International Journal of Biological Macromolecules 54 (2013) 144–154

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Fenugreek seed mucilage-alginate mucoadhesive beads of metformin HCl:

Design, optimization and evaluation
Amit Kumar Nayak a,∗ , Dilipkumar Pal b , Jyotiprakash Pradhan a , M. Saquib Hasnain a
a
Seemanta Institute of Pharmaceutical Sciences, Mayurbhanj 757086, Odisha, India
b
Department of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya, Koni, Bilashpur 495 009, C.G., India

a r t i c l e i n f o a b s t r a c t

Article history: The work investigates the development and optimization of fenugreek (Trigonella foenum-graecum L.)
Received 11 October 2012 seed mucilage (FSM)-alginate mucoadhesive beads containing metformin HCl through ionotropic gela-
Received in revised form tion using 32 factorial design. The effect of polymer-blend ratio (sodium alginate to FSM) and cross-linker
22 November 2012
(CaCl2 ) concentration on the drug encapsulation efficiency (DEE, %), and cumulative drug release after 10 h
Accepted 5 December 2012
(R10h , %) was optimized. The DEE (%) of all these beads was within the range of 71.63 ± 2.32 to 95.08 ± 3.73%
Available online 12 December 2012
with sustained in vitro drug release of 69.78 ± 2.43% to 95.70 ± 4.26% over 10 h. The in vitro drug release
from these beads was followed controlled-release (zero-order) pattern (R2 = 0.9910 to 0.9953) with super
Keywords:
Fenugreek seed mucilage
case-II transport mechanism. The average size of these beads was within the range of 0.92 ± 0.05 to
Trigonella foenum-graecum L. 1.30 ± 0.14 mm. The beads were also characterized by SEM, FTIR and 1 H NMR. The swelling and degrada-
Alginate tion of FSM-alginate beads containing metformin HCl were influenced by pH of the test medium. These
Mucoadhesion beads also exhibited good mucoadhesivity in wash-off test. The optimized FSM-alginate mucoadhesive
Controlled drug release beads containing metformin HCl showed significant hypoglycemic effect in alloxan-induced diabetic rats
Metformin HCl over prolonged period after oral administration.
Factorial design © 2012 Elsevier B.V. All rights reserved.

1. Introduction prepared by ionotropic gelation [8,10,11]. In previous literature,

Over the past few decades, a great deal of attention has
been paid to the development of polysaccharide-based hydrogel
beads through ionotropic gelation technique, which are useful as
potential carriers in controlled drug delivery [1,2]. The advan-
tage of physical cross-linking by ionotropic gelation is that the
drug encapsulation in the beads could be achieved in an eco-
friendly environment avoiding the possible toxicity of reagents
associated with chemical cross-linking [1]. Among various ionic
natural polysaccharides, sodium alginate has been investigated
widely for its unique nature of forming hydrogel beads with the
influence of various metal ions like Ca2+ , Ba2+ , Zn2+ , Al3+ , etc.
due to an ionic interaction between the carboxylic acid groups
located on the polymer backbone and these cations [3–5]. Sodium
alginate has mucoadhesive property, but the cross-linked algi-
nates are usually fragile [6–8]. To formulate various cross-linked
alginate mucoadhesive systems for controlled-release drug deliv-
ery, use of mucoadhesive polymer-blend with alginate is one
of the widely accepted approaches [8,9]. Again, blending with
suitable polymer can improve drug encapsulation and stability,
which are found lower in cross-linked alginate hydrogel beads
∗ Corresponding author. Tel.: +91 9583131603.
E-mail address: amitkrnayak@yahoo.co.in (A.K. Nayak).
a few investigations have been carried out on the develop-
ment of mucoadhesive beads using sodium alginate and other
natural mucoadhesive polymer blend [12–16]. However, not a
single report is available in the literature regarding the use of
fenugreek seed mucilage (FSM) as a mucoadhesive material for
the development of alginate-based beads for mucoadhesive drug
delivery.
FSM is isolated from Trigonella foenum-graecum L. seeds known
as methi and is a commonly available material in nature [17].
Fenugreek seeds contain a high percentage of mucilage. The phar-
maceutical utility of FSM is already established as mucoadhesive
gelling agent [17], binding agent [18], and disintegrating agent [19].
It is also reported that the FSM has antidiabetic property [20]. In the
present study, the utility of FSM, as a possible natural mucoadhe-
sive polymeric-blend with sodium alginate for the development
of a novel mucoadhesive beads by ionotropic gelation technique
for the use in oral delivery was evaluated using metformin HCl as
model drug.
Metformin HCl is an oral hypoglycemic agent, which belongs
to the class of biguanide-derivatives, widely used as one of the
mainstays in the management of type-II diabetes mellitus [21].
Metformin HCl has been reported its absolute bioavailability of
50–60%, when administered orally [22]. The biological half-life is
1.5–1.6 h, and the main site of absorption is the proximal small
intestine of the GIT [22]. Therefore, the study was planned with

0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijbiomac.2012.12.008
 A.K. Nayak et al. / International Journal of Biological Macromolecules 54 (2013) 144–154
an aim of developing a natural polysaccharide-based multiple-
unit mucoadhesive system of metformin HCl using sodium
alginate-FSM polymer-blend for controlled-release oral drug
delivery.
Traditionally, pharmaceutical formulators develop formulations
by changing one variable at a time and the method is time consum-
ing. Again, many experiments not succeed in their purpose because
they are not properly thought out and designed, and even the best
data analysis cannot compensate lack of planning. Therefore, it is
essential to understand the influence of formulation variables on
the quality of formulations with a minimal number of experimental
trials to develop an optimized formulation using established statis-
tical tools for optimization such as factorial designs [23]. Factorial
designs, where all the factors are studied in all possible combina-
tions, are considered the most efficient in estimating the influence
of individual variables and their interactions performing minimum
numbers of experiments [24]. A computer aided optimization tech-
nique using 32 (two-factors and three-levels) factorial design was
employed to investigate the effects of two independent process
variables (factors), i.e., sodium alginate to FSM ratio, and cross-
linker (CaCl2 ) concentration on the properties of FSM-alginate
mucoadhesive beads containing metformin HCl like drug encap-
sulation, and drug release.
2. Experimental
2.1. Materials
Metformin HCl (Abhilash Chemicals Pvt. Ltd., India), sodium
alginate (Loba Chemie, India), calcium chloride (Merck Ltd., India)
were used. Fenugreek (Trigonella foenum-graecum L.) seeds were
collected from Jharpokharia market in the month of September
2011. All other chemicals and reagents were commercially avail-
able and of analytical grade.
2.2. Isolation of FSM from fenugreek seeds
FSM was isolated from fenugreek seeds according to the previ-
ously reported literature [17] with little modifications. Fenugreek
seeds (200 g) were soaked in distilled water (1.5 l) at room temper-
ature for 12 h, and boiled using water bath until the preparation of
slurry. After cooling, the slurry was cooled and kept in refrigera-
tor overnight to settle out undissolved materials. The upper clear
solution was decanted and concentrated at 60 ◦ C by water bath to
one-third of its original volume. The solution was cooled at room
temperature and poured into thrice the volume of acetone with
continuous stirring. The precipitate was washed repeatedly with
acetone and dried at room temperature for 24 h. The isolated mate-
rial was passed though sieve number 80 and stored in desiccators
until further use.
Table 1
Experimental plan of 32 factorial design (coded values in bracket) with observed response values for different formulations.
Experimental formulations Normalized levels of factors
SA to FSM ratio
(X1 )
F-1 4.00 (+1)
F-2 4.00 (+1)
F-3 4.00 (+1)
F-4 2.50 (0)
F-5 2.50 (0)
F-6 2.50 (0)
F-7 1.00 (−1)
F-8 1.00 (−1)
F-9 1.00 (−1)
DEE (%) = drug encapsulation efficiency (%); R10h (%) = Cumulative drug release from FSM-alginate beads containing metformin HCl after 10 h.
145
2.3. Preparation of FSM-alginate beads containing metformin HCl
FSM-alginate beads containing metformin HCl were prepared
using calcium chloride (CaCl2 ) as cross-linking agent by ionotropic
gelation method. Briefly, sodium alginate and FSM aqueous dis-
persions were prepared separately using distilled water. These
dispersions were well mixed with stirring for 10 min at 1000 rpm
using a magnetic stirrer (Remi Motors, India). Afterwards, met-
formin HCl was added to the dispersion mixture maintaining
the ratio of drug to polymer 1:2 in all formulations. The final
FSM-alginate dispersion mixture containing metformin HCl was
homogenized for 20 min at 1000 rpm using a homogenizer (Remi
Motors, India) and ultrasonicated for 5 min for debubbling. The
resulting dispersion was then added via a 21-G needle. The added
droplets were retained in the CaCl2 solution for 15 min to com-
plete the curing reaction and to produce rigid beads. The wet beads
were collected by decantation, and washed two times with distilled
water and dried at 40 ◦ C for 24 h. The prepared dried FSM-alginate
beads containing metformin HCl were stored in a desiccator until
used.
2.4. Experimental design for optimization
A two-factor, three-level factorial design (32 ) was employed for
optimization with polymer-blend (sodium alginate to FSM) ratio
(X1 ), and concentration of cross-linker (CaCl2 ) (X2 ) as the prime
selected independent variables, which were varied at three levels,
low (−1), medium (0), and high (+1) levels. The drug encapsu-
lation efficiency (DEE, %), and cumulative drug release after 10 h
(R10h , %) were used as dependent variables (responses). Design-
Expert® Version 8.0.6.1 software (Stat-Ease Inc., USA) was used
for the generation and evaluation of the statistical experimental
design. The matrix of the design including investigated responses
i.e., DEE (%), and R10h (%) are shown in Table 1. The effects of
independent variables were modelled using following quadratic
mathematical model generated by 32 factorial design is following
[23]:
Y = b0 + b1 X1 + b2 X2 + b3 X1 X2 + b4 X12 + b5 X22
where, Y is the response; b0 is the intercept, and b1 , b2 , b3 ,
b4 , b5 are regression coefficients. X1 and X2 are individual
effects; X12 and X22 are quadratic effects; X1 X2 is the inter-
action effect. One-way ANOVA was applied to estimate the
significance of models (p < 0.05). Individual response param-
eters were evaluated using the F-test. The surface response
plots, and contour plots were analyzed to reveal the effect
of independent factors (sodium alginate to FSM ratio and
CaCl2 concentration) on the measured responses (DEE, and
R10h ).
Responses (Mean ± S.D., n = 3)
CaCl2 (% w/v) DEE (%) R10h (%)
(X2 )
10.00 (+1) 84.37 ± 2.97 80.12 ± 3.10
7.50 (0) 76.03 ± 2.82 89.56 ± 3.97
5.00 (−1) 71.63 ± 2.32 95.70 ± 4.26
10.00 (+1) 86.98 ± 3.08 76.32 ± 3.02
7.50 (0) 78.12 ± 3.00 84.67 ± 3.16
5.00 (−1) 74.61 ± 2.63 90.18 ± 4.04
10.00 (+1) 94.86 ± 4.25 69.78 ± 2.43
7.50 (0) 86.82 ± 3.87 78.45 ± 3.20
5.00 (−1) 81.26 ± 2.91 83.20 ± 3.33
146 A.K. Nayak et al. / International Journal of Biological Macromolecules 54 (2013) 144–154
2.5. Determination of DEE
100 mg of beads were taken and were crushed using pestle and
mortar. The crushed powders of drug containing beads were placed
in a 250 ml volumetric flask and the volume was made up to 250 ml
by phosphate buffer, pH 7.4, and kept for 24 h with occasionally
shaking at 37 ± 0.5 ◦ C. After the stipulated time, the mixture was
stirred at 500 rpm for 20 min using a magnetic stirrer (Remi Motors,
India). The polymer debris formed after disintegration of bead was
removed filtering through Whatman® filter paper (No. 40). The
drug content in the filtrate was determined using a UV–vis spec-
trophotometer (Shimadzu, Japan) at 233 nm against appropriate
blank. The DEE (%) of these prepared beads was calculated by the
following formula:
Actual drug content in beads
DEE (%) = × 100
Theoretical drug content in beads
2.6. Bead size measurement
Particle size of 100 dried beads from each batch was measured
by optical microscopic method for average particle size using an
optical microscope (Olympus). The ocular micrometer was previ-
ously calibrated by stage micrometer.
2.7. Surface morphology analysis by scanning electron
microscopy (SEM)
FSM-alginate beads containing metformin HCl were gold coated
by mounted on a brass stub using double-sided adhesive tape and
under vacuum in an ion sputter with a thin layer of gold (3–5 nm)
for 75 s and at 20 kV to make them electrically conductive and their
morphology was examined by scanning electron microscope (ZEISS
EVO 40, Japan).
2.8. Fourier transform-infrared (FTIR) spectroscopy
Samples were reduced to powder and analyzed as KBr pellets
by using a Fourier transform-infrared (FTIR) spectroscope (Perkin
Elmer Spectrum RX I, USA). The pellet was placed in the sam-
ple holder. Spectral scanning was taken in the wavelength region
between 4000 and 400 cm−1 at a resolution of 4 cm−1 with scan
speed of 1 cm/s.
2.9. Nuclear magnetic resonance (NMR) spectroscopy
1 H NMR (600 MHz, 25 ◦ C) spectra of solution of various sam-
ples in dimethyl sulfoxide were recorded on a Bruker AvanceTM III
500 spectrometer (Bruker Biospin Gmbh, Germany) operating at
500.13 MHz using a 4-mm CP-MAS probe head.
2.10. Evaluation of swelling behavior
Swelling behavior evaluation of FSM-alginate beads containing
metformin HCl were carried out in two different aqueous media:
0.1 N HCl (pH 1.2), and phosphate buffer (pH 7.4). 100 mg beads
were placed in vessels of dissolution apparatus (Campbell Electron-
ics, India) containing 500 ml respective media. The experiment was
carried out at 37 ± 1 ◦ C under 50 rpm paddle speed. The swelled
beads were removed at predetermined time interval and weighed
after drying the surface by using tissue paper. Swelling index was
determined using the following formula:
Weight of beads after swelling − Dry weight of beads
Swelling index (%) =
Dry weight of beads
2.11. Ex vivo mucoadhesion testing
The mucoadhesive properties of FSM-alginate beads contain-
ing metformin HCl were evaluated by ex vivo wash-off method
[14]. Freshly excised pieces of goat intestinal mucosa (2 cm × 2 cm)
(collected from slaughterhouse) were mounted on glass slide
(7.5 cm × 2.5 cm) using thread. About 50 beads were spread onto
the wet tissue specimen, and the prepared slide was hung onto a
groove of disintegration test apparatus. The tissue specimen was
given a regular up and down movement in a vessel containing
900 ml of 0.1 N HCl (pH 1.2) and phosphate buffer (pH 7.4), sepa-
rately, at 37 ± 0.5 ◦ C. After regular time intervals, the machine was
stopped and the number of beads still adhering to the tissue was
counted.
2.12. In vitro drug release studies
The release of metformin HCl from various FSM-alginate beads
was tested using dissolution apparatus USP (Campbell Electron-
ics, India). The baskets were covered with 100-mesh nylon cloth
to prevent the escape of the beads. The dissolution rates were
measured at 37 ± 1 ◦ C under 50 rpm speed. Accurately weighed
quantities of beads containing metformin HCl equivalent to 100 mg
were added to 900 ml of 0.1 N HCl (pH 1.2). The test was carried
out for 2 h and then continued in phosphate buffer (pH 7.4) for
next 8 h. 5 ml of aliquots was collected at regular time intervals,
and the same amount of fresh dissolution medium was replaced
into dissolution vessel to maintain the sink condition throughout
the experiment. The collected aliquots were filtered, and suitably
diluted to determine the absorbance using a UV–vis spectropho-
tometer (Shimadzu, Japan) at 233 nm against appropriate blank.
2.13. Analysis of in vitro drug release kinetics and mechanism
In order to predict and correlate the in vitro release behaviour of
metformin HCl from formulated FSM-alginate beads, it is necessary
to fit into a suitable mathematical model. The in vitro drug release
data were evaluated kinetically in important mathematical models
[25]:
Zero-order model: Q = kt + Q0 ; where Q represents the drug released
amount in time t, and Q0 is the start value of Q; k is the rate
constant.
First-order model: Q = Q0 ekt , where Q represents the drug released
amount in time t, and Q0 is the start value of Q; k is the rate
constant.
1/3
Hixson-Crowell model: Q 1/3 = kt + Q0 , Q represents the drug
released amount in time t, and Q0 is the start value of Q; k is the
rate constant.
Weibull model: m = 1 − exp[−(t)b /a], where m represents the drug
released amount in time t, a is the time constant and b is the shape
parameter.
Baker-Lonsdale model: 3/2[1 − (1 − Q )2/3 ] − Q = kt, where Q rep-
resents the drug released amount in time t, and k is the rate
constant.
Higuchi model: Q = kt0.5 , where Q represents the drug released
amount in time t, and k is the rate constant.
Korsmeyer-Peppas model: Q = ktn , where Q represents the drug
released amount in time t, k is the rate constant and n is the
diffusional exponent, indicative of drug release mechanism.
The accuracy and prediction ability of these models were com-
pared by calculation of squared correlation coefficient (R2 ) and
× 100 root mean squared error (RMSE) using KinetDS 3.0 Rev. 2010 soft-
ware [25]. Again, The Korsmeyer-Peppas model was employed
 A.K. Nayak et al. / International Journal of Biological Macromolecules 54 (2013) 144–154
in the in vitro drug release behaviour analysis of these formu-
lations to distinguish between competing release mechanisms:
Fickian release (diffusion-controlled release), non-Fickian release
(anomalous transport), and case-II transport (relaxation-controlled
release). When n is ≤0.43, it is Fickian release. The n value between
0.43 and 0.85 is defined as non-Fickian release. When, n ≥ 0.85, it
is case-II transport [11].
2.14. Pharmacodynamic evaluation of FSM-alginate beads
containing metformin HCl in alloxan-induced diabetic rats
In vivo pharmacodynamic studies were performed in alloxan-
induced diabetic male albino rats [8,9,12] of either sex (weighing
315–364 g). The acclimatized rats were kept fasting for 24 h with
water ad libitum. All experiments were performed between 8 AM to
12 PM to minimize circadian influences. The experimental proto-
col was subjected to the scrutiny of the Institutional Animal Ethical
Committee and was cleared before starting. The experimental ani-
mals were handled as per guidelines of committee for the purpose
of control and supervision on experimental animals (CPCSEA). All
efforts were made to minimize both the suffering and number of
animals used.
The male albino rats were made diabetic by intraperitoneal
administration of freshly prepared alloxan solution at a dose of
150 mg/kg dissolved in 2 mM citrate buffer (pH 3.0). After one
week of alloxan administration, alloxanized rats with fasting blood
glucose of 300 mg/dl or more were considered diabetic and were
employed in the study for 10 h. After initial collection of blood
samples from the alloxan-induced diabetic rats, they were divided
randomly into 2 groups of 6 rats each and treated as follow: Group
A was administered with pure metformin HCl (100 mg/kg body
weight) in suspension form and Group B were administered with
optimized FSM-alginate beads containing metformin HCl, both at
a dose equivalent to 100 mg metformin HCl/kg body weight using
oral feeding needle. Blood samples were withdrawn (0.1 ml) from
tail tip of each rat at regular time intervals under mild ether anes-
thesia, and were analyzed for blood glucose by oxidase-peroxidase
method using commercial glucose kit. Comparative in vivo blood
glucose level in alloxan-induced diabetic rats after oral adminis-
tration of pure metformin HCl and optimized FSM-alginate beads
containing metformin HCl were evaluated.
2.15. Statistical analysis
Statistical optimization was performed using Design-Expert®
Version 8.0.6.1 software (Stat-Ease Inc., USA). The R2 and RMSE
for evaluation of accuracy and prediction ability of various kinetic
models were calculated using KinetDS 3.0 Rev. 2010 software. The
in vivo data was tested for significant differences (p < 0.05) by paired
samples t-test. All other data was analyzed with simple statistics.
The simple statistical analysis and paired samples t-test were con-
ducted using MedCalc software version 11.6.1.0.
3. Results and discussion
3.1. Isolation of FSM and preparation of FSM-alginate beads
containing metformin HCl
The yield of mucilage isolated from fenugreek seeds was
17.36% w/w. The FSM-alginate beads containing metformin HCl
was prepared by ionotropic gelation technique using CaCl2
aqueous solution as cross-linker. When dispersion mixture of
sodium alginate, isolated FSM, and metformin HCl was dropped
into the solutions containing calcium ions, ionotropically gelled
FSM-alginate beads containing metformin HCl were formed instan-
taneously. In presence of divalent calcium ions in the cross-linking
147
solution, the calcium is ionically substituted at the carboxylic site
present the alginate strands and attaches two alginate strands
together. This leads to the formation of solid gel [26].
3.2. Optimization
For the 32 factorial design, 9 trial formulations were suggested
by Design-Expert® Version 8.0.6.1 software (Stat-Ease Inc., USA) for
two independent variables: sodium alginate to FSM ratio (X1 ), and
concentration of CaCl2 as cross-linker (X2 ), which were varied at
three levels: low level (−1), medium level (0), and high level (+1).
The DEE (%), and R10h (%) were evaluated as dependent variables
(responses). Design-Expert® Version 8.0.6.1 software (Stat-Ease
Inc., USA) was used for the generation and evaluation of the statis-
tical experimental design. According to this trial plan, FSM-alginate
beads containing metformin HCl were prepared by ionotropic
gelation technique. Overview of matrix of the design including
investigated responses (i.e., DEE and R10h ) is presented in Table 1.
The values of investigated responses measured for all trial formu-
lations were fitted in the 32 factorial design to get model equations
for responses analyzed in this investigation.
The model equation relating DEE (%) as response became:
DEE (%) = 91.61 − 8.76X1 − 1.98X2 − 0.06X1 X2 + 1.15X12 + 0.31X22
[R2 = 0.9979; F-value = 286.92; p < 0.05].
The model equation relating R10h (%) as response became:
R10h (%) = 76.49 + 6.90X1 + 1.53X2 –0.14X1 X2 –0.41X12 –0.27X22
[R2 = 0.9995; F-value = 1296.90; p < 0.05].
Model simplification was carried out by eliminating non-
significant terms (p > 0.05) in model equations resulting from the
multiple regression analysis [27], giving:
DEE (%) = 91.61 − 8.76X1 − 1.98X2 + 1.15X12 + 0.31X22
R10h (%) = 76.49 + 6.90X1 + 1.53X2 − 0.14X1 X2 − 0.41X12 − 0.27X22
The influences of main effects (factors) on responses investigated
(here, DEE, and R10h ) were further elucidated by response surface
methodology. Response surface methodology is a widely proficient
approach in the development and optimization of drug delivery
devices [23]. Based on the design of experiments, response sur-
face methodology encompasses the generation of model equations
of the investigated responses over the experimental domain to
determine optimum formulation(s) [11,23]. The three-dimensional
response surface plots and corresponding two-dimensional con-
tour plots relating DEE and R10h are presented in Figs. 1 and 2,
respectively. The three-dimensional response surface graph is very
useful in learning about the main and interaction effects of the
independent variables (factors), whereas two-dimensional contour
plot gives a visual representation of values of the response [26].
The three-dimensional response surface plot relating DEE (Fig. 1a)
depicts increasing of DEE with the decreasing of sodium alginate to
FSM ratio (X1 ), and increasing CaCl2 concentration (X2 ). However,
an increase in R10h values with the increasing of sodium alginate
to FSM ratio (X1 ), and decreasing CaCl2 concentration (X2 ) is indi-
cated by the three-dimensional response surface plot relating R10h
(Fig. 2b). All the contour plots relating measured responses (Fig. 2a
and b) indicate nonlinear relationships between two independent
variables (here, sodium alginate to JFSM ratio, and CaCl2 concen-
tration) on all measured responses, investigated in this study.
A numerical optimization technique using the desirability
approach was employed to develop new formulations with desired
response (optimum quality). The desirable ranges of the unde-
pendable variables (factors) were restricted to 1 ≤ X1 ≤ 2, and
8 ≤ X2 ≤ 12%; whereas the desirable ranges of responses were
restricted to 90 ≤ DEE ≤ 100%, and 50 ≤ R10h ≤ 70%. The optimal val-
ues of responses were obtained by numerical analysis using the
148 A.K. Nayak et al. / International Journal of Biological Macromolecules 54 (2013) 144–154

Fig. 1. Effect of sodium alginate to FSM ratio, and concentration of CaCl2 on DEE (a),
and R10h (b) presented by response surface plots.

Fig. 2. Effect of sodium alginate to FSM ratio, and concentration of CaCl2 on DEE (a),
and R10h (b) presented by contour plots.

Design-Expert® Version 8.0.6.1 software and one of them was

selected based on the criterion of desirability. In order to evalu-
ate optimization capability of models generated according to the
results of the full 32 factorial design, optimized FSM-alginate beads
containing metformin HCl was prepared by ionotropic gelation
technique using these optimal process variable settings. The opti-
mized FSM-alginate beads containing metformin HCl (F-O) was
evaluated for DEE (%), and R10h (%). Table 2 lists the results of exper-
iments with predicted responses by the mathematical model and
those actually observed. The optimized FSM-alginate beads con-
taining metformin HCl (F-O) showed DEE of 95.08 ± 3.73%, and
R10h of 71.34 ± 2.89% with small error-values (−1.07 and −4.73%,
respectively). Percentage error evaluation is helpful in establishing
the validity of generated model equations to describe the domain
of applicability of optimization model. The percentage error
values indicate that mathematical models obtained from the full
32 factorial design were well fitted.
3.3. DEE
The DEE (%) of all these FSM-alginate beads containing met-
formin HCl were within the range between 71.63 ± 2.32 (F-3) and
95.08 ± 3.73 (F-O) % w/w (Tables 1 and 2). It was observed that
DEE (%) was increased with the decreasing of sodium alginate to
FSM ratio in polymer-blend. This phenomenon might be due to the
increase in viscosity of the polymeric solution by the increasing of
FSM addition, and it might have been prevented drug leaching to
the cross-linking solution. Again, the DEE (%) of FSM-alginate beads
was increased with increasing CaCl2 concentration in cross-linking

Table 2
Results of experiments for confirming optimization capability.

Code Factors Responses

SA to FSM ratio CaCl2 (%w/v) DEE (%) R10h (%)

Predicted value Observed value Error (%) Predicted value Observed value Error (%)

F-O 1.43 10.83 96.11 95.08 ± 3.73 −1.07 68.12 71.34 ± 2.89 4.73

DEE (%) = drug encapsulation efficiency (%); R10h (%) = Cumulative drug release from FSM-alginate beads containing metformin HCl after 10 h; Observed response values:
mean ± S.D., n = 3; Error (%) = [difference between predicted value and observed value/predicted value] × 100.
 A.K. Nayak et al. / International Journal of Biological Macromolecules 54 (2013) 144–154 149

Table 3
Mean diameter of FSM-alginate beads containing metformin HCl.

Formulation codes Mean diameter (mm) (Mean ± S.D.)

F-1 1.11 ± 0.14

F-2 1.22 ± 0.12
F-3 1.30 ± 0.14
F-4 0.96 ± 0.06
F-5 1.09 ± 0.10
F-6 1.24 ± 0.12
F-7 0.88 ± 0.04
F-8 1.01 ± 0.05
F-9 1.12 ± 0.08
F-O 0.92 ± 0.05

solution, which could be due to high degree of cross-linking by the

interaction between sodium alginate in the polymer blend, and Ca2+
present in the cross-linking solution.

3.4. Bead size

Particle size of metformin HCl loaded various FSM-alginate

beads was within the range of 0.92 ± 0.05 to 1.30 ± 0.14 mm
(Table 3). Increasing the size of beads was found with the increasing
incorporation of FSM in the polymer solutions with sodium algi-
nate. This could be explained due to the increase in viscosity of the
polymer-blend solution with the incorporation of FSM in increasing
ratio that in turn increased the droplet size during addition of the
polymer-blend solution to the cross-linking solution. On the other
hand, with the increasing amount of FSM in the polymer-blend, the
number free sites available for cross-linking could be less so that
the size of these beads prepared with decreasing sodium alginate to
FSM ratio was increased. Again, the decrease in particle size of FSM-
alginate beads was observed, when concentrated CaCl2 solution
was used as cross-linking solution. This could be due to shrinkage
of polymeric gel by higher degree of cross-linking with the high
concentration of cross-linker (i.e. CaCl2 ) [14].
3.5. Bead surface morphology
The SEM photograph of the surface of the FSM-alginate bead
metformin HCl (Fig. 3) exhibited very rough surface with charac-
teristic large wrinkles and cracks. These cracks and wrinkles may
be caused by partly collapsing the polymeric gel network during
drying [28]. However, polymeric derbies were seen on the bead
Fig. 4. FTIR spectra of sodium alginate (a), isolated FSM (b), FSM-alginate blank bead
(c), FSM-alginate bead containing metformin HCl (d), and metformin HCl (e).
surface, which could be due to the method of preparation (i.e.,
simultaneous gel bead preparation and formation of the polymer
blend matrix). The drug crystals observed on the bead surface were
probably formed a result of their migration along with water to the
surface during drying.
3.6. FTIR spectroscopy
The FTIR spectra of sodium alginate, isolated FSM, FSM-alginate
blank bead, FSM-alginate beads containing metformin HCl, and
metformin HCl are shown in Fig. 4. The FTIR spectra of sodium
alginate showed the band around 3445.2, 1614.6, 1417.3 and
1032.5 cm−1 , which are due to the stretching of –OH, –COO (asym-
metric), –COO (symmetric), and C–O–C, respectively. In the FTIR
spectrum of isolated FSM showed characteristic peaks of –OH
between 3511.6 and 3154.3 cm−1 , –CH3 at 2925.48 cm−1 , –CH
stretching between 2920.0 and 2852.4 cm−1 , ether linkage at
1455–1400 cm−1 and–CO stretching at 1017.7 cm−1 . The FTIR spec-
trum of blank FSM-alginate beads showed characteristic peaks of
both sodium alginate and FSM without any significant interaction.
In the FTIR spectrum of metformin HCl, the principal absorption
peaks appeared at 3169 cm−1 due to the N-H stretching of the pri-
mary amine group (–NH2 ) and at 1063 cm−1 due to C-N stretching,
and a peak at 1584 cm−1 occurs due to N-H bending vibrations of
the primary amine group. In the FTIR spectrum of FSM-alginate
beads containing metformin HCl, various characteristic peaks of
sodium alginate, FSM, and metformin HCl were appeared without
any significant shifting of these peaks. In short, the FSM-alginate
beads containing metformin HCl prepared with FSM-sodium algi-
nate polymer blend had significant characters of metformin HCl in
the FTIR spectrum, suggesting, there were no interaction between
the drug, metformin HCl and the polymers used (sodium alginate
and FSM).

3.7. 1H NMR spectroscopy

The 1 H NMR spectra of FSM-alginate blank beads, FSM-alginate

beads containing metformin HCl and metformin HCl, are shown
Fig. 3. Scanning electron microphotograph of the surface of optimized FSM-alginate in Figs. 5–7, respectively. In the 1 H NMR spectra of FSM-alginate
beads containing metformin HCl (F-O) at 1.50k× magnification. blank beads and FSM-alginate beads containing metformin HCl,
150 A.K. Nayak et al. / International Journal of Biological Macromolecules 54 (2013) 144–154

1
Fig. 5. H NMR spectra of FSM-alginate blank beads.

1
Fig. 7. H NMR spectra of metformin HCl.

1
Fig. 6. H NMR spectra of FSM-alginate beads containing metformin HCl.

typical characteristic signals of polysaccharides were crowded in

a narrow region between 3 and 5 ppm and indicates the presence
of many similar sugar residues [29]. Characteristic signals present
in 1 H NMR spectra of metformin HCl were also appeared in that of
FSM-alginate beads containing metformin HCl indicating no inter-
action between the metformin HCl and the polymer blend (sodium
alginate and FSM) used.

3.8. Swelling behavior

The swelling behavior of various FSM-alginate beads containing

metformin HCl was evaluated in 0.1 N HCl, pH 1.2, and phosphate
buffer, pH 7.4. The swelling behaviors of these beads in both the
pH, 0.1 N HCl (pH 1.2), and phosphate buffer (pH 7.4) are shown in
Figs. 8 and 9, respectively. The swelling index of FSM-alginate beads Fig. 8. Swelling behavior of FSM-alginate beads containing metformin HCl in 0.1 N
containing metformin HCl was lower in 0.1 N HCl in comparison HCl, pH 1.2 [Mean ± S.D., n = 3].
with that of in phosphate buffer, initially. This was occurred due to
 A.K. Nayak et al. / International Journal of Biological Macromolecules 54 (2013) 144–154
Fig. 9. Swelling behavior of FSM-alginate beads containing metformin HCl in phos-
phate buffer, pH 7.4 [Mean ± SD, n = 3].
shrinkage of alginate at acidic pH. Maximum swelling of beads was
noticed at 2–3 h in phosphate buffer, pH 7.4 and after which, ero-
sion and dissolution took place. It has been previously reported that
the swelling of calcium alginate in presence of calcium ion captur-
ing agent depends on the progressive displacement of calcium ions
within calcium alginate-based beads [14]. It has been also reported
that the swelling of calcium alginate-based beads can be enhanced
by the presence of phosphate ions (in phosphate buffer), which
act as calcium sequestrant [30]. Therefore, the swelling behavior of
FSM-alginate beads containing metformin HCl in phosphate buffer,
pH 7.4 could be explained by the ion exchanging between cal-
cium ion of the ionotropically cross-liked beads and the sodium
ions present in phosphate buffer with the influence of calcium
sequestrant phosphate ions. This might result disaggregation of the
FSM-alginate matrix structure leading to matrix erosion and disso-
lution of the swollen beads. These results clearly suggest that these
FSM-alginate beads containing metformin HCl may swell slightly
in the stomach as they subsequently move to the upper intestine,
where the metformin HCl is to be absorbed, and these beads begin
to swell more.
3.9. Mucoadhesion
The ex vivo wash off test using goat intestinal mucosa for
assessing mucoadhesivity of FSM-alginate beads containing
metformin HCl was performed at both gastric pH (0.1 N HCl, pH
1.2) and intestinal pH (phosphate buffer, pH 7.4) for 8 h. The
wash off was faster at intestinal pH than at gastric pH. In 0.1 N
HCl, the percentage of beads adhering to the goat intestinal
Fig. 10. Result of ex vivo wash-off test to assess mucoadhesive properties of FSM-
alginate beads containing metformin HCl in 0.1 N HCl, pH 1.2. [Mean ± S.D., n = 3].
151
Fig. 11. Result of ex vivo wash-off test to assess mucoadhesive properties of FSM-
alginate beads containing metformin HCl in phosphate buffer, pH 7.4 [Mean ± S.D.,
n = 3].
mucosal tissue varied from 56.53 ± 2.52 to 73.85 ± 2.86%
(Fig. 10) over 8 h, whereas, this was varied from 38.55 ± 3.25
to 48.60 ± 0.86% in phosphate buffer (Fig. 11). The decreased
mucoadhesion of FSM-alginate beads containing metformin HCl
in phosphate buffer may be resulted due to the erosion of calcium
ion [13]. Therefore, the results of the wash off test indicated that
the FSM-alginate beads containing metformin HCl possessed good
mucoadhesivity.
3.10. In vitro drug release
The in vitro drug release studies were carried out for FSM-
alginate mucoadhesive beads containing metformin HCl in the 0.1 N
HCl (pH, 1.2) for first 2 h and then, in phosphate buffer (pH, 7.4) for
next 8 h. All these mucoadhesive beads containing metformin HCl
showed prolonged drug release over 10 h (Fig. 12). Metformin HCl
release from these mucoadhesive beads in the acidic medium was
slow (less than 15.50% after 2 h) due to the shrinkage of alginate
at acidic pH (as alginate is pH sensitive). The trace amount of drug
release could probably be due to the surface adhered drug. After
that, drug release was observed faster in phosphate buffer (pH 7.4)
comparatively, due to the higher swelling rate of these beads in
phosphate buffer. The percentage drug released from FSM-alginate
mucoadhesive beads containing metformin HCl in 10 h (R10h , %)
was within the range of 69.78 ± 2.43% (F-7) to 95.70 ± 4.26% (F-
3), and this was found to be higher with the decreasing sodium
alginate to FSM ratio as polymer blend and increasing CaCl2
concentration in cross-linking solution. In case of comparatively
higher FSM containing beads, the more hydrophilic property of FSM
Fig. 12. In vitro drug release from various FSM-alginate mucoadhesive beads con-
taining metformin HCl [Mean ± S.D., n = 3].
152 A.K. Nayak et al. / International Journal of Biological Macromolecules 54 (2013) 144–154

could bind better with water to form viscous gel-structure, which
may blockade the pores on the surface of beads and sustain the drug
release profile. Again, the drug (metformin HCl) release from FSM-
alginate mucoadhesive beads prepared using higher with higher
CaCl2 concentration was comparatively sustained than the beads
formulated with that of lower concentration. The higher concentra-
tion of CaCl2 (cross-linker) can produce high degree of cross-linking
and thereby slower the drug release from highly cross-linked FSM-
alginate mucoadhesive beads containing metformin HCl.
The in vitro drug release data from various FSM-alginate
mucoadhesive beads containing metformin HCl were evaluated
kinetically using various mathematical models like zero-order,
first-order, Hixson-Crowell, Weibull, Baker-Lonsdale, Higuchi, and
Korsmeyer-Peppas models. The R2 and RMSE values of these mod-
els were determined for evaluation of accuracy and prediction
ability of these models using KinetDS 3.0 Rev. 2010 software.
The result of the curve fitting into various mathematical mod-
els is given in Table 4. When the respective R2 of FSM-alginate
mucoadhesive beads containing metformin HCl were compared,
it was found to follow the zero-order model (R2 = 0.9910–0.9953)
as the best-fit model over a period of 10 h amongst others.
This was also observed to be closest to some other models like
Weibull model (R2 = 0.9741–0.9912), and Korsmeyer-Peppas model
(R2 = 0.9850–0.9910). Finally, the best fitting of the zero-order
model was verified by comparing RMSE values for each tested mod-
els and minimum RMSE values for the zero-order model was found
between 1.53 and 2.34. The best fit of zero-order model indicates
that the drug release from these beads followed controlled-release
pattern. The values of diffusional exponent (n) determined from
Korsmeyer-Peppas model ranged from 1.04 to 1.10, indicating
the drug release from these beads followed the super case-II
transport mechanism controlled by swelling and relaxation of
polymeric-blend (FSM-alginate) matrix. This could be attributed
due to polymer dissolution and polymeric chain enlargement or
relaxation.
3.11. Pharmacodynamic evaluation
Pharmacodynamic efficiencies of optimized FSM-alginate
mucoadhesive beads containing metformin HCl (F-O) were per-
formed in alloxan-induced diabetic Albino rats and estimated by
measuring blood glucose level. The comparative in vivo blood glu-
cose level and the mean percentage reduction in blood glucose
level in alloxan-induced diabetic rats after oral administration
of pure metformin HCl and optimized FSM-alginate mucoadhe-
sive beads containing metformin HCl is presented in Fig. 13 (a
and b, respectively). In case of the group treated with pure met-
formin HCl (Group A), a rapid reduction in blood glucose level was
observed up to 3 h, and after that, the blood glucose level recov-
ered rapidly towards the normal. In case of the group (Group B)
treated with optimized FSM-alginate mucoadhesive beads contain-
ing metformin HCl, the reduction in blood glucose level reached
more than 25% in between 1 to 2 h, and remained more than 30%
reduction in blood glucose level over 10 h. A 25 percent reduc-
tion in glucose level was considered as significant hypoglycemic
effect [8,12]. There were significant differences (p < 0.05) between
the blood glucose level after oral administration of pure metformin
HCl and optimized FSM-alginate mucoadhesive beads contain-
ing metformin HCl (F-O). Therefore, the significant hypoglycemic
effect by the optimized FSM-alginate mucoadhesive beads con-
taining metformin HCl (F-O) was observed over 10 h. However,
the attaining of significant hypoglycemic effect (25% reduction in
blood glucose level) in case of optimized FSM-alginate mucoadhe-
sive beads containing metformin HCl (F-O) after oral administration
was impressive in comparison with the results of our previous
reports on gliclazide-loaded methyl cellulose-alginate microcap-
sules, where the significant hypoglycemic effect was attained
within 3–4 h. In addition, the combined effect of metformin HCl and
FSM for assessing hypoglycemic effect in alloxan-induced diabetic
rats could not be ruled out, as FSM possess antidiabetic property
[20].

Table 4
Results of curve fitting of the in vitro metformin HCl release data from FSM-alginate mucoadhesive beads.

Models Code

F-1 F-2 F-3 F-4 F-5 F-6 F-7 F-8 F-9 F-O

Zero order
R2 0.9910 0.9921 0.9926 0.9914 0.9940 0.9955 0.9945 0.9953 0.9918 0.9943
RMSE 2.28 2.34 2.41 2.08 1.94 1.76 1.53 1.58 2.19 1.59

First order
R2 0.8473 0.8375 0.8453 0.8324 0.8440 0.8510 0.8451 0.8474 0.8323 0.8445
RMSE 11.38 15.84 16.02 13.71 14.75 14.62 11.96 13.13 14.31 12.30

Hixson-Crowell
R2 0.9158 0.9119 0.9164 0.9087 0.9186 0.9240 0.9199 0.9218 0.9096 0.9205
RMSE 7.72 8.63 8.86 7462 7.83 7.73 6.27 6.91 7.76 6.40

Weibull
R2 0.9874 0.9876 0.9741 0.9836 0.9890 0.9827 0.9907 0.9912 0.9881 0.9909
RMSE 1.85 2.45 3.64 1.39 2.23 3.15 1.44 1.87 2.42 1.60

Baker-Lonsdale
R2 0.9217 0.8963 0.8663 0.9212 0.9012 0.8783 0.9150 0.9045 0.8957 0.9123
RMSE 45.49 49.91 53.53 42.52 46.71 49.46 38.18 43.02 45.83 38.96

Higuchi
R2 0.6216 0.6056 0.6271 0.6027 0.5890 0.6041 0.5815 0.5443 0.5994 0.5753
RMSE 11.46 16.58 17.10 14.18 16.07 16.59 13.33 14.68 15.39 13.76

Korsmeyer-Peppas
R2 0.9863 0.9887 0.9899 0.9881 0.9891 0.9910 0.9877 0.9903 0.9850 0.9881
RMSE 2.99 3.72 3.39 3.41 3.12 2.66 2.44 2.55 3.45 2.54

N 1.03 1.07 1.04 1.08 1.08 1.06 1.05 1.09 1.08 1.10
2
R : squared correlation coefficient; RMSE: root mean squared error; n: diffusional exponent.
All results were obtained using KinetDS 3.0 Rev. 2010 software.
 A.K. Nayak et al. / International Journal of Biological Macromolecules 54 (2013) 144–154
Fig. 13. (a) Comparative in vivo blood glucose level in alloxan-induced diabetic rats after oral administration of pure metformin HCl and FSM-alginate mucoadhesive beads
containing metformin HCl (F-O) [Mean ± S.D., n = 6]. The data were analyzed for significant differences (p < 0.05) by paired samples t-test. The statistical analysis was conducted
using MedCalc software version 11.6.1.0. (b) Comparative in vivo mean percentage reduction in blood glucose level in alloxan-induced diabetic rats after oral administration
of pure metformin HCl and FSM-alginate mucoadhesive beads containing metformin HCl (F-O).
4. Conclusion
Novel mucoadhesive beads containing metformin HCl made of
FSM-alginate polymer-blend by ionotropic gelation technique was
successfully developed and evaluated. The above studies suggested
that the FSM-alginate mucoadhesive beads containing metformin
HCl swelled slowly in the stomach and accordingly adhered to the
stomach mucosa allowing more drug to be absorbed minimizing
the diffusion barriers to increase the absorption period by prolong-
ing the gastric residence time. Then, these beads were subsequently
move to the upper part of the intestine, where they swelled more
and released drug through the polymeric gel layer, formed at the
matrix-periphery. The results clearly demonstrated that the abil-
ity of the developed system to maintain tight blood glucose level
and improved patient compliance by enhancing, controlling and
prolonging systemic absorption of metformin HCl. Thus, FSM is
proved as a potential mucoadhesive excipient in the development
of controlled-release mucoadhesive beads for oral use.
Acknowledgements
The first, third and fourth authors are thankful to Principal and
Management of Seemanta Institute of Pharmaceutical Sciences,
Mayurbhanj-757086, India for providing necessary facilities.
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