Vol 3, Issue 3, 2021 (139-148)
http://journal.unpad.ac.id/idjp
*Corresponding author,
e-mail yanni.dhiani@bku.ac.id (Y. D. Mardhiani)
https://doi.org/10.24198/idjp.v3i3.36777
2021 Y. D. Mardhiani et al
Astaxanthin Nanoemulsion Formulation and Evaluation
Yanni D. Mardhiani*1, Deny Puriyani A*1, Lailatul Fadilah1
1Research Group of Pharmaceutics, Bhakti Kencana University, Jl. Soekarno Hatta No.754,
Bandung city, West Java, Indonesia.
email: yanni.dhiani@bku.ac.id
Submitted :24/11/2021, Revised :21/01/2022, Accepted :25/01/2022, Published :07/02/2022
Abstract
Astaxanthin has antioxidant activity ten times higher than carotenoids such as -
carotene and a hundred times higher than vitamin E. However, its utilization is still
limited because its solubility in water is very low which results in low absorption
by the skin, resulting in low bioavailability. In this case, to increase the potency of
astaxanthin, this research was aimed at the formulation and characterization of
astaxanthin nanoemulsions using polysorbate 80 and polyethyleneglycol 400 as a
mixture of surfactants with a ratio of 7:1; 8:1 and 9:1 with the method of making a
combination of low and high energy emulsification. The data obtained were
analyzed using the Kruskal-Wallis test for data on the pH of the preparation and the
efficiency of adsorption while the pH test during freeze-thaw stability was analyzed
by the Wiloxon test. Based on the test results, it was found that the nanoemulsion
preparation with the smix (surfactant mixture) 9:1 formula is the most optimum
formula among other formulas, which is to produce preparations with quite good
characteristics organoleptically and give a light orange color appearance, clear,
distinctive smell with a pH value that meets the SNI standard 16-164399-1996 with
pH values ranging from 7.13 to 7.15 and based on the centrifugation test gave stable
results and had particle size, polydispersity index and zeta potential values,
respectively, 22.9 ± 9.4 nm, 0.435 and -21. ,4 mV and the value of entrapment
efficiency ranges from 93.87% to 94.32%. However, the thermodynamic stability
is not good enough. This is indicated by the instability of the preparation during the
freeze-thaw test with the results of changes in color, transparency and changes in
pH.
Keywords: Nanoemulsion, Astaxanthin, Polyethyleneglycol 400, Polysorbate 80,
Surfactants
1. Introduction
The potential pharmacological
effects of astaxanthin are very large,
including anti-cancer, anti-diabetic, anti-
inflammatory and antioxidant activities as
neuro protective, cardiovascular protective,
ocular protective and skin protective [1].
However, the utilization of the potential
benefits of astaxanthin in cosmetics and
pharmaceuticals is still limited because the
solubility of astaxanthin in water is so low
that it cannot be absorbed by the skin,
resulting in low bioavailability. In addition,
astaxanthin is known to be easily degraded
Y. D. Mardhiani et al / Indo J Pharm 3 (2021) 139-148
140
both during storage and processing.
Therefore, it is necessary to develop an
efficient and effective delivery strategy.
Several studies have shown a functional
increase in astaxanthin formulated in
nanodispersions, liposomes and
nanoemulsions [2]. Although the
nanoemulsion system can improve the
function of astaxanthin, its success is highly
dependent on the selection and composition
of the surfactant used [3, 4]. Tween 80 is a
non-ionic hydrophilic surfactant that works
by increasing the solubility of one
substance with another [5]. Meanwhile,
Polyethyleneglycol (PEG) 400 is also a
non-ionic hydrophilic surfactant that can
support the function of tween 80 in
increasing the solubility of astaxanthin. In
this study, the formulation and
characterization of astaxanthin
nanoemulsion preparations will be carried
out using variations of tween 80 and
polyethylene glycol (PEG) 400 as a mixture
of surfactants.
2. Method
In the optimization stage, two steps
were carried out, the first by making
variations of Smix (a mixture of
surfactants) namely tween 80 as a
surfactant and polyethyleneglycol 400
(PEG 400) as a co-surfactant with a ratio of
9:1; 8:1 and 7:1, respectively, the ratio of
Smix was optimized with sunflower oil as
the oil phase. Optimization of Smix and oil
phase is done by making five variations of
each ratio of Smix, where the ratio of Smix:
oil phase is F1 (9:1); F2 (8:2); F3 (7:3); F4
(6:4) and F5 (5:5) with a total Smix and oil
phase of 40% (w/w).
Manufacture of astaxanthin nanoemulsion
using spontaneous nanoemulsification
method, namely by adding an oil phase,
surfactant, and co-surfactant into a mixture
with the ratio selected to incorporate
astaxanthin as much as 5% (w/v). The
selection of the amount of astaxanthin to be
incorporated is based on research [6]
related to the manufacture of lotions from
astaxanthin where astaxanthin has the best
antioxidant activity at a concentration of
5% with an IC50 of 87.571 ppm so that in
this study 5% (w/w) astaxanthin was used.
Then stirred using a magnetic stirrer at 200
rpm for 30 minutes then sonicated for 1
hour to form a stable and clear
nanoemulsion [7].
3. Results
3.1 Optimization Results
Astaxanthin nanoemulsion was made based
on the optimization results of 27
nanoemulsion formulas with various
concentrations of surfactant, cosurfactant
and oil. The selected formula is F1 from
each ratio of smix (9:1; 8:1 and 7:1).
Furthermore, each formula is made in
triples to get accurate and valid results. The
temperature in the manufacture of
astaxanthin nanoemulsions is very
important, where the temperature for the
manufacture of astaxanthin should not
exceed the temperature of 50oC because it
will cause the astaxanthin to undergo
structural isomerization and completely
degrade within 32 hours [8]. Therefore, the
process of making astaxanthin
nanoemulsions using a low energy
combination method using a magnetic
stirrer is carried out at room temperatures
ranging from 25-30oC. A high energy
sonicator is used with a final temperature
not exceeding 50oC. Sonification using a
probe sonicator aims to reduce particle size
by utilizing ultrasonic waves that can
convert electrical energy into physical
vibrations that can reduce particle size to a
nanometer size range of 20-200 nm.1.
Y. D. Mardhiani et al / Indo J Pharm 3 (2021) 139-148
141
Table 1. Smix formulation
Furthermore, astaxanthin is incorporated
into the base using the same manufacturing
method.
Characterization
3.2 Organoleptic Test
Organoleptic test was carried out visually
by observing the color, shape and smell of
the preparation.
3.3 pH test
The pH test was carried out in triples to get
accurate and valid results. The pH test for
the astaxanthin nanoemulsion preparation
has met the required range of topical
preparations as can be seen in table 2.
Table 2. pH test for astaxanthin nanoemulsion
Formulation
Batch
average pH ± SD
9:1
1
7,13 ± 0,020
2
7,15 ± 0,026
3
7,13 ± 0,011
8:1
1
7,14 ± 0,023
2
7,13 ± 0,015
3
7,11 ± 0,015
7:1
1
7,13 ± 0,035
2
7,13 ± 0,015
3
7,12 ± 0,012
3.4 Particle Size Test and
Polydispersion Index
Particle size test was carried out to
determine the particle size of astaxanthin
nanoemulsion preparations. According to
[9] which states that a preparation is said to
be a nanoemulsion if it has a size of 20-200
nm. The results of particle size testing
carried out at the Print-G Laboratory of
Universitas Padjadjaran using the Horiba S
Z-100 Particle Size Analyzer measured at a
temperature of 25oC can be seen in Table
3.
Nama Bahan
Smix 9:1
(% b/v)
Smix 8:1
(% b/v)
Smix 7:1
(% b/v)
Polisorbat 80
32,4
32
31,5
PEG 400
3,6
4
4,5
Sunflower oil
4
4
4
Aquadest
Ad 100
Ad 100
Ad 100
Y. D. Mardhiani et al / Indo J Pharm 3 (2021) 139-148
142
Table 3. Particle Size Test and Polydispersion Index
Formulation
Particle Size
Polydispersion Index
9:1
22,9 ± 9,4 nm
0,435
8:1
28,8 ± 13,8 nm
0,475
7:1
28,2 ± 15,2 nm
0,541
3.5 Zeta Potential Test
The zeta potential test was carried out to
determine the overall charge of a particle
that could describe the stability of a
nanoemulsion. Zeta potential can describe
the stability of a system containing
dispersed particles because this potential
regulates the degree of repulsion between
dispersed particles of the same charge and
close to each other [10]. The results of the
potential zeta test were carried out at the
Print-G Laboratory of the University of
Padjadjaran using the Horiba S Z-100
Particle Size Analyzer which was measured
at a temperature of 25oC.
Zeta potential measurements were carried
out only on samples of astaxanthin smix 9:1
nanoemulsion which were selected based
on the results of the most optimum particle
size and polydispersity index test results.
The results of the zeta potential
measurement on the astaxanthin smix 9:1
nanoemulsion sample showed a zeta
potential value of -21.4 mV.
3.6 Centrifugation Test
Based on the test results that all formulas
did not experience changes in either phase
separation, precipitation, creaming or
cracking after the test which indicated a
picture of the stability of the preparation
against gravity.
3.7 Adsorption Efficiency Test
The adsorption efficiency test was carried
out to measure the amount of drug adsorbed
in the nanoemulsion system as a carrier.
The measurement of the absorption
efficiency of the active substance in the
nanoemulsion system is carried out
indirectly by measuring the amount of
astaxanthin that is not adsorbed in the
nanoemulsion where the amount can be
calculated from the results of the
absorbance measurement which can be
calculated by the equation for the
percentage of adsorption efficiency:
Note:
Qt: The number of drugs used Qs : The amount of drug that is not
adsorbed in the nanoemulsion
[11]
The results of the adsorption efficiency test
on the astaxanthin nanoemulsion can be
seen in table 4.
Table 4. Adsorption Efficiency Test
Formula
Batch
% EE 1
% EE 2
% EE 3
Rata-rata ± SD
9:1
I
94,55 %
94,55 %
93,87 %
94,32% ± 0,003925982
II
93,87 %
93,87 %
93,87 %
93,87% ± 0
143
III
94,55 %
93,87 %
93,87 %
94,10% ± 0,003925982
8:1
I
92,49 %
92,49 %
93,18 %
92,72% ± 0,003983717
II
93,18 %
92,49 %
93,18 %
92,95% ± 0,003983717
III
93,18 %
93,18 %
92,49 %
92,95% ± 0,003983717
7:1
I
91,80 %
91,80 %
92,49 %
92,03% ± 0,003983717
II
93,18 %
92,49 %
92,49 %
92,72% ± 0,003983717
III
91,80 %
92,49 %
92,49 %
92,26% ± 0,003983717
3.8 Freeze-thaw Stability Test
Freeze thaw test was conducted to
determine the effect of temperature on the
physical stability of nanoemulsion
preparations. This test was carried out for
six cycles, where one cycle consisted of
storage at a temperature of 4˚C ± 2˚C for 24
hours and at a temperature of 40˚C ± 2˚C
for 24 hours. The results of the freeze-thaw
stability test can be seen in Table 5.
Table 5. Freeze-thaw stability test on organoleptic results
Batch
Formula
Parameter
Cycle
1
2
3
4
5
6
I
F1 (9:1)
Discoloration
Phase separation
Transparency
-
-
-
-
-
-
-
-
-
+
-
-
+
-
-
+
-
-
F1 (8:1)
Discoloration
Phase separation
Transparency
-
-
-
-
-
-
-
-
-
+
-
+
+
-
+
+
-
+
F1 (7:1)
Discoloration
Phase separation
Transparency
-
-
-
-
-
-
-
-
-
+
-
+
+
-
+
+
-
+
II
F1 (9:1)
Discoloration
Phase separation
Transparency
-
-
-
-
-
-
-
-
-
+
-
+
+
-
+
+
-
+
F1 (8:1)
Discoloration
Phase separation
Transparency
-
-
-
-
-
-
-
-
-
+
-
+
+
-
+
+
-
+
F1 (7:1)
Discoloration
Phase separation
Transparency
-
-
-
-
-
-
-
-
-
+
-
+
+
-
+
+
-
+
III
F1 (9:1)
Discoloration
Phase separation
Transparency
-
-
-
-
-
-
-
-
-
+
-
+
+
-
+
+
-
+
F1 (8:1)
Discoloration
-
-
-
+
+
+
144
Note :
(-) : No change
(+) : There is a change
In addition, a freeze thaw stability test was also carried out on pH
Figure 1. Freeze-thaw stability on pH results
4. Discussion
Characterization
4.1 Organoleptic test
The resulting astaxanthin nanoemulsion is
a clear and transparent single-phase liquid
with a light orange color and a special odor.
This shows that the nanoemulsion
formulation process is running well.
4.2 Ph measurement
The results of statistical analysis showed
that in the normality test, the data
distribution was not normal in the smix 9:1
formula for the second batch and smix 7:1
for the third batch with a significance value
of 0.000 < 0.05, in addition to the
homogeneity test, a significance value of
0.001 < 0.05 so that the data is also said to
be inhomogeneous. Therefore, the
statistical test used is the Kruskal-Wallis
test, where the results of the Asymp test are
obtained. Sign 0.010 < 0.05 so it can be
concluded that there is a significant
difference in pH in each formula. This
indicates that there is a significantly
different effect between increasing the
concentration of smix on the pH value of
the preparation.
4.3 Particle Size Test and
Polydispersion Index
Based on the results of particle size
measurements, the astaxanthin
nanoemulsion formula with a ratio of 9:1
smix; 8:1 and 7:1 are in the range of particle
sizes required for nanoemulsion
preparations, where astaxanthin
nanoemulsion with 9:1 smix has the
smallest particle size, this is due to the
composition in smix where the amount of
polysorbate 80 surfactant used is more than
Phase separation
Transparency
-
-
-
-
-
-
-
+
-
+
-
+
F1 (7:1)
Discoloration
Phase separation
Transparency
-
-
-
-
-
-
-
-
-
+
-
+
+
-
+
+
-
+
145
the two. other formulas so as to further
lower the surface tension, stabilize the new
surface during the homogenization process
and produce smaller particles [9]. Particle
size measurement is a very important factor
to determine the stability of a nanoemulsion
preparation. The stability of nanoemulsions
depends on the size of the particles in the
dispersed phase. The smaller the particle
size, the smaller the rate of incorporation so
that the nanoemulsion is not easily
creamed. In addition, the small particle size
results in good optical clarity and can be
stored longer, is not easily damaged, does
not change easily and is easily absorbed by
the body [12].
The polydispersity index test was carried
out to describe the level of uniformity of
particle size in a nanoemulsion, the value of
the polydispersity index is important to
know because it is related to the uniformity
of the size of the nanoemulsion, a small
polydispersity index value indicates better
size uniformity, this test was carried out at
the Print-G Laboratory of the University
Padjadjaran using the Particle Size
Analyzer Horiba S Z-100 measured at a
temperature of 25oC, the results of the
polydispersity index test measurements can
be seen in Table 3. Based on the
measurement results of the three
astaxanthin nanoemulsion formulas with
smix 9:1; 8:1 and 7:1 have polydispersity
indexes of 0.435, respectively; 0.475 and
0.541. These results according to [13] fall
into the range of 0.3-0.7 where the
polydispersity index value of a
nanoemulsion from 0.3-0.7 is
polydispersity, which indicates that the
particle size is uniform but has different
shapes and distributions. the particles are
wide.
4.4 Zeta Potential Test
The results of the zeta potential
measurement on the astaxanthin smix 9:1
nanoemulsion sample showed a zeta
potential value of -21.4 mV. The results of
the measurement of the zeta value which is
negative (-) indicates that the majority of
the surface charge of the droplet is anionic,
resulting in a decrease in the surface charge
of the droplet to be negative [14]. The zeta
potential value is -21,4 mV which is greater
than -30mV so it is possible that the
preparation has a repulsive force that is not
good enough to achieve colloid physical
stability as stated by [15] that a good zeta
potential value to produce a stable
preparation is less than -30 mV or more
than +30 mV because it can avoid particle
aggregation with repulsive forces
exceeding the attractive forces of the
dispersed particles so that they can stabilize
themselves, on the contrary if the potential
zeta value is outside What is required is the
possibility of the preparation to produce
aggregation and flocculation of very large
particles because there is a van der Waals
force that produces physical instability in
the preparation. The low value of zeta
potential can be caused by the surfactant
and the amount of surfactant used, in this
study non-ionic surfactants were used
which have a constant hydrophobic group
and tend to reduce the potential zeta value
[16].
4.5 Centrifugation Test
The centrifugation test was carried out to
determine the stability of the preparation
against the force of gravity and as an
illustration of the stability of the
preparation against shocks during
distribution with the test parameters being
the occurrence of phase separation,
precipitation, creaming or cracking [17,
146
18]. Based on the test results that all
formulas did not experience changes in
either phase separation, precipitation,
creaming or cracking after the test which
indicated a picture of the stability of the
preparation against gravity. This shows that
all formulas are stable and there is no phase
separation
4.6 Adsorption Efficiency Test
Based on Table 4, it was found that the
average adsorption efficiency of the
astaxanthin nanoemulsion was more than
90% in each formula made. This high
adsorption efficiency can be attributed to
the high solubility of astaxanthin in
sunflower oil as the oil phase, which
according to [6], sunflower oil can dissolve
astaxanthin well up to a concentration of
100 mg/L. The results showed that
nanoemulsion with smix 9:1 had greater
adsorption efficiency than nanoemulsion
with smix 8:1 and 7:1 so that it could also
be related to the amount of surfactant used
which was associated with increasing
surfactant concentration causing lower
surface tension between droplets so that
prevent incorporation which can thus
increase the solubility of astaxanthin in
nanoemulsions[19] .
The results of statistical analysis showed
that in the normality test, the data
distribution was not normal in the entire
formula with a significance value of 0.000
< 0.05 so that the data distribution was
concluded to be abnormal, in addition to the
homogeneity test, the significance value
was 0.106 > 0.05 so that the data was said
to be homogeneous, Therefore, the
statistical test used is the Kruskal-Wallis
test because the data are not normally
distributed even though it is homogeneous,
where based on the results of the Kruskal-
Wallis test, the Asymp test results are
obtained. Sign 0.005 < 0.05 so it can be
concluded that there is a difference in the
value of the adsorption efficiency in each
formula. This indicates that there is a
significantly different effect between
increasing the concentration of smix on the
adsorption efficiency.
4.7 Freeze-thaw Stability Test
Results in table 5 state that all formulas
have changed either in color change or
transparency, the stability of the resulting
astaxanthin nanoemulsion can be predicted
beforehand by looking at the potential zeta
value, where the 9:1 formula has a particle
size and the best polydispersity index but
has a zeta potential value that is not good
enough, causing the preparation to have a
repulsion force that is not good enough to
achieve colloid physical stability, as
evidenced by the test results that the
preparation is unstable after the third cycle
is characterized by a change in color in the
preparation caused by the presence of an
oxidation reaction during the test, as well as
the other formulations. In addition to the
color change, phase separation and
transparency, other parameters that can be
seen are the pH of each formula, and it can
be concluded that there is a change in the
pH of each formula from the 1st cycle to the
6th cycle. Changes in pH during the test
were caused by hydrolysis after the
preparation was stored at 40oC, but when
viewed based on the requirements for the
pH range during the test, it was still
acceptable according to SNI 16-4399-1996
which stated that the ideal pH for topical
preparations was between pH 4.5- 8.0.
The results of statistical analysis
from figure 1 showed that in the normality
test, the data distribution was not normal in
the 3rd batch smix 9:1 formula, smix 8:1
batch 1, smix 7:1 batch 1, smix 7:1 batch 3
147
before the freeze-thaw test test and there
was an abnormal distribution of data in the
2nd batch of smix 9:1 formula and smix 7:1
batch 3 on the data after the freeze-thaw test
with a significance value of 0.000 <0.05 so
that the data distribution was concluded
abnormal. The statistical test used was the
Wiloxon test to compare the effect of the
freeze-thaw test on the initial pH (cycle 0)
and final pH of the preparation (cycle 6) in
each formula. The Wiloxon test was used
because the data distribution was not
normal, the Wiloxon test results showed the
asymp value. Sig of 0.000 <0.05 so it can
be concluded that there is a significant
difference in the pH value of each formula
between before and after the freeze-thaw
test
5. Conclusions
Nanoemulsion preparation with the smix
9:1 formula is the most optimum formula,
which is to produce preparations with quite
good characteristics organoleptically and
give a light orange color appearance, clear,
distinctive smell with a pH value that met
the SNI standard 16-164399-1996 with pH
values ranging from 7.13 to 7.15 and based
on the centrifugation test gave stable results
and had particle size, polydispersity index
and zeta potential values, respectively, 22.9
± 9.4 nm, 0.435 and -21,4 mV and the value
of entrapment efficiency ranges from
93.87% to 94.32%. However, the
thermodynamic stability is not good
enough. This is indicated by the instability
of the preparation during the freeze-thaw
test with the results of changes in color,
transparency and changes in pH.
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