Characterization and In vitro Penetration Testing of Gel p-sinefrin Loaded in

Transfersome Using Franz Diffusion Cells

Amelia Luthfiah1, Iskandarsyah1, Erny Sagita1
Fakultas Farmasi Universitas Indonesia, Depok, Indonesia
E-mail: amelialuthfiaha

@gmail.com

ABSTRACT
p-Sinefrin has lipolytic activity, however it has a low oral bioavailability, and also hydrophylic characteristic which
is difficult to penetrate the epidermis if it is made into transdermal peparation. The purposes of this research were
to increase the penetration of p-sinefrin by preparing into transfersome gel. In this research three transfersome
formulas were prepared, e.g. F1, F2 and F3 with the use of surfactants respectively: tween 80, span 80, and
combination of tween 80 and span 80 with ratio 1:1. The result showed that F1 is the best formula with the highest
entrapment efficiency 64.058 0.754%, particle size average 103.3 nm, polydispersity index 0.269 0.05 and zeta
potential = -36.2 0.64 mV, so the best formula was incorporated into gel formulation. There were two gel
formulas prepared in this research, gel transfersome (GT) and non transfersome gel (GNT). Both of gels were
evaluated for their physical stability, and also in vitro penetration test using Franz diffusion cells using rat male
Sprague Dawley skin. The result showed the physical stabilty test of GT was more stable than GNT. Cumulative
penetration of p-sinefrin GT was higher than GNT, which value for GT was 1955.4 9.36 g.cm-2 and GNT was
892.87 31.79 g.cm-2. It can be concluded that GT can increase penetration of p-sinefrin compared to GNT.
Keyword : transfersom; gel, p-sinefrin; transdermal; penetration in vitro test.

INTRODUCTION

Overweight

are

tissue (1). If p-sinefrin is made into

a health problem its

transdermal preparations as the anti-

prevalence is always increased. The data

cellulite gel, a problem will happen

from WHO in 2014 showed more than 1.9

because

considered as

and

obesity

million adults were overweight (BMI 25

kg/m2). The physical condition that is not
acceptable because of overweight is
cellulite.

is

hydrophilic

compound. The lipid bilayer in the

stratum corneum of the epidermis
skin

has

hydrophobic

characteristic, so it would be an
obstacle to an active compound that

One of compound that useful to

decrease

p-sinefrin

cellulite

Synephrine

has

is

p-synephrine. p-

lipolysis

is hydrophilic to penetrate into the

deepest skin layers (2).

effect. The

The solution for this problem is

mechanism of this compound is binding to

made the active ingredients into

-3 adrenoceptor so it can increase

tarnsfersome.

thermogenesis and lipolysis in adipose

vesicle that can change the shape,

Transfersome is a

elastic, flexible and it has the ability

to increase entrapment of the drugs
and also the penetration of drugs
into the skin tissue to pass through
the stratum corneum layer of the
skin.

Transfersom

ultradeformable

(very

is

easy

to

deform) because of surfactant (3).

The surfactant is a molecule
that has a hydrophilic group and a
lipophilic group which can unite a
mixture of water and oil (4). There
are three classification of surfactan
which are cationic, nonionic and the
amfoter surfactant (5). In this study,
we

used

non-ionic

surfactant

because the surfactant has a low

toxicity.

Tween 80 is used as high

HLB value surfactant, while span 80

is used as a low HLB value and the
third formulation is used a mixture
thereof.

The

purpose

of

using

surfactants in different HLB value is

to

show

the

differences

characteristics of the transfersome

suspension that is produced, and
also

the

entrapment

efficiency

percentage.

TOOLS AND MATERIALS

Tools
Glass tools, analytical balance
(Sartorius), Zetasizer (Malvern Zetasizer),
Transmission Electron Microscopy (Tecnai
F20, The Netherlands) and (JEOL JEM
1400), Brookfield viscometer (Brookfield,
USA),
Uv-Vis
spectrophotometer
(Shimadzu UV-1800, Japan), mini extruder

set (Avanti Polar Lipids), Franz diffusion

cell test equipments (Bengkel gelas,
Indonesia),
centrifuge
(Zentrifugen,
Germany), rotary evaporator (Hahn Shin
HS-2005s-N), the vacuum evaporator
(Janke and Kunkel IKA, Germany),
ultrasonicator, homogenizer (Edmund
Bhler, Germany), pH meter (Eutech
Instruments pH 510, Singapore), vortex
(Digisystem
Laboratory,
Taiwan),
glassbeads, oven (Memmert, Germany),
autoclave, mini extruder set (Avanti Polar
Lipids),
polycarbonate
membrane
(Whattman), stopwatch, centrifugation
tube with a filter.
Material
p-Sinefrin
(Green
Spring
Technology, China), phosphatidylcholine
(Lipoid, Germany), dichloromethane,
methanol (Merck, Germany), tween 80
(Croda, Singapore), span 80 (Croda,
Singapore), carbomer (Lubrizol, Korea),
triethanolamine (Petronas, Malaysia),
NaOH, potassium dihydrogen phosphate,
aqua
demineralisata
(BRATACO,
Indonesia).
METHODS
Transfersom is made using thinlayer method. Phospholipids and surfactant
are used in the ratio of 85:15 (7).
Formulations which are using span, its
dissolved in dichloromethane and mixed
with phospholipids which had been
dissolved in dichloromethane, and then the
mixture is rotated by rotary evaporator at
50-150 rpm with a vacuum. After a thin
layer formed, it is flowed by N 2 then
left in cool place up to 24 hours in the
refrigerator. After that, a thin layer is
hydrated using aquadem.

When the hydration process

formulations that containing tween, its
dissolved in aquadem with p-sinefrin.
Then thin layer is rotated at 50-150 rpm
using glass beads without vacuum. After
the suspension is formed, it is placed in
cool place about 24 hours and then the
particle size in the suspension is reduced
by ultrasonication. Here is a transfersom
formulation:

Entrapment Efficiency

F1

F2

As much as (2-3 ml) suspension is

filled Using centrifugation tube with filter,
with speed 4500 rpm for 48 hours. Add
methanol to the suspension residue on top
of the tube, centrifugate again. The
resulted solution is at the bottom of the
tube (entrapped medicine concentration).
transfersome suspension add methanol and
vortex, after that add the mixture of
phosphate buffer:ethanol (1:1) and vortex
again (total medicine concentration),
measure the absorption of both
concentrations using spectrophotometer
UV-Vis. Entrapment efficiency percentage
is the concentration of entrapped medicine
divided by the total of medicine
concentration multiplied by 100%.

Fosfatidilkolin Kedelai

2,5

2,5

Deformability Index (3)

Tween 80

0,441

0,6

Span 80

12,5

p-sinefrin

aquades

Ad 100

Ad 100

Table 1. The formulation of pSynephrine Transfersome

Bahan

Konsentrasi % (b/b)

rv
D=J
rp

( )

D = deformability index, J = the

amount of suspension transfersome that
passed the membrane for 5 minutes (mL),
rv = transfersome particle size that passed

Transfersome Characteristics

the membrane (nm), rp = the size of

Morphology Characteristics

membrane pore (nm).

Using
Transmission
Electron
Microscopy. A drop of transfersome
suspension dissolved in 50 drops of pH 7.4
phosphate buffer, and then dropped it on
the grid, wait until dry, and then the
vesicle's morphology is observed in TEM
Particle Size and
Characteristics

Zeta

Potential

Using particle size analyzer with

dynamic light scattering method (Malvern
zetasizer) (3).

Gel Manufacture
Carbomer

is

dissolved

in

aquademineralisata until expanded, then

homogenized with speed of 1500 rpm, add
propylene glycol and homogenized again,
add

TEA

and

homogenized.

The

suspension is add in to the gel, and then

homogenized with speed of 1500 rpm (6).
Gel Evaluation

Organoleptic observation to check

the

form,

color,

and

smell.

All of three formulations showed

the results are appropriate to the desired
characteristics which are PDI value in the
range of 0 to 0.6, the value of the zeta
potential is more positive than + 30 mV or
more negative than -30 mV. The results
showed the smallest particle size were
suspensions that were used a tween as a
surfactant (F1)

pH

measurement in order to the same as the

skins

pH

range,

and

viscosity

measurement to check the rheology of gel

(7).
Stability Test
It is conducted in low temperature

Tabel 2. Results of particle size,

polydispersity index and zeta potential from psynephrine transfersome

(42oC), room temperature (272oC), and

high temperature (42oC) (8).
RESULT AND DISCUSSION
Characterization of Transfersome

(a)

(b)

Figure 1. The transmission electron microscope

result of cp-sinephrine transfersome suspension
(a) magnifications of 9900 x, 200 kV
(b) magnifications of 38000 x, 200kV

Particle Size and Potential Zeta

Zeta

Polidispersita

Potensial

Sampe
l

Partikel

(mV)

101,93 0,55

0,264 0,011

-36,20 0,69

0,358 0,009

-33,761,88

0,248 0,006

-43,130,86

Morphology Characteristics
The result of TEM showed that
suspension contains spherical shape
vesicle, with particles size were around
100-200 nm. These clearly visible on the
outside was a wrapping layer as a
phospholipid bilayer and they were the
single lamellar vesicles.

Indeks

Ukuran

F1
F2
F3

171,00

14,06
110,03 3,82

ENTRAPMENT EFFICIENCY
Entrapment efficiency test results
by the direct method were obtained. The
average
percentage
of
entrapment
efficiency for p-sinefrin in F1 was 64.05
0.75%, 46.05 0.24% for F2 and F3 was
56.21 1.55%. The highest percent of
entrapment was F1, thats used tween as a
surfactant. Things that affects to
entrapment efficiency was the alkyl chain
that is contained in surfactants (9). The
longer alkyl chain of the surfactant in span
80, the lower the capacity of entrapment
will be. If the HLB value is 8.6 or more it
will have a high entrapment efficiency,
while the value of HLB is around 8.6 to
1.7, entrapment efficiency will be
decreased.

Gels Evaluation and Stability Test

Gels Evaluation results showed
that the color of GT was white and for
GNT was transparent, gel smelled like
carbomer's aroma. The pH of GT and GNT
are respectively 5.48 and 5.70. Results of
viscosity at 20 rpm is 8920 cps for GT
and 8200 cps for GNT. Transfersom gel
has a higher viscosity because it contained
phospholipid. Both of gels have a plastic
thixotropic flow. The results for
organoleptic and homogeneity in the
physical stability test of GT and GNT at
low temperature (4 2 C), room
temperature (25 2 C), and high
temperature (40 2C) was not changed,
the color of GT and GNT were still same.
The smell for the GT and GNT during
storage also not changed. Gel's pH
changed but they were still at a pH range
of the skin. In the test cycling test, the gel
does not change the organoleptic and
syneresis did not occur.

5. Salager , J.L. 2002. Surfactants

types and uses. los andes:
laboratory
of
formulation,
interfaces rheology and processes.
J. Am Oil Chem Soc, Vol. 65 (6):
1000-1006.
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