III.

RESULTS AND DISCUSSIONS

A successful ate mpt was made to formulate Metformin microspheres using
different drug polymer ratios, composition of which was showed. Effect of polymer
ratio in formulation development was assessed. The formulations are subjected to
evaluation parameters which are discussed below:
3.1 Preformulation studies:
3.1.1Melting point determination:
Melting point of the Metformin was determined by using capillary method. The
melting point of Metformin was found to be in the range of 128°C which indicates the
purity of the sample
3.1.1.2Compatability studies:
IR-Spectroscopy: From the FTIR spectra of pure drug and combination of drug with the
polymers, it was observed that all the characteristic peaks of drug are present in the
combination spectra as well as indicating the compatibility of the drug with the polymers
used. The individual spectra of pure drug were shown in the Fig.3.1 and optimized
formulation of IR spectrum was shown in the Fig.3.2.

Fig.3.1. FT-IR spectra of pure drug.

Page 47
Fig.3.2. FT-IR Spectra of optimized formulation.

Page 48
3.1.1.3 FT-IR Spectro scopy:
The FT-IR spectrum of the metformin pure drug showed the following functional
groups at their frequencies mentioned in Table 3.1
Table 3.1. Metformin functional groups and their frequencies

Functional group Absorbance cm-1

C=O stretching 1770.85

1718.58

OH stretching 2970.64

CH stretching 2935.68
superimposed on OH
stretching

NH stretching 3317.56

N-O 669.50

S=O 1266.05

C-N 1581.31

Infra red spectroscopy (FTIR)

The prepared microspheres were characterized by FTIR spectroscopy to

find out any chemical interaction between Metformin and polymers used. The
FTIR spectra of Metformin, polymers and selected formulations is shown in
the figure-3.1,3.2 Our experimental results were assessed on the basis of
physical data obtained for drug and polymers as well as formulations. The
FTIR spectra of microspheres of Metformin using ethyl cellulose and HPMC
are as follows. The drug Metformin has exhibited CH absorptions from
cm-1 -1
2995.68 to 3055 cm. indicating the presence of aromatic as well as
aliphatic CH vibrations. The strong S=O absorption peak is noticed at 1266.05
-1 -1
cm . The FTIR spectra show the pick at 3317.56 cm the pick in this range
N-H stretching indicated that presence of amine group. The spectrum also
Page 49
 -1
shows the pick at 1581.31cm . The pick in this range is due to C-N stretching
When the polymer ethyl-cellulose a single product was taken for IR
measurements when strong hydroxyl primary OH peak was noticed and
-1
2 9 7 0 . 6 4 cm may be due to the OH absorption which is present in the
cellulose molecule. The aliphatic CH absorptions were noticed at 2995.68
-1 -1
cm . The C=O peak was isolated has given its absorption at 1770.85 cm .
-1
Another polymer HPMC exhibited primary OH absorption at 2 9 7 0 . 6 4 cm
due to the presence of anticipated functional group. In this case also the
-1 -1
aliphatic OH absorption peaks were noticed 2978 cm and 2879 cm . This
-1
molecule showed C=O absorption peak at 1756 cm . The IR spectrum
obtained of Metformin, ethyl cellulose and HPMC were identical and there
was no change in the functional group absorption of any molecule present in
formulated product. The FTIR spectra were shown in fig no.3.2

3.1.2. Determination of max:

The absorption spectrum of pure drug was scanned over the range of 200-400nm
with 20 µg/ml concentration prepared in pH 7.4 phosphate buffer. The absorption
spectra of Metformin showed only one peak at 274-77nm, which represents the
maximum absorption (max) of drug in phosphate buffer pH 7.4

3.1.3. Calibration curve of Metformin in PH 7.4 phosphate

buffer
Table.3.1 Shows the absorbance of standard solutions of Metformin in pH 7.4
phosphate buffer at 275nm. Fig.4.3 shows the standard calibration curve for pure drug.
The curve was found to be linear in the range of 1-20µg/ml.
3.3. Characterization of microspheres
The prepared Metformin microspheres formulation (F1-F6) were evaluated for
variable parameters such as bulk density, tapped density, Carr's compressibility index,
Hausner's ratio and angle of repose and the evaluation parameters of six formulations
(F1-F6) were shown in Table.3.3
The Carr's compressibility index for formulations F1-F6 was found to be in the
range of 14.8 ± 2.15 - 20.0 ± 1.22 which indicates good flow characteristics.

Page 50
 The value of Hausner's ratio for the formulationsF1 to F6 was below 1.17 to
1.25 which indicates good flow property.
The values of angle of repose for formulations F1 to F6, was found to be
below 20º which indicates excellent flow of all the formulations.
4. Particle size determination:
The mean particle size of the microspheres containing Metformin was found to be in
the range of 95.03 ± 1.17 to 156.01± 2.1 μ. The particle size of all formulations were
shown in Table.11 particle size comparison of different formulations were shown in
Fig.3.2

5. Percentage yield:
The percentage yields of different formulations F1 to F6 were calculated and the yield
was found to be in the range of 67.68 ± 3.06 to 80.47±1.02%. The loss of material
during preparation of microspheres may be due to process parameters as well as
during filtration of microspheres. Percentage yield of all the batches is shown in
Table 3.5. Percentage yield comparisons of different formulation were shown in
Fig.3.3.
6. Estimation of drug loading and encapsulation efficiency:
The drug loading was found to be in the range of 18.18 ± 1.10% to 25.00 ±
2.31% for formulations F1 to F6.
The percentage encapsulation efficiency of Metformin microspheres in all the
formulations was found to be in the range of 50.30 ± 2.23 to 87.20 ± 2.18 %. The
microspheres of batch F1 showed maximum drug encapsulation of 87.20 ± 2.18 %.
The F4,F6 batch microspheres showed lowest drug encapsulation of 50.30 ± 2.23 %.
From the results it was seen that as the polymer concentration increased, viscosity of
the dispersed phase increased, encapsulation efficiency decreased.
The percentage encapsulation efficiency, percentage drug loading was
shown in Table 3.6 Fig.3.4 shows the comparison of % drug loading of different
formulations Fig.3.5 Shows the comparison of % encapsulation efficiency of
different formulations.

Page 51
Chapter-III Result&Discussion

7. In vitro drug release:

The in vitro drug release characteristic were studied in pH 7.4 phosphate buffer
for a period of 12 hrs using USP XXXIII dissolution apparatus , type-II. The microsphere
containing Metformin (F1-F6) were prepared. The results of the dissolution studies
indicated that the formulations F1, F2, F3, F4, F5 and F6 released 99.53%, 99.76%,
97.45%, 94.56%, 92.65% and 90.56 %, of Metformin at the end of 12hrs respectively.
The change in polymer concentration may also affect the in vitro drug release
mechanism of drug from the microspheres. By increasing the polymer concentrations
the rate of drug release was decreased due to the unavailability of drug molecules at
the surface of Metformin microspheres.
Further, by increasing the concentration of polymer in the microsphere
formulation, a point will be reached where the pores or channels formed by the drug
particles within the polymer matrix were diminished. i.e., the diffusion of drug
molecules from the channels of the matrix was disturbed by the increased
concentration of polymer. In other words, increased polymer concentration affects the
drug leaching and diffusion process from the matrix, by making it less porous and
slower drug release rate occurs.
8. Data analysis:

In order to understand the kinetics and mechanism of drug release, the result of
the in vitro dissolution study of Metformin microspheres were fitted with various kinetic
equations like zero order as cumulative percentage released Vs Time, First order as log
percentage of drug remaining to be released Vs. Time, and Higuchi’s model, cumulative
percentage drug released Vs Square root of time. The r 2 values were calculated for the
linear curves obtained by regression analysis of the above plots.
The curve fitting results of the release rate profile of the designed formulation
shown in Figures 3.8, 3.9 and 3.10 gave an idea on the release rate profile and the
mechanism of the drug release. The regression coefficients for different drug release
kinetic models are shown in Table3.7. Models with the highest regression coefficient
were judged to be the most appropriate model for the dissolution data.
Peppas model equation is given as: -
% R = K tn

Or Log % R = log K + log t

Department of Pharmaceutics, Sri Shivani College of Pharmacy Page 57

Chapter-III Result&Discussion

Where R = drug release, k =constant, n= slope, t=time.

n' Mechanism

<0.5 Fickian Diffusion (Higuchi matrix )

0.5 < n < 1 Non Fickian Diffusion or anomalous release

>1 Case II Transport

The data of the various models revealed that formulation F1to F9 follows Higuchi
matrix release kinetics.
In controlled or sustained release formulations diffusion, swelling and erosion
are the three most important rate controlling mechanism followed. The drug release
from the polymeric system is mostly by diffusion and best described by fickian
diffusion. The in vitro release profile of the drug from the formulations can be
expressed by Higuchi’s kinetics, as it indicates diffusion, Korsmeyer-peppas’s
kinetics, as the ‘n’ value between 0.45 and 0.89 indicates that diffusion is coupled
with erosion and hence this mechanism is called anomalous diffusion and zero order
kinetics as it indicates that prepared microspheres drug release was controlled by
diffusion.
Table.3.2. Calibration curve of Metformin in pH 7.4 Phosphate buffer.

Concentratio Absorbance
n (µg/ml)

0 0

2 0.110

6 0.283

8 0.352

12 0.552

16 0.744

20 0.922

Department of Pharmaceutics, Sri Shivani College of Pharmacy Page 58

Chapter-III Result&Discussion

Graph.3.1. Calibration curve of Metformin

Calibration curve of Tinidazole in pH 7.4

phosphate buffer
1
0.9
f(x) = 0.0459513274336284 x
0.8 R² = 0.999642912591213
0.7
Absorbance

0.6
0.5
0.4
0.3
0.2
0.1
0
0 5 10 15 20 25
Concentration (μg/ml)

Table.3.3 Micromeritic propertic parameters of

microspheres

Formulatio Bulk density Tapped density Carr’s Hausner’s Angle of

n (gm/cm3) (gm/cm3) index (%) ratio (%) repose (ө)

F1 0.16 ± 1.30 0.20 ± 1.70 20.0 ± 1.22 1.25 ± 3.11 19°03' ± 0.50

F2 0.44 ± 2.11 0.53 ± 2.12 16.9 ± 0.64 1.20 ± 1.82 17°01' ± 0.41

F3 0.42 ± 2.50 0.52 ±1.97 19.5 ± 2.12 1.24 ± 0.41 16°04' ± 0.19

F4 0.16 ± 1.21 0.19 ± 2.21 16.5 ± 4.78 1.18 ± 0.81 21°02' ± 1.80

F5 0.46 ± 4.10 0.54 ± 2.80 14.8 ± 2.15 1.21 ± 1.32 20°04' ± 0.21

F6 0.43 ± 2.84 0.52 ± 1.94 17.3 ± 1.83 1.17 ± 0.86 18°01' ± 1.31

The values represents mean ± SD, n=3.

Department of Pharmaceutics, Sri Shivani College of Pharmacy Page 59

Chapter-III Result&Discussion

Table.3.4. Mean particle size of different formulations of Metformin

microspheres

Sr. no Formulation code Particle size (μ)

1 F1 95.03 ± 1.7

2 F2 117.21 ± 0.8

3 F3 124.17 ± 1.0

4 F4 135.51 ± 1.3

5 F5 143.21 ± 1.8

6 F6 156.01 ± 2.1

The values represents mean ± SD,n=3.

Fig.3.2 Mean particle size comparison of different formulations.

180 Particle size comparison of different formulations

160
P 140
a
r 120
t
i 100
c
l 80
e
s 60
i
z 40
e
20
0
F1 F2 F3 F4 F5 F6
Formulation code

Department of Pharmaceutics, Sri Shivani College of Pharmacy Page 60

Chapter-III Result&Discussion

Table.3.5. Percentage yield comparison of microspheres.

Formulation
Practical yield Percentage yield
code

F1 643.76 ± 0.12 80.47 ± 1.02

F2 696.90 ± 1.24 77.40 ± 3.01

F3 712.5 0 ± 0.21 75.00 ± 6.53

F4 728.00 ± 2.13 72.82 ± 1.04

F5 735.50 ± 2.11 70.05 ± 0.12

F6 744.48 ± 0.32 67.68 ± 3.06

The values represents mean ± SD, n=3.

Fig.3.3. Percentage yield comparison of different formulations.

85 Percentge yield of different formulations

P
e 80
r
c
e
n 75
t
a
g
e 70
y
i
e 65
l
d

60
F1 F2 F3 F4 F5 F6
Formulation code

Department of Pharmaceutics, Sri Shivani College of Pharmacy Page 61

Chapter-III Result&Discussion

Table.3.6. % Drug loading and % Drug encapsulation of different

formulations.

Formulation %Drug loading %Encapsulation

efficiency

F1 25.00 ± 2.31 87.20 ± 2.18

F2 22.22 ± 1.70 83.33 ± 1.23

F3 21.0 0 ± 0.23 64.00 ± 0.85

F4 20.00 ± 1.90 58.00 ± 1.96

F5 19.04 ± 2.76 51.33 ± 1.28

F6 18.18 ± 1.10 50.30 ± 2.23

The values represents mean ± SD, n=3.

Fig.3.4. Comparison of % drug loading of different formulations.

P 30 Percentge drug loading of different formulations

e
r
c 25
e
n
t
a 20
g
e
15
d
r
u
g 10

l
o 5
a
d
i
n 0
g F1 F2 F3 F4 F5 F6
Formulation code

Department of Pharmaceutics, Sri Shivani College of Pharmacy Page 62

Chapter-III Result&Discussion

Fig.3.5. Comparison of % drug encapsulation of different formulations.

P 100 Percentge of drug encapsulations in different formu -

e 90
r lations
c 80
e 70
n
t
60
a 50
g 40
e
30
o 20
f
10
d 0
r F1 F2 F3 F4 F5 F6
u Formulation code
g

e
n
c
a
p
s
u
l
a
t
i
o
n

Fig.3.6. Cumulative % drug release Vs time for formulations F1 –F3.

Dissolution profile of Tinidazole microspheres (F1-F3)

120
% of drug release

F1
100

80 F2

60 F3

40

20

0
0 2 4 6 Time (hr)
8 10 12 14

Department of Pharmaceutics, Sri Shivani College of Pharmacy Page 63

 Chapter-III Result&Discussion

Dissolution profile of Tinidazole microspheres (F4-F6)

100
% of drug release
80

60 F4
F5
40 F6

20

0
0 2 4 6 Time(hr)8 10 12 14

Fig.3.7. Cumulative % drug release Vs time for formulations F1 –F6.

Table.3.7. Cumulative % drug release Vs time for formulations F1 –F6

Cumulative percentage drug release for F1-F6.

Time F1 F2 F3 F4 F5 F6
(hr)

0 0±0 0±0 0±0 0±0 0±0 0±0

0.5 15.02 ± 0.12 12.25 ± 1.01 11.45 ± 0.12 10.12 ± 0.42 10.15 ± 1.03 09.95 ± 0.12

1 33.30 ± 0.23 24.58 ± 2.01 23.56 ± 0.14 22.35 ± 0.32 20.25 ± 0.12 21.25 ± 1.08

2 50.03 ± 1.02 42.55 ± 0.32 40.65 ± 0.01 40.35 ± 0.11 38.14 ± 1.01 35.35 ± 1.03

4 65.21 ± 0.12 60.61 ± 0.21 57.25 ± 0.12 57.54 ± 0.05 54.65 ± 0.51 50.95 ± 0.08

6 83.12 ± 0.31 72.6 ± 0.53 70.58 ± 1.02 70.85 ± 0.23 68.55 ± 1.03 65.55 ± 1.06

10 99.9 ± 0.23 86.24 ± 0.21 83.25 ± 1.04 82.25 ± 0.12 80.15 ± 1.03 78.95 ± 0.32

12 - 99.95 ± 0.32 97.45 ± 0.03 94.15 ± 0.32 92.65 ± 0.12 90.54 ± 0.06

The values represents mean ± SD, n=3

Department of Pharmaceutics, Sri Shivani College of Pharmacy Page 64

Chapter-III Result&Discussion

Table.3.8. Model fitting release profile of formulations F1 to F6.

Formulation r2 values

Zeroored First order Higuchi matrix Kroesmeyers peppas

r2 n

F1 0.943 0.903 0.969 0.912 0.452

F2 0.956 0.964 0.9774 0.903 0.516

F3 0.948 0.992 0.984 0.891 0.521

F4 0.944 0.909 0.986 0.923 0.512

F5 0.934 0.922 0.993 0.912 0.613

F6 0.930 0.992 0.993 0.962 0.632

Fig.3.8. Plots of Log cumulative % drug retained Vs Time for formulations

F1-F6 (First order kinetics).

Comparative drug release profile of different formulations (First

order)
2.5
Log cumulative percentage drug retained

F1

F2
2
F3
1.5
F4

F5
1
F6

0.5

0
0 2 4 6 8 10 12 14
Time (hr)

Department of Pharmaceutics, Sri Shivani College of Pharmacy Page 65

Chapter-III Result&Discussion

Fig.3.9. Plots of cumulative % drug release Vs Sq. root time for formulations F1-
F6. (Higuchi model)

Comparative drug release profile of different formulations

(Higuchi model)
Cumulative percentage drug release

120 F1

100 F2

F3
80
F4
60
F5
40 F6

20

0
0 0.5 1 1.5 2 2.5 3 3.5 4
Suare root of time

Fig.3.10. Plots of log cumulative % drug release Vs log time for formulations F1-
F6. (Peppas model)

Comparative drug release profile of different formulations

(peppas model)
2.5
F1
Cumulative percentage drug release

F2
2
F3
1.5 F4

F5
1
F6

0.5

0
0 0.2 0.4 0.6 0.8 1 1.2
Log time

Scanning electron microscopy

Department of Pharmaceutics, Sri Shivani College of Pharmacy Page 66

Chapter-III Result&Discussion

(SEM):
Morphology of microspheres was examined by scanning electron
microscopy. The view of the microspheres showed a hollow spherical structure
with a smooth surface morphology (Fig29) Some of the microspheres showed a
dented surface structure but they showed good floating ability on the surface of
the medium, indicating intact surface. The outer surface of the
microspheres was smooth and dense, while the internal surface was porous.
The shell of the microspheres also showed some porous structure (Fig.29). It
may be caused by the evaporation of solvent entrapped within the shell of
microspheres after forming a smooth and dense skin layer.

Figure 3.3.Scanning electron microphotograph of floating

microspheres of Metformin

Department of Pharmaceutics, Sri Shivani College of Pharmacy Page 67