and measure the total
biomass (Duong et al., 2013). SRB is a bright-
pink amino xanthene dye that can form an
electrostatic complex with basic amino acid
residues of proteins in slightly acidic conditions
but it can dissociate under basic conditions
(Mauludin et al., 2009).
*Address for Corresponding Author:
Mr. Sagar Kishor Savale,
Department of Pharmaceutics,
R. C. Patel Institute of Pharmaceutical
Education & Research, Shirpur, 425405,
Dist. Dhule, MS, India.
Mobile No: +91 9960885333
Email ID: avengersagar16@gmail.com
It has been widely used for drug toxicity
screening against different types of cancerous
and non-cancerous cell lines (Martin et al.,
1987). In addition, this assay is independent of
cell metabolic activity and therefore should
show less interference by the testing
compounds. Since the binding of SRB is
stoichiometric, the incorporated dye released
from stained cells after washing is directly
proportional to the cell biomass and can be
measured at 565 nm. SRB cell cytotoxicity
assay kit is simple, accurate, reproducible and
sensitive. This kit offers an excellent and
efficient method for in vitro cytotoxicity studies
as well as high-throughput drug screening that
can detect between 5,000-50,000 cells per well.
Nanoemulsion is modern colloidal drug delivery
system in which polyphenolic curcumin (CRM)
was incorporated by spontaneous-emulsification
method (Savale et al., 2015; Savale et al., 2017).
Curcumin loaded nanoemulsion (CRM-NE)
shows anti-angiogenesis activity. It can prevent
angiogenesis by inhibiting vascular endothelial

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growth factor. CRM-NE subjected to SRB assay
for estimation of cytotoxic effect and total
control growth (Skehan et al., 1990; Vichai et
al., 2006).
Material and Method
Material
Curcumin (CRM) were purchased from Sigma
Chemical Company (Sigma, St, Louis, USA)
and All compounds were initially dissolved in
dimethyl sulfoxide (DMSO) (Sigma, St, Louis,
USA).
Method
Sulforhodamine B (SRB) assay of Curcumin
(CRM) and Curcumin Nanoemulsion (CRM-
NE) in (U373MG) Human Glioblastoma Cell
line
The cell lines were grown in RPMI 1640
medium containing 10% fetal bovine serum and
2 mM L-glutamine. For present screening
experiment, cells were inoculated into 96 well
microliter plates in 100 µL at plating densities
as shown in the study details above, depending
on the doubling time of individual cell lines.
After cell inoculation, the microliter plates were
incubated at 37° C, 5 % CO2, 95 % air and 100
% relative humidity for 24 h prior to addition of
experimental drugs. Experimental drugs and
formulation were initially solubilized or added
in dimethyl sulfoxide at 100 mg/ml and diluted
to 1 mg/ml using water and stored frozen prior
to use. At the time of drug addition, an aliquote
of frozen concentrate (1 mg/ml) was thawed and
diluted to 100 μg/ml, 200 μg/ml, 400 μg/ml and
800 μg/ml with complete medium containing
test article. Aliquots of 10 µl of these different
drug and formulation dilutions were added to
the appropriate microliter wells already
containing 90 µl of medium, resulting in the
required final drug and formulation
concentrations i.e.10 μg/ml, 20 μg/ml, 40 μg/ml,
80 μg/ml. After compound addition, plates were
incubated at standard conditions for 48 hours
and assay was terminated by the addition of cold
TCA. Cells were fixed in situ by the gentle
addition of 50 µl of cold 30 % (w/v) TCA (final
concentration, 10 % TCA) and incubated for 60
minutes at 4°C. The supernatant was discarded;
the plates were washed five times with tap water
and air dried. Sulforhodamine B (SRB) solution
(50 µl) at 0.4 % (w/v) in 1 % acetic acid was
added to each of the wells, and plates were
incubated for 20 minutes at room temperature.
After staining, unbound dye was recovered and
the residual dye was removed by washing five
times with 1 % acetic acid. The plates were air
dried. Bound stain was subsequently eluted with
10 mM trizma base, and the absorbance was
read on a plate reader at a wavelength of 540 nm
with 690 nm reference wavelength. Percent
growth was calculated on a plate-by-plate basis
for test wells relative to control wells. Percent
Growth was expressed as the ratio of average
absorbance of the test well to the average
absorbance of the control wells * 100. Using the
six absorbance measurements [time zero (Tz),
control growth (C), and test growth in the
presence of drug at the four concentration levels
(Ti)], the percentage growth was calculated at
each of the drug concentration levels.
Percentage growth inhibition was calculated as:
[Ti/C] x 100 % (Skehan et al., 1990; Vichai et
al., 2006).
Result and Discussion
Sulforhodamine B (SRB) assay of Curcumin
(CRM) and Curcumin Nanoemulsion (CRM-
NE) in (U373MG) Human Glioblastoma Cell
line
The freeze-dried CRM-NE as the untreated
equal volume of solvent to control (A) with
CRM-NE formulation (B) to sample B-1 (C)
positive control (adrenomycin) (D) (Figure 1)
and the graph of % inhibition of astrocytoma-
glioblastoma cell line (U373MG) incubated with
the CRM-NE (P); incubated sample B-1 (Q) and
adrenomycin (R) versus different drug
concentrations (Figure 2). From the results, we
can see that at the different concentration,
CRM-NE produced higher inhibition on the
cells. Graph of % inhibition of astrocytoma-
glioblastoma cell line (U373MG) incubated with
the CRM-NE and adrenomycin vs different drug
concentrations. From results, we conclude that

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at the different concentration, CRM-NE
produced higher cell inhibition on the cells
when the drug concentration is 10 µg/ml. The
pure drug CRM is not cause cell inhibition and
does not show cytotoxicity. To explain the
cytotoxic effect of CRM-NE, it was
hypothesized that NE absorbed to cell surface
and released CRM close to the membrane,
leading to a high local drug concentration
gradient or was brought in the cells and then
released from the NE. The IC50 values were
found to be 38.8 µg/ml for CRM-NE and < 10
µg/ml for adrenomycin, demonstrating
effectiveness of CRM-NE against the
gliblastoma cell line. Thus, CRM-NE show less
cellular viability which means that it increases
the cytotoxicity of formulation towards cells.
CRM-NE shows significant effect against
glioblastoma cell line having higher cytotoxicity
and minimum cell viability.

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Figure 1: Sulforhodamine B (SRB) assay: control (A); CRM-NE formulation (B); sample B-1 (C);
positive control (adrenomycin) (D)

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Figure 2: Sulforhodamine B (SRB) assay: % Inhibition of astrocytoma-glioblastoma cell line
(U373MG) incubated with the CRM-NE (P); incubated sample B-1 (Q) and adrenomycin (R) versus
different drug concentrations
Conclusion
CRM-NE shows higher cytotoxicity and less
cellular viability, hence CRM-NE effective
against treatment of human glioblastoma cell
line U373MG and it is used for treatment of
brain tumor. These practical advances make the
SRB assay an appropriate and sensitive assay to
measure drug-induced cytotoxicity even at
large-scale application.
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