Preparation and Characterization of Valsartan Loaded Alginate Mucoadhesive
Microcapsules
Sandhya
Raj S.*, Sundramoorthy. K. and Vetrichelvan. T.
Department of Pharmaceutics, Adhiparasakthi College of Pharmacy, Melmaruvathur,
Kanchipuram Dst.-603391, Tamilnadu. India.
ABSTRACT:
Sustained
release alginate microcapsules of valsartan were
prepared by orifice-ionic gelation method using Hydroxy Propyl Methyl Cellulose,
(viz, 50
cps, K4M) as mucoadhesive polymer. Microcapsules were
discrete spherical and free flowing. Encapsulation efficiency varied from
68.42% to 85.46%. Microcapsules were evaluated for % yield, drug content
uniformity, particle size distribution, surface morphology(scanning electron
microscopy), percentage moisture loss, in
vitro drug release profile, and mucoadhesion
study by in vitro wash off test,
short term stability ,and drug excipients
interactions (DSC and IR spectroscopy). The formulation prepared by using
alginate – Hydroxy propyl
methyl cellulose (K4M) in the ratio of 5:1 along with magnesium stearate, emerged as the overall best formulation based
upon their drug release characteristics (in phosphate buffer 6.8). This
formulation showed slow release up to 12 hrs. In vitro drug release followed first order kinetics, fickian diffusion mechanism (n<0.5) and the results had
proven the release of the best formulation had extended up to 12 hrs according
to t50%, t70% and t90% and the values. All the
microcapsules exhibited good mucoadhesive property in
the in –vitro wash off test. Accelerated stability studies were carried out for
short term studies of formulations according to ICH and Q 1 A
(R2) guidelines. These mucoadhesive microcapsules are
thus suitable for oral controlled release of valsartan.
KEYWORDS: Valsartan, mucoadhesive microcapsules, orifice ionic gelation method, Hydroxy Propyl Methyl Cellulose (50 cps, K4M)
INTRODUCTION:
Micro-encapsulation is a process in which tiny particles or droplets are surrounded by a
coating to give small capsules many useful properties. In a relatively
simplistic form, a microcapsule
is a small sphere with a uniform wall around it. The novel design of an oral
controlled drug delivery system should primarily be aimed at a achieving more
predictable and increased bioavailability of drugs. But there are several
difficulties, which include restraining/ localizing the drug delivery system
within the regions of the gastro intestinal tract and the highly variable
nature of gastro emptying process (few minutes to 12 hrs and above)1.
The
major absorption zone (stomach or upper part of the intestine), can result in
incomplete drug release from the drug delivery system leading to diminished
efficacy of the administered dose. Therefore, restraining a drug delivery
system in a specific region of the gastro intestinal tract due to its mucoadhesiveness increases the intimacy and duration of
contact between a drug containing polymer and mucus surface. Such a drug
delivery system offers numerous advantages, especially for drugs exhibiting an
absorption window or for drugs with a stability problem. Alginate is easily
gelled by the addition of Ca2+ to an aqueous solution of sodium
alginate, since insoluble calcium alginate is formed by cations
exchange between Na+ and Ca2+.
Then
an aqueous solution of sodium alginate was added drop wise to an aqueous
solution of calcium chloride, a spherical gel is termed as “Alginate Bead”1.
Valsartan
(VAL) is a potent and specific competitive antagonist –II –AT1 receptor.
It is used orally for the treatment of hypertension and has a low
bioavailability of 23%, because of its poor absorption in lower gastro
intestinal tract. It undergoes little or no hepatic metabolism and its
elimination half life is 6 hrs. Therefore, it is selected as a suitable drug
for the design of mucoadhesive microcapsules with a
view to improve its oral bioavailability and increase its drug release in a
sustained manner.
MATERIALS AND
METHODS:
Valsartan
(VAL) is a gift sample from Aurobindo pharmaceutical
(Hyderabad, India), Hydroxy propyl methyl cellulose (K4M) obtained from ParasChem Suppliers, Pune, Hydroxy propyl methyl cellulose
(50 cps), sodium alginate, calcium chloride and magnesium stearate
was provided by Qualigens, Mumbai. The concentration of valsartan
was measured with UV – visible spectrophotometer, Shimadzu. All the reagents
used were in analytical grade.
PREPARATION OF MICROCAPSULES:
Orifice ionic gelation
method :( syringe method)
Orifice
ionic gelation method is also been successfully used to prepare large sized alginate
beads. In this method microcapsules are prepared by employing sodium alginate
in combination with different mucoadhesive polymers
like Hydroxy propyl methyl
cellulose (K4M), Hydroxy propyl
methyl cellulose(50 cps) in the different polymer ratios like 1:1, 3:1, 5:1,
9:1are dissolved in purified water to form a homogenous polymer solution.1
A
homogenous polymer solution was prepared by coating material (sodium alginate)
and mucoadhesive polymer was dissolved in 32 ml to
form a homogenous polymer solution. The core material valsartan
(1 g) was added to the polymer solution, it was properly stirred in homogenous
solution at a 500 rpm. The resulting dispersion was dropped drop wise to a
calcium chloride (10% w/v) [40 ml] through a syringe fitted with a needle of 18
gauge. The added droplets were retained in the calcium chloride solution for 30
min to complete the curing reaction and to produce spherical rigid
microcapsules. The microcapsules were collected by decantation and the product
was thus separated and washed repeatedly with water and dried at 45º for 12
hrs. Since the microcapsules prepared by the above method sustain the release
up to 8 hrs to 9 hrs only, further modifications were made in this method.
These modifications include (i) increasing the
cross-linking time for further 3 to 6 hrs (Formulations MC 5 to MC 8). (ii)
Addition of magnesium stearate (#200 mesh) in 2 to
4%w/w concentration (Formulation MC 7 to MC 8)after thoroughly mixing with the
drug valsartan by spatulaion
(on a butter paper).The prepared microcapsules were stored in a desiccators for
further use2 (Table 1).
EVALUATION PARAMETERS FOR MICROCAPSULES:
i) Percentage
yield: 3, 4
The
total amount of microcapsules obtained were weighed and evaluated for
percentage yield and the yield was calculated as per the equation.
Percentage
yield = Practical yield × 100
Theoretical
yield
ii) Size analysis:
For
size distribution analysis, different sieves in a batch were separated by
sieving, using a set of standard sieves (IP). The amount retained on different
sieves was weighed.
iii) Drug content estimation:5
Valsartan
microcapsules (25mg) from each batch initially stirred in 3ml of sodium citrate
solution (1%w/v) until complete dissolution. Methanol (7ml) was added to above
solution to gel the solubilised calcium alginate and
further solubilize valsartan.
This solution was filtered through what man filter no
1 filter paper. The filtrate was assayed for drug content by measuring the
absorbance at 250nm after suitable dilution.
iv)Encapsulation
efficiency:6
Encapsulation efficiency was calculated using the
equation:
Encapsulation
efficiency =
Estimated
practical % drug content in microcapsules
Estimated
theoretical %drug content in microcapsules
v) Percentage moisture loss:7
The
valsartan loaded microcapsules was evaluated for
percentage moisture loss, which sharing an idea about its hydrophilic nature.
The microcapsules weighed initially kept in a desicator
containing Cacl2 at 37ºC for 24 hours. The final weight was noted
when no further change in weight of sample was observed.
Moisture
loss = Initial weight – final weight × 100
Final weight
vii) Scanning electron microscopy (SEM):
The
microcapsules were observed under a scanning electron microscopy (SEM-3400 N,
SEM HITACHI). They were mounted directly onto SEM sample stub using
double-sided sticking tape and coated with gold film with ion spillter with gold target with resolution 3 nm(30 KV HV
Mode),10 nm (30 KV HV Mode),40 nm (30 LV Mode) and a vacuum system is fitted to it.(Fig 2)
viii) In
vitro wash off test for mucoadhesion:8,
9
The mucoadhesive
property of the microcapsules was evaluated by an in vitro adhesion testing method known as the wash –off method.
Freshly excised pieces of intestinal mucosa (4×5 cm) from sheep were mounted
onto glass slides (3×1inch) with cyanoacrylate glue.
TABLE 1: FORMULATION TABLE OF MICROCAPSULES
|
FORMULATION CODE
|
SODIUM ALGINATE:HPMC RATIO
|
VALSARTAN (MG)
|
SODIUM ALGINATE (MG)
|
MAGNESIUM
STEARATE (MG)
|
HPMC(50CPS) (MG)
|
HPMC(K4M) (MG)
|
|
MC 1
|
1:1
|
1000
|
500
|
-
|
500
|
-
|
|
MC2
|
3:1
|
1000
|
750
|
-
|
250
|
-
|
|
MC3
|
5:1
|
1000
|
800
|
-
|
200
|
-
|
|
MC4
|
9:1
|
1000
|
900
|
-
|
100
|
-
|
|
MC5
|
5:1
|
1000
|
800
|
-
|
-
|
200
|
|
MC6
|
9:1
|
1000
|
900
|
-
|
-
|
100
|
|
MC7
|
5:1
|
1000
|
800
|
2%
|
-
|
200
|
|
MC8
|
5:1
|
1000
|
800
|
4%
|
-
|
200
|
HPMC 50 cps= Hydroxyl Propyl Methyl Cellulose, HPMC K4M= Hydroxyl Propyl Methyl Cellulose.
TABLE 2: EVALUATION OF MICROCAPSULES
|
SL. NO:
|
FORMULATION CODE
|
YIELD (%)
|
MICROCAPSULES WITH MEAN DIAMETER IN MICRONS (%)
|
MEAN % DRUG CONTENT (X±SD)* Average of three
determinations
|
ENCAPSULATION EFFICIENCY (%)
|
PERCENTAGE MOISTURE LOSS (%)
(X±S.D).
|
|
1204(22/25) Mesh
|
710(14/16) mesh
|
|
1
|
MC1
|
77.4
|
23.20
|
76.80
|
34.21±0.03
|
68.42
|
7.273±0.155
|
|
2
|
MC2
|
73.2
|
15.20
|
84.80
|
38.12±0.058
|
76.25
|
4.837±0.078
|
|
3
|
MC3
|
82.2
|
20.05
|
79.95
|
38.47±0.046
|
75.95
|
2.876±0.090
|
|
4
|
MC4
|
82
|
20.26
|
79.94
|
39.41±0.014
|
78.83
|
3.258±0.141
|
|
5
|
MC5
|
71.6
|
14.93
|
85.06
|
36.99±0.018
|
73.90
|
2.529±0.050
|
|
6
|
MC6
|
65.6
|
13.94
|
86.06
|
40.77±0.015
|
81.54
|
1.838±0.063
|
|
7
|
MC7
|
87.95
|
11.19
|
88.81
|
41.61±0.063
|
83.22
|
2.516±0.040
|
|
8
|
MC8
|
88.65
|
23.20
|
76.80
|
42.73±0.0004
|
85.46
|
1.244±0.130
|
TABLE 3:
RESULTS OF IN VITRO WASH OFF
TEST TO ASSESS MUCOADHESIVE PROPERTIES OF MICROCAPSULES
|
%of microcapsules adhering to the tissue at various
time interval* in hours
|
|
Phosphate buffer (6.8)
|
|
Formulations
|
pH used
|
1
|
2
|
4
|
6
|
8
|
|
MC1
|
6.8
|
97.3 ±0.9
|
90.3±0.9
|
87.6±2.0
|
60.5±1.2
|
44.5 (2.0)
|
|
MC2
|
6.8
|
96±1.07
|
91
|
87±2.0
|
72.3±1.2
|
52.3 (1.8)
|
|
MC3
|
6.8
|
98±2.0
|
95.6±1.2
|
85.3±3.3
|
79±2.0
|
53.6 (2.2)
|
|
MC4
|
6.8
|
98±2.1
|
95.2±1.2
|
90.5±2.0
|
78.7±1.9
|
51.2±1.5
|
|
MC5
|
6.8
|
98.6±2.0
|
96.6±1.2
|
92.6±1.2
|
72.9±1.2
|
59.1±1.5
|
|
MC6
|
6.8
|
98.4±2.0
|
97±1.2
|
90.3±2.0
|
62.9±1.2
|
49.5±3.5
|
|
MC7
|
6.8
|
99± 1.3
|
97
|
91.6±1.2
|
79.9±1.9
|
61.3±1.2
|
|
MC8
|
6.8
|
100
|
97.3±0.9
|
92.6±2.0
|
84.2±3.1
|
64.9±1.2
|
*average of three
determinations (S.D)
TABLE 4: IN – VITRO DRUG RELEASE DATA FOR
MICROCAPSULES
|
SR. NO.
|
FORMULATION CODE
|
t 50%(hrs)
|
t70%(hrs)
|
t 90%(hrs)
|
AVERAGE PERCENT DRUG RELEASE(12 hrs)*
|
|
1
|
MC 1
|
3.7
|
5.8
|
8.1
|
92.423±0.20
|
|
2
|
MC 2
|
4.4
|
6.1
|
7.9
|
91.377±0.32
|
|
3
|
MC 3
|
3.7
|
5.4
|
7.2
|
94.531±0.97
|
|
4
|
MC 4
|
3.7
|
5.8
|
8.0
|
90.545±0.19
|
|
5
|
MC 5
|
4.5
|
6.3
|
8.2
|
86.126±0.07
|
|
6
|
MC 6
|
4.1
|
6.3
|
8.7
|
90.308±0.24
|
|
7
|
MC 7
|
4.6
|
7.3
|
10.3
|
78.937±0.53
|
|
8
|
MC 8
|
4.3
|
7.6
|
12.6
|
71.179±0.26
|
*Average of three
determinations
Two
glass slides were connected with a suitable each wet rinsed tissue specimen,
and immediately thereafter the support were hung onto the arm of a USP tablet
disintegrating test machine. When the disintegrating test machine was operated,
the tissue specimen was given a slow, regular up and down movement in the test
fluid (400ml) at 37ºC contained in a 1000 ml vessel of the machine. At the end
of 1 hr and at hourly interval up to 12 hr, the machine was stopped and the
number of microcapsules still adhering to the tissue was counted. The test was
performed both in simulated gastric fluid (pH 1.2) and simulated intestinal fluid
(pH6.8 phosphate buffer).The data of in
vitro wash off test are shown in Table 3.
ix) In
vitro drug release studies: 10, 11
Drug
release study was carried out in USP basket type dissolution test apparatus (Veego – VDR-8DR, USP standards).A quantity of microcapsules
equivalent to 80 mg of valsartan was used for the
test. Dissolution medium was phosphate buffer having a pH 6.8. Volume of
dissolution medium was 900 ml, and bath temperature was maintained at 37±0.5ºC
throughout the study. At specified time intervals, 2ml samples were withdrawn
by means of a syringe fitted with prefilter and
replaced immediately with 2ml of fresh medium. The absorbance of sample was
measured at 250nm after suitable dilution with the medium. All the studies are
conducted in triplicate (n=3) (Table 4, Figure 1).
Figure 1: Percentage drug release of microcapsule
formulations
Percentage drug release of
microcapsule formulations (n=3) for formulations O (
),
MC1 (
),
MC2 (
),
MC3 (×), MC4 (
),
MC5 (
),
MC6 (l), MC7 (-),
MC8(-)
xi) Drug release mechanism and release:12, 13
The
in vitro drug release data was fitted
into four popular models of data treatment for the matrix formulations as
follows:
(1)Zero
order kinetics (2) first order kinetics (3) Higuchi’s square root model, (4)Peppas model the data obtained
from the stability studies was subjected to the statistical (student “t” in
order to find out any significant in the drug content and dissolution
parameters of the promising formulation(MC8)after the storage for 3 months at a
temperature of 40º±1ºC ambient humidity conditions (Table-5).
The
criteria for selecting more appropriate model was based on the goodness of the
test, Koresmeyer and peppas
equation; Mt/Mά=ktn;
whereas Mt/ Mά is the fraction of drug released
at time “t” k=constant. Incorporating of structural and geometric
characteristics of controlled release device n=diffusion release exponent
indicative of release mechanism. The best fit model was determined statically
employing comparison of correlation coefficient. The drug release rate from the
formulations and the respective half lives were calculated. The preparation of
graphs and statistical calculations were carried out with the help of computer.
RESULT AND
DISCUSSION:
All
the prepared microcapsules were found to be spherical, discrete and free
flowing with yellowish white color. The SEM studies of microcapsules (MC-8)
reveals that the microcapsules were perfectly spherical in shape and show a
rough porous surface with free drug crystals (Fig.2). Percent yield was in the
range of 65.6% to 88.65% (Table 2). The mean diameter of microcapsules was
found to be in the range of 750.631µm to 823.28 µm (Table 2). The mean percent
drug content of the microcapsules ranged from 34.21% - 42.73% (Table 2).The low
values of standard deviation indicates that uniform distribution of the drug
within the various batches of microcapsules prepared. Encapsulation efficiency
ranges approximately from 68.42% - 85.46% (Table 2). Results reveal that the
encapsulation efficiency of microcapsules increases with an increase in
alginate concentration.
Figure 2:
Scanning electron photomicrography of formulation MC 8, magnification
5To300.000v. Resolution 30 KV HV Mode, 10nm (30 KV HV Mode, 40nm (30 KV LV
Mode), Detector –standard detector SE, BSC, Vacuum system based –TMP/RP based
.Model S-3400 N SEM –HITACHI.
Microcapsules
with a coat consisting of alginate and a mucoadhesive
polymer exhibited good mucoadhesive properties in the
in vitro wash off test .The wash off
test was faster at a intestinal pH than at a gastric pH.
It was observed that the pH of the medium was critical for the degree of
hydration, solubility and mucoadhesion of the
polymers. The rapid wash off test observed at intestinal pH is due to
ionization of carboxyl and other functional groups in the polymers at this pH,
increases their solubility and reduces adhesive strength. The results of the
wash off test indicated that the microcapsules had very good mucoadhesive properties with more than 70% retention for 8
hrs.
TABLE5: DRUG RELEASE KINETIC STUDIES OF MICROCAPSULE
FORMULATIONS
|
Formulation
|
Zero order Kinetics
|
First order kinetics
|
Higuchi square root equation
|
Korsemeyer-Peppas model
|
Best fit model
|
|
R
|
r
|
r
|
r
|
n
|
|
MC 1
|
0.9873
|
0.9199
|
0.9916
|
0.9927
|
0.4539
|
Peppas
|
|
MC 2
|
0.9877
|
0.9165
|
0.9803
|
0.9853
|
0.4437
|
Zero order
|
|
MC 3
|
0.9902
|
0.9388
|
0.9903
|
0.9925
|
0.5002
|
Peppas
|
|
MC 4
|
0.9877
|
0.9612
|
0.9628
|
0.9934
|
0.4771
|
Peppas
|
|
MC 5
|
0.9989
|
0.9447
|
0.9531
|
0.9947
|
0.4226
|
Zero order
|
|
MC 6
|
0.9835
|
0.9223
|
0.9562
|
0.9855
|
0.4583
|
Peppas
|
|
MC 7
|
0.9755
|
0.9691
|
0.9697
|
0.9906
|
0.3744
|
Peppas
|
|
MC 8
|
0.9408
|
0.9949
|
0.9935
|
0.9971
|
0.3481
|
Peppas
|
Valsartan
release from microcapsules was studied in phosphate buffer 6.8 (pH 6.8). Due to
the slight solubility of drug in water and alginate HPMC (50cps) formulation
demonstrated a drug release of 90- 97% in the 8 hrs and required release was
not obtained and the release profile was very fast. Microcapsules of
alginate-HPMC (K4M) were observed that drug release from the microcapsules
produces a sustained release, it was evident that followed a diffusion
controlled from the best fit model data obtained (n<0.5). The formulation
prepared by alginate-HPMC (K4M) in the ratio of 5:1 along with 4% magnesium stearate, emerged as the best formulation(t50%=4.4h,t70%=7.6h,t90%=12.5h),
based upon drug release characteristics(in pH 6.8 phosphate buffer). This
formulation shows slow and extended release up to 12.5 hrs according to t90%
values.
Accelerated
stability studies were carried out using the ICH and Q1 A
(R2) guidelines meant for storing the product for 90 days. Products have been
kept in the stability chamber. Products were kept in stability chamber with the
temperature of 25±2ºC (60% ±5%), 40ºC ±2ºC(75%RH±5%).
The sample time points were 30, 60 and 90 days. The formulations will be
monitored for changes in particle size, physical changes, morphology, drug
content, entrapment efficiency and drug release profile.
CONCLUSION:
The
formulation MC8 containing drug: polymer in the ratio of 1:1 and polymer ratio
5:1 with the combination of sodium alginate, HPMC K4M and magnesium stearate (4%) was concluded to be the best
formulation(released 68.168% drug in 8hrs, t90% was found to be 12.5 hrs), regarding
all the studies evaluated in order to achieve objective of this study. The
novel formulation design facilitated the successful development of valsartan microcapsules formulations. Our data concluded
that combination of sodium alginate along with HPMCK4M and on addition
magnesium stearate may be an effective strategy for
the designing and development of valsartan mucoadhesive microcapsules for easy, reproducible and cost
effective method to prove its potential for safe and effective controlled for
oral drug delivery.
ACKNOWLEDGEMENT:
The
authors are thankful to Aurobindo Pharmaceuticals, Hyderabad
for providing the gift sample of Valsartan, Qualigens, Mumbai for providing
sodium alginate, and HPMC (K4M, 50cps) by Paraschem
suppliers, Pune. We wish to thank the Principal, Mr. T.
Vetrichelvan, Adhiparasakthi
College of Pharmacy for providing facilities to carry out the present work.
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