Vol 2, Issue 2, 2020 (34-42)
http://journal.unpad.ac.id/idjp
Formulation and Stability Testing of Griseovulfin Microemulsion
Iyan Sopyan
1,2
, Dolih Gozali
1
, Eka Paramudya
1
1
Departement of Pharmacutics and Technology of Pharmacy, Faculty of Pharmacy, Universitas Padjadjaran.
Sumedang, Indonesia
2
Research Center of Drugs discovery and Drugs Development Faculty of Pharmacy, Universitas
Padjadjaran, Bandung, Indonesia.
Received : 10 Dec 2018/Revised : 30 Dec 2018/Accepted : 2 Jan 2019/ Published : 23 Jun 2020
ABSTRACT
The use of drugs that are less soluble in water will become clinically less efficient, this is caused
by the low penetration of the drug into the body. A microemulsion is a dispersion system such as
an emulsion that can increase the solubility of drugs that are sometimes difficult to dissolve in
water. Microemulsions have long-term stability, clear, transparent, and good penetration
capabilities. In this study, a microemulsion formulation with active ingredients griseofulvin and
virgin coconut oil was conducted as an oil phase. The resulting microemulsion evaluates
physical stability during 35 storage days. The results showed that the microemulsion preparation
remained stable during storage time without changes in color, odor, and consistency, while the
pH and dosage viscosity experienced less significant changes. The consequences of the
centrifugation test at 3700 rpm for 5 hours and freezing tests for 24 days showed stable
preparation and cannot be separated. The diffusion test results obtained by FG2 had the largest
permeation percentages of 3.6136%, FG3 2.8724%, and the smallest FG1 2.0477%.
Keywords: microemulsion, griseofulvin, stability, diffusion test
1. Introduction
The solubility of a nutritious substance plays an
important role in the formulation of a
pharmaceutical preparation [1]. More than 50% of
new chemical compounds found today are
hydrophobic. The clinical use of hydrophobic drugs
becomes inefficient with low solubility, resulting in
a small penetration of the drug in the body[2].
Griseofulvin is a substance that is very difficult to
dissolve in water and ethanol (95%), but easily
dissolved in tetrachloroethane [3]. Griseofulvin is
produced by Penicillium griseofulvin and it was
uesd for oral route as fungistatic against many
dermatophytes [4,5] The dosage forms that are
widely used for griseofulvin are tablets, oral
suspensions, capsules, and ultramicrosize tablets
with this approach the absorption is very limited so
efforts are needed to increase the dissolution,
especially in local route administration. The effort
that can be done is to use enhancers and reduce
particles such as microemulsions.. One way to
increase the solubility of a drug that is lipophilic or
hydrophobic is to make an emulsion preparation.
Emulsion is a preparation containing liquid drug
ingredients or drug solutions, dispersed in a carrier
liquid, stabilized with an emulsifying agent or a
suitable surfactant [3]. The purpose of emulsion
formation is that the medicinal material has an
unpleasant taste and composition which can be
made more comfortable in oral administration if it is
*Corresponding author,
e-mail :
i.sopyan@unpad.ac.id
(I. Sopyan)
https://doi.org/10.24198/idjp.v2i2.27574
2020 I. Sopyan et al
I. Sopyan et al / Indo J Pharm 1 (2020) 34-42
35
formulated in the form of an emulsion, and is easily
absorbed [6]. Emulsions are thermodynamically
unstable systems that consist of at least two liquid
phases which are not mixed, one of which is
dispersed in the form of drops (dispersed phase) in
the other liquid phase (continuous phase), stabilized
by an emulsifying agent. This dispersion is unstable,
these grains combine to form two separate layers of
water and oil [7].
A dispersion system developed from an emulsion
preparation. When compared to emulsions, there are
many characteristics of microemulsions that make
these preparations attractive for use as a drug
delivery system. Among others, it has a long-term
stability, clear and transparent, can be sterilized by
filtration, low manufacturing costs, has a high
solubility and has good penetrating ability. These
characteristics make microemulsion have an
important role as an alternative in formulas for
active substances that are not soluble [8].
Microemulsions have great solubility in both
water and oil and microemulsions can be formed for
sustained release preparations. It is known that
microemulsions are used to improve bioavailability
in drug administration, for example in topical and
oral administration. Microemulsions are commonly
used for topical preparations where there is an
increase in drug absorption [9].
In this research we use VCO in formulation as
enhancer and propylene glycol as cosolvent to form
emulsion to be microemulsion, so microemulsion
will be performed an enhancing of
griseofulvin.Absorption especially in topical
administration.
2. Materials and Methods
2.1 Materials
The materials used in the study were
Aquadestilata, Aquabidestilata, Griseofulvin was
purchased from PT Kimia Farma, potassium
dihydrogen phosphate (Quadrant), sodium
hydroxide (Quadrant), propilenglikol (Bratachem),
span 80 (Quadrant),t 80 (Bratachem), and virgin
coconut oil (PT Wuwung Prada Pratama
Yogyakarta).
2.2 Equipments
The equipment used in this study was the Stirrer
motor RZR 2021 (Heidolph), the UV
spectrophotometer (Specoord. The Jena analytic),
the pH meter (pH meter 744 Methrom),
centrifugation (Hettich Zentrifugen)
2.3 Methods
The stages in the research method used are the
collection of ingredients, Griseofulvin
microemulsion preparations, evaluation of dosage,
permeation test, analysis, and data processing.
2.3.1 Microemultion Formulation
The preparation of the microemulsion is to
dissolve the Tween 80 in the water and the
constantly stirred using an ultraturax stirrer motor at
a temperature of 50
o
C at 1200 rpm so that a clear
solution is obtained. Griseofulvin dissolved in
Propilenglikol then inserted into the dispersion span
in the VCO that serves as the oil phase. This
dispersion is inserted into the water phase, stirring
with an ultrathurax mixer for 30 minutes at a speed
of 2200 rpm to a clear and transparent
microemulsion.
2.3.2 Dosage evaluation
Some tests were conducted to determine the
stability of microemulsion preparations and
Griseofulvin emulsions, among others.
1. Organoleptic Observation
Organoleptic observations of microemulsions
and griseofulvin emulsions were examined by
observing changes in consistency, color, odor, and
homogeneity during storage.
2. pH observation
PH measurement using a pH indicator.
Measurements were taken at room temperature on
days 1, 3, 5, 7, 14, 21, 28, and 35 days.
3. Determintion of viscosity
Viscosity measurements were carried out using a
Brookfield Viscometer. The trick is that the spindle
is mounted in place and arranged so that the spindle
boundary is immersed into the preparation that has
I. Sopyan et al / Indo J Pharm 1 (2020) 34-42
36
been placed on the beaker. The tool is turned on and
the spindle is left to rotate until the needle shows a
constant number [10].
4. Centrifugation test
Griseofulvin microemulsion preparations of
approximately 10 ml were put into centrifugation
tubes then centrifuged for 5 hours at 3700 rpm.
5. Freeze-thaw studies
Evaluation of physical preparations using the
freeze-thaw method was carried out at low
temperature (4
0
C) and high temperature (40
0
C) for 6
cycles. One cycle consists of 48 hours at low
temperatures and 48 hours at high temperatures.
6. In vitro permeation testing of microemulsion
preparations using cellulose membranes. The stages
of testing are as follows:
2.4.1 Material Preparation
a. Preparation of receptor fluids (phosphate buffer
pH 7.4).
Making phosphate buffer pH 7.4 according to
Pharmacopoeia Indonesia edition III by mixing 50
ml of potassium dihydrogen phosphate (KH2PO4)
0.2 M and then adding 39.1 ml of sodium hydroxide
(NaOH) 0.2 N and diluted with enough carbon
dioxide water to 200 mL[3]
b. Spangler Fluid Preparation
All ingredients such as cholesterol, stearic acid,
palmitic acid, oleic acid, vaseline albumin, paraffin
liquid, and squalene stir homogeneously and are
melted in a water bath. After everything is mixed,
Whatman paper number 1 is put into the liquid then
impregnated for 15 minutes. Dry by placing
Whatman paper on filter paper [11].
2.4.2 Determination of analysis method of
griseofulvin microemulsion permeation study
a. Making griseofulvin raw solution
The standard solution is made at a concentration
of 800 ppm by weighing 80 mg of griseofulvin
dissolved in 100 ml phosphate buffer.
b. Determination of griseofulvin maximum
absorption wavelength
From the standard solution of griseofulvin,
dilution of 2 ppm is made and
themaximumwavelength is sought.
c. Making the griseofulvin raw curve
Making the griseofulvin standard curve by using
dilutions of various concentrations from 0.5 ppm, 1
ppm, 1.5 ppm, 2 ppm, and 2.5 ppm.
2.4.3 Permeation study of griseofulvin
microemultion
Permeation tests were carried out using a Franz
diffusion cell device consisting of cellulose
membrane permeation cells, peristaltic pumps,
magnetic stirrers, beaker glass, hot plates, receptor
receptors, thermometers, and silicone hoses. The
test preparation was weighed 1 gram and then
leveled over the membrane. The temperature of the
media is 370C with a total volume of 100 ml of
receptor fluid. The peristaltic pump sucks the
receptor fluid from the beaker and is then pumped to
the cell so that a hydrodynamic flow takes place.
Samples were taken from the permeation media at
30, 60, 90, and 120 minutes [12].
3. Results and Disscusion
3.1 Results of griseofulvin microemulsion
formulation
In the microemulsion formulation process, a
preliminary experiment involves determining the
conditions of the experiment and determining the
composition of the appropriate material to produce
clear microemulsion preparations. Based on these
preliminary experiments, the results of griseofulvin
microemulsion formulations resulted in clear and
stable preparations as listed in the following Table
1.
The active substance which is used as a model
for making microemulsion preparations is
griseofulvin. Microemulsion preparation uses virgin
coconut oil which is pure coconut oil derived from
fresh coconut meat as the oil phase. The surfactants
used are Tween 80 and Span 80 [13]. Propylene
glycol used in this formula functions as a cosolvent.
I. Sopyan et al / Indo J Pharm 1 (2020) 34-42
37
Table 1: The results of griseofulvin microemulsion with variations in surfactant concentrations
Materials FG
0
FG
1
FG
2
FG
3
Griseofulvin
-
0.5%
0.5%
0.5%
Tween 80
30%
30%
35%
40%
Span 80
20%
20%
15%
10%
Propilenglikol
25%
25%
25%
25%
VCO
5%
5%
5%
5%
Aquadest
20%
Clear yellow
20%
Clear yellow
20%
Clear yellow
20%
Clear yellow
Notice:
FG0: Microemulsion formula with Tween 80 as much as 30%, Span 80 as much as 20% without the addition of
Griseofulvin.
FG1: Microemulsion formula with Tween 80 as much as 30%, Span 80 as much as 20%, and Griseofulvin 0.5%.
FG2: Microemulsion formula with Tween 80 as much as 35%, Span 80 as much as 15%, and Griseofulvin 0.5%.
FG3: Microemulsion formula with Tween 80 as much as 40%, Span 80, as much as 10% and Griseofulvin 0.5%.
Cosolvent can increase the solubility in water and
oil and can reduce the interface tension by
stabilizing the layer formed between two phases.
The addition of cosurfactants can reduce the oil-
water interface voltage [14].
The surfactant used in this microemulsion system
is a combination of nonionic surfactants, Tween 80,
and Span 80 [13]. The use of these surfactants is
relatively safe for oral administration because of
their low toxicity. However, nonionic surfactants,
especially those with polyoxyethylene groups, are
sensitive to temperature so that they will affect the
stability of the system thermodynamically. As the
temperature increases, nonionic surfactants will be
more lipophilic, this is due to the polyoxyethylene
group that functions as a polar group or the head
will become dehydrated with increasing
temperature. The addition of Span 80 as a second
emulsifier aims to assist Tween 80 in isolating the
oil phase from microemulsion. Tween 80 with the
ability as a surfactant and its chemical structure can
jointly reduce the surface tension of the
microemulsion system formed and increase the
hydrophilic-lipophilic balance (HLB) value of Span
80 [15].
3.2 Dosage evaluation
3.2.1 Results of griseofulvin microemulsion stability
evaluation.
The results of physical stability evaluation
include organoleptic observations, pH observations,
viscosity tests, centrifugation tests, freeze-thaw
methods, and diffusion test microemulsion
preparations.
3.2.2 Organoleptic observation results
The results of organoleptic observations of
griseofulvin microemulsions with various surfactant
concentrations include observations of changes in
consistency, color, and odor can be seen in Table 2.
Based on Table 2, it can be seen that the
griseofulvin microemulsion preparation is stable and
has no physical appearance changes during the 35-
day storage period at room temperature [10]. The
dosage remains homogeneous, clear yellow, and has
a distinctive odor derived from virgin coconut oil.
3.2.3 Results of pH observation
The results of observing the pH of griseofulvin.
Microemulsions with various concentrations of
surfactants can be illustrated in Figure 1.
The results of checking the pH of the four
microemulsion formulas measured for 35 days
showed that the pH of the four microemulsion
formulas did not change drastically, despite a
decrease and an increase in pH during storage [16]
This shows that the preparation is chemically stable,
no chemical reactions or interactions occur either
with the storage container or between the
ingredients contained in the preparation. pH
measurements still indicate the pH that can be used
by the skin (4.2-7.0) Based on statistical analysis
I. Sopyan et al / Indo J Pharm 1 (2020) 34-42
38
Table 2: Results of organoleptic observation of griseofulvin microemulsion during 35 Days of storage
Days of Observation
Formula Parameters
1 3 5 7 14 21 28 35
F0
Homogenity
Color
Smell
H
Y
S
H
Y
S
H
Y
S
H
Y
S
H
Y
S
H
Y
S
H
Y
S
H
Y
S
F1
Homogenity
H
H
H
H
H
H
H
H
Color
Y
Y
Y
Y
Y
Y
Y
Y
Smell
S
S
S
S
S
S
S
S
F2
Homogenity
H
H
H
H
H
H
H
H
Color
Y
Y
Y
Y
Y
Y
Y
Y
Smell
S
S
S
S
S
S
S
S
F3
Homogenity
H
H
H
H
H
H
H
H
Color
Y
Y
Y
Y
Y
Y
Y
Y
Smell S S S S S S S S
Notice: H = Homogen ; Y = Clear Yellow; S = Specifik (oily smell)
Figure 1. Graph of griseofulvin microemulsion pH values with variations in surfactant concentration.
showed that the microemulsion preparations of
using a randomized complete block design with a
confidence level of 99% or α = 0.01, it is found that
the f Table value calculated results are greater than
α, meaning that H0 is accepted. This means the
length of time of storage has no effect on the pH of
the preparation.
3.2.4 Viscosity observation results.
The results of observations of the viscosity of
griseofulvin microemulsions in various surfactant
and cosurfactant concentrations can be seen in
Figure 2.
The results of measurements of the viscosity of
microemulsion preparations for 35 days using
Viscometer Brookfield at room temperature (27
0
C)
formula FG0, FG1, FG2, and FG3 had viscosity
which tended to increase in the first week and
decreased viscosity from day 14 to day to 35.
Viscosity produced is not too large, indicating that
the microemulsion preparation contains particles
that can disperse well so that they have a good flow
rate (300-1000 Cps). Besides, statistical analysis
was also carried out on the length of time of storage
and its relationship with the viscosity of
microemulsion preparations [17]. Based on
statistical analysis using a randomized complete
block design with a confidence level of 99% or α =
0.01, it was found that the f Table value calculated
was smaller than α, meaning H
0
was rejected. It
means that the storage time affects the viscosity of
I. Sopyan et al / Indo J Pharm 1 (2020) 34-42
39
Figure 2: Graph of griseofulvin microemulsion dosage values with various surfactant concentrations.
the preparation because each formula gives a
different viscosity value.
3.2.5 Evaluation centrifugation result
This process is an accelerated test to see the
stability of preparation within a certain period. The
results obtained from the four formulas tested did
not occur. This shows that the preparation is still a
single and clear solution. The centrifugation process
is carried out for 5 hours at a speed of 3700 rpm to
find out the preparation is stable within a period of
one year[18].
3.2.6 Uji freeze-thraw
a. Cycle at temperature 4
o
C
At the time of physical stability test using the
freeze-thaw method, when the preparation is stored
at a low temperature (4
o
C) it appears that the
preparation has undergone a physical change ie the
preparation becomes milky white and becomes
viscous, and even looks solid. This is because the oil
phase tends to freeze at low temperatures. As a
result, the particles tend to combine to form a bond
between particles that is denser which causes the
preparation to become milky white because the
structure becomes denser and more orderly.
b. Cycle at temperature 60
o
C
The four microemulsion preparations stored at
60
o
C did not show any changes such as
precipitation, rupture, or lumps which showed stable
preparations at high temperatures. This shows that
the water and oil phases with the help of surfactants
can form a single solution that is well dispersed.
c. Cycle at room temperature (27
0
C)
Storage at room temperature (27
0
C) shows that
the four microemulsion preparation formulas remain
stable and show no significant physical changes.
The four microemulsion formulas remained clear,
homogeneous, the smell and color did not change.
The observation of the four microemulsion formulas
stored at 4
o
C and 60
o
C for 6 cycles showed that the
four formulas remained stable, did not occur in
phase separation, did not break, did not form lumps,
and did not undergo precipitation [11].
3.2.7 Results griseofulvin microemulsion
permeation (in vitro)
a. Results of griseofulvin maximum wavelength
determination in phosphate buffer solution pH 7.4.
Determination of the maximum wavelength of
griseofulvin in phosphate buffer pH 7.4 obtained the
maximum wavelength at 296 nm (figure 3)[20].
Based on the measurement results of absorption
of griseofulvin solution, various concentrations in
phosphate buffer pH 7.4 can be made graph of the
relationship of absorption to the concentration in the
form of a straight line. This shows that with the
increase in concentration the amount of absorption
will also increase with the linear regression equation
y = 0.175 + 0.397 with r
2
= 0.995. its proved that
I. Sopyan et al / Indo J Pharm 1 (2020) 34-42
40
Figure 3: Maximum absorption of griseofulvin in phosphate buffer pH 7.4
Table 3. Results of griseofulvin absorption results in phosphate buffer solution at a wavelength of 296 nm
Concentration (ppm)
A1
A2
A3
Mean
0.5
0.4738
0.4742
0.4753
0.4738
1
0.5753
0.5759
0.5755
0.5755
1.5
0.6796
0.6791
0.6796
0.6794
2
0.7453
0.7456
0.7532
0.7480
2.5
0.8254
0.8256
0.825
0.8253
Notice: A:
Absorbance
method for griseovulvin assay from emulsionwas
validated [21].
In vitro permeation microemulsion test in vitro
was carried out using Franz diffusion cells Base on
data of the study it was found that the formula FG2
had the highest permeation percentages at 3.6136%,
while the formula FG1 had a small percent
permeation at 2.0477% [22]. Whereas FG3 has a
permeation percentage of 2.8724% (Table 4 and
figure 4). Based on statistical analysis using a
randomized complete block design with a
confidence level of 99% or α = 0.01 it is found that f
Table is greater than f arithmetic then Ho is
accepted. It means that each formula provides the
same permeation rate to the cellulose membrane
[23].
Figure 4: Profile for Microemulsion Release for 2 hours
I. Sopyan et al / Indo J Pharm 1 (2020) 34-42
41
Table 4: Griseofulvin microemulsion permeation percentage for 2 hours.
Time (minutes) FG1 %
FG2 %
FG3 %
concentration concentration concentration
30
0.0079
0.0096
0.0003
60
0.0497
0.0059
0.023
90
0.8494
1.3970
1.6112
120
2.0477
3.6136
2.8724
4. Conclusion
Base on the results of the study, several
conclusions were obtained: microemulsion
preparations could be made using griseofulvin as a
hydrophobic drug model using virgin coconut oil oil
phase and tween 80 and span 80 surfactant mixtures
with various concentrations, evaluation of physical
stability of the four preparations was quite stable
during 35 days storage, and base on permeation
studi FG2 preparations have the highest percent
levels compared to FG1 and FG3 for two hours.
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