350 JOURNAL OF COSMETIC SCIENCE
Construction of pseudo-ternary phase diagram to prepare ketoconazole loaded microemulsion. The
pseudo-ternary phase diagram was constructed using ternary plot software (TernaryPlot.
com) and the microemulsion region was located.7 Solvents that exhibited the maximum
solubility of ketoconazole were selected for preliminary study: Omix (Omix-TTO and
coconut oil at weight ratios of 1:1, 1:2, and 2:1) and Smix (Tween 80 and propylene glycol
at weight ratios of 1:1, 1:2, and 2:1). The above solutions were titrated in distilled water (0.5
to 10 mL) with gentle stirring at 37 ± 0.5°C. The clarity and consistency of the mixtures
were observed visually after the addition of water. Next, a clear, transparent, and yellowish
color mixture assured the formation of microemulsion. Lastly, the microemulsion region
was plotted at a ternary phase diagram using weight percentages of the water, oil, and
surfactant phases. Finally, the above microemulsions were kept at room temperature for 48
hours to check for any phase separation.1
Composition of microemulsion. As ketoconazole is poorly water soluble, larger amounts of
oil and surfactant phase were used than water phase to formulate microemulsion. This
ratio was selected from the phase diagram. The combination of the ketoconazole loaded
microemulsion is shown in Table I. The microemulsion was prepared as follows: oil and
surfactant phases were homogeneously mixed, and ketoconazole was completely dissolved
in it using a magnetic stirrer.14 Next, sodium benzoate was added in water phase and
mixed with the above oil-surfactant-ketoconazole solution using a magnetic stirrer. The
best composition was 45.77% Omix, 43.82% Smix, 10.41% water, and 2% ketoconazole
(Table I).
CHARACTERIZATION OF MICROEMULSION
Viscosity and pH. Viscosity of the microemulsion was measured using a LVDVE Brookfield
digital viscometer (Brookfield Engineering Corporation, Massachusetts, USA). The sample
was filled in a suitable container and was subjected to the viscometer. The spindle number
64 was attached to the viscometer and then lowered into container, until dipping to the
till mark on the spindle shaft and was then run at 50 rpm. The pH was determined with a
digital pH meter (Labman Scientific Instruments Pvt. Ltd., Tamil Nadu, India) that used
a glass electrode at room temperature.4
Determination of cloud point. Cloud point is the temperature at which transparent
microemulsion become cloudy and phase separation starts.15 It was conducted as follows:
Table I
Composition and Characterization of Ketoconazole Loaded Microemulsion (2:1)
Batch Ketoconazole
(%w/w)
Omix
(TTO +coconut)
(2:1)
Smix (Tween
80 +propylene
glycol) (2:1)
Viscosity
(cp)
pH Cloud
point
(°C)
Drug
content (%)
F1 2 8.744% 10.65% 154 5.32 ± 0.21 53 ± 0.4 84% ± 0.02
F2 2 45.77% 43.82% 958 5.56 ± 0.20 82 ± 1.1 89% ± 0.03
F3 2 19.40% 41.17% 635 5.52 ± 0.34 73 ± 0.7 85% ± 0.01
F4 2 3.88% 8.97% 102 5.30 ± 0.18 51 ± 1.5 33% ± 0.03
F5 2 50.44% 25.46% 249 5.75 ± 0.09 57 ± 0.3 75% ± 0.01
F6 2 30.03% 25.17% 457 5.43 ± 0.16 54 ± 1.3 64% ± 0.04

351 Treatment of Dandruff
a microemulsion sample was put in the water bath with gently stirring, followed by
increasing the temperature of the water bath at 2°C/minute. The turbidity point was noted
and then again subjected to cool and heat to check the reproducibility of the process.16
The test was performed three times. The cloud point should be greater than 30°C for the
desired formulation, which indicates good stability during storage and availability of drug
in a solubilized from at the site of the application.17
Drug content of microemulsion. Microemulsion (equivalent to 200 mg ketoconazole) was
dissolved in methanol, the volume was made up to 100 mL, and was then stirred for 10
minutes. After suitable dilutions, the resultant solution was filtered through Whatman
filter paper, and absorbance was measured at 244 nm using a UV-1800 spectrophotometer
(Shimadzu Corporation, Kyoto, Japan).18 The drug content was calculated as shown
in Eq. 1:
Drug content(%) Actual amount of drug
Therotical amount of =drug ×100 (1)
Analysis of particle size, PDI, and zeta potential. The particle size and polydispersity index
(PDI) were performed to study the colloidal characteristics of a ketoconazole-loaded
microemulsion. PDI is used to investigate the degree of homogeneity of the globule
size. Analysis of particle size, zeta potential, and PDI of the best batch was performed as
follows: microemulsion (0.1 mL) was suitably diluted with 10 mL of Milli-Q deionized
water (Direct-Q@, Mecrk KGaA, Darmstadt, Germany). The particle size and PDI were
estimated using a SZ-100 zeta potential/particle sizer (HORIBA Scientific, Kyoto, Japan) at
25°C, with a scattering angle of 90° based on dynamic light scattering technique (DLS).19
DLS measures the variations in light scattering intensity brought on by Brownian motion
of particles in a continuous phase, and the instrument can determine the average particle
size and PDI. The zeta potential indicates the stability of microemulsion, for which the
optimal value must be greater than ±30 mV.20
Antifungal study of ketoconazole loaded microemulsion. A ketoconazole-loaded microemulsion
(10 mg/mL) and simple microemulsion (without ketoconazole) were tested against microbial
stain M. furfur (MONERA Lab, Pune, India). Antifungal activity of above formulation
was determined by cup-plate method using a Sabouraud dextrose agar medium. Briefly,
sterilized medium was poured into a sterilized petri dish to get a depth of 3–4 mm under
aseptic condition. After solidification, a loop from the subculture (M. furfur) was added
on to the surface of solidified agar medium and spread uniformly with help of a sterile
spreader. After stabilization of culture, with the help of sterile cork borer, well (each 7 mm)
diameters were punched and scooped out from the petri dish. Wells were next filled with
0.5 mL ketoconazole-loaded microemulsion (10 mg/mL). Finally, petri plates were then
incubated at 37°C for 24 hours. The zone of inhibition was measured and reported as
mean ± S.D.21
Scanning electron microscopy of microemulsion. The surface morphology of the microemulsion was
characterized using scanning electron microscopy (SEM). One drop of the microemulsion
was placed on carbon tape on an AI stub and air-dried. It was then examined using a SEM
Quanta-200-EDX system (ICON labs, Mumbai, India), outfitted with a digital camera at
a voltage of 20Kv.22