Formulation Optimization of Myristica fragrans (Nutmeg) Seed Oil Nanoemulgel

Ika Yuni Astuti, Retno Nur Utami

Department of Pharmaceutical Technology, Faculty of Pharmacy, Universitas

Muhammadiyah Purwokerto, Purwokerto, Indonesia

Corresponding author: ikayuniastuti@ump.ac.id

Introduction: Nutmeg seed oil with a concentration of 10% showed high activity as an
antimicrobial against Staphylococcus aureus cause of acne. The research on nutmeg seed
oil nanoemulgel has never been reported, whereas this dosage form can provide high
penetration of active substances into the skin. Objectives: the objectives of this research
were to acquire the optimum composition of nutmeg nanoemulgel oil and know the
validity of the model depicting the effect of composition on the responses. Materials and
Methods: The formulation optimization used a design expert program with the general
factorial design method. The factors were carbopol 940 and tween 80: PEG 400 (3:2). The
responses, namely the viscosity and spreadability. Results: The optimum composition of
nutmeg seed oil nanoemulsion consists of 10% nutmeg seed oil, 0.5% carbopol 940, 20%
tween 80: PEG 400 (3:2). Both of the responses followed the main effect model. The
predicted viscosity and spreadability of the optimum formulation were 5.28 cm and
32,477.8 cps respectively. These values were not significantly different from the
experimental response values (p>0.05), so the models were valid. Conclusion: By using
general factorial design, the optimum composition of nutmeg seed oil nanoemulgel was
successfully obtained with a valid equation model.

Keywords: nutmeg seed oil, nanoemulgel, general factorial design

INTRODUCTION
Acne is caused by four main factors, namely overactive sebaceous glands, blocked skin pores
due to hyperkeratosis, the activity of commensal skin bacteria (Staphylococcus aureus and
Propionibacterium acnes) and inflammation. Fixed oil of nutmeg seed contains trimyristin,
together with myristic acid, myristicin, and elemicin, which have anti-oxidant, anti-
convulsion, analgesic, anti-inflammatory, antidiabetic and anti-fungal activity. It also has
antibacterial activity on Escherichia Coli, S. Aureus dan P. acnes (Panggabean, 2016;
Agustinisari, 2014; Huang, 2010).
To apply nutmeg seed oil to the skin, it is necessary to make a topical preparation.
Nanoemulsion consists of an oil phase that is nanodispersed in the aqueous phase. The oil
phase has function as an emollient or occlusive that prevents evaporation from the skin. The
nanodroplets allow the diffusion barriers to be reduced, while gel can provide a cool feeling
in the skin with a water content high enough to be comfortable to use. Therefore, the presence
of an emulsion system in the form of a gel preparation (referred to as nanoemulgel) will
provide high penetration in the skin and comfort in use (Salih, et al, 2017).
The quality of nanoemulgel is influenced by the composition of the gelling agent and
surfactant. The composition must be optimized effectively. It can be reached with a design
expert software. A general factorial design is a design for 1 to 12 factors where each factor
may have a different number of levels, so it provides a more flexible model than simplex
lattice design. Thus, this research aims to optimize the nanoemulgel formulation of nutmeg
seed oil using general factorial design.

MATERIALS AND METHODS

Materials
Materials used were nutmeg seed oil from PT. Nusaroma Essential Indonesia. Carbopol 940,
Tween 80, PEG 400, TEA, and aquadest were the technical grade (Merck).
Methods
Experimental design
The experimental design was made using and general factorial design method of Design
Expert 7.0.0 software. The two independent variables (factors) used in this design were
Carbopol 940 (0.25-0.75%) and a mixture of Tween 80:PEG 400 (6:5) in the range of 20-
40%. Meanwhile, the two dependent variables were viscosity and dispersability.
Nanoemulgel Preparation
The formulas produced by the experimental design became the basis for preparing the
emulgel. The general formula of the emulgel is shown in table 1. The mixture of oil and
aqueous phase was homogenized using High Shear Homogenizer (Ultra Turax IKA T25)
with 14,000 rpm in 3 minutes. Then, it was added to the Carbopol 940 which had been added
by TEA so that a nanoemulgel product was obtained.

Formula Optimization
The viscosity tested using viscometer (Brookfield DV2T) with spindle no. 4 and 10 rpm in 30
seconds. Dispersability test was done using circle glass. The optimization parameter for each
response was maximum viscosity and maximum for dispersability.
Cycling Test
This test was done through freeze-thaw cycle method by storing nanoemulgel in low
temperature (4° C) and room temperature, 27° C in 24 hours in 6 cycles with its viscosity and
dispersability being observed.
Data Analysis
The effect of the variables on the responses was analyzed using the design expert. The
optimum formulation was determined based on the desirability function and made
experimentally. The viscosity and dispersability of the optimum formulation which was
obtained experimentally were analyzed using a single sample T-Test method by SPSS 17.0.0.
Then, the cycling test result of the optimum formulation was tested statistically using SPSS
17.0.0 with the One Way ANOVA method with 95 % confidence level. If there are any
significant differences, the Least Significance Difference test (LSD) is conducted using the
Tukey test method. If it does not meet the criteria for the ANOVA test, the result will be
analyzed using the Kruskal-Walls test.
RESULT AND DISCUSSION
Experimental design
With the help of the Design Expert 7.0.0 formula, 9 formulas were produced. The
experimental design can be seen in table 2.
Nutmeg seed oil nanoemulgel preparation
The emulsions resulting from the oil, surfactant, cosurfactant, and aquadest in table 1 showed
a mean of droplet size of 41.4±0.1 nm, so it was classified as nanoemulsion. The
nanoemulsion was added with a gel base consist of a gelling agent (Carbopol 940) and the
neutralizing agent (TEA) to form a nanoemulgel. Each nanoemulgel that had been prepared
was tested for its viscosity and spreadability. The result is shown in table 2.
Formula optimization
The optimization parameters were the viscosity and dispersability of the nanoemulgel. The
viscosity result is shown in table 2. From table 2, it can be seen that the viscosity of
nanoemulgel varies from 2,120 to 57,520 cps. This value provides a fairly watery to viscous
consistency.
The dispersability in the range of 3-5 cm is ideal for topical preparation because the product
will be easy to be applied without spilling too easily. Based on the result at table 2, the
dispersibility was 3.74 to 8.15 cm.
To find out the right model in describing the effect of the factors on the responses, the design
expert carried out the analysis of variance (ANOVA) on various models. The ANOVA results
showed that the effect of factors on viscosity and dispersability was most appropriate to be
explained with the main effect model. The ANOVA calculation of viscosity and spreadability
is shown in table 3.
At table 3, the P-values <0.05, which means this model was significant in predicting viscosity
and spradability response. From the fit statistic summary, the R-squared obtained were
0.9895 and 0.9588 for viscosity and dispersability respectively. The R-squared value
indicated that the response result was following the model because it was not far from 1. The
Predicted R-squared value showed not so differents result from the Adjusted R-squared, i.e
0.09468 vs 0.9790 for viscosity and 0.7915 vs 0.9176 for dispersability. The Adeq Precision
Value for the viscosity response was 23.969 and 12.941 for dispersability, which were greater
than 4. That means an adequate signal to noise ratio and the model can be used as the design
space guide (Mulyawanti et al, 2016).
The main effect model for viscosity was described by equation 1.
Yviscosity = -28,175.56 A1 + 4,471.11 A2 – 3,955.56 B1 + 11.11 B2 (Eq. 1)
Where : Y = response; [A1] = Carbopol 940 at low level ; [A2] = Carbopol 940 at high level ; [B1]=Tween
80:PEG 400 (6:5) at low level ; [B2]=Tween 80:PEG 400 (6:5) at high level .
The negative sign on the coefficient A1 indicated the carbopol 940 at the low level decrease
the viscosity. Otherwise, the positive sign on the coefficient A2 indicated the effect of
increasing viscosity of carbopol 940 at a high level. Once dispersed in water, the carbopol
940 molecules begin to hydrate and partially uncoil and swelling, cause an increase in
viscosity. The degree of swelling increases with increasing numbers of carbopol 940
molecules and as the pH approaches neutral. The concentration of TEA as a neutralizer was
in excess because normally neutralization is achieved at a TEA: carbopol ratio of 1.5-1. The
visual description of the effect of Carbopol 940 at the low, medium and high levels of Tween
80:PEG 400 (6:5) on viscosity can also be seen in Figure 1.
 Fig. 1. The Influence of Carbopol 940 on viscosity at the low, medium and high levels of
Tween 80:PEG 400

The Eq.1 also explained that Tween 80:PEG 400 (6:5) at a low level decreased the viscosity
and vice versa. The lack of surfactant-cosurfactant concentration cause the lack of
stabilization of nanoemulgel since the amount of surfactant-cosu rfactant was insufficient to
envelop the oil, which could lead to breaking/cracking and decrease the viscosity. The
graphic of the relationship between Tween 80:PEG 400 (6:5) and viscosity of low, medium
and high level of Carbopol 940 is showed in figure 2.

Fig. 2. The Influence of Tween 80:PEG 400 on viscosity at the low, medium and high
level of Carbopol 940

Dispersability is shown in table 2. The main effect model resulted from the dispersability
response was explained by equation 2.
Ydispersability = +1,87 A1 – 0,64 A2 + 0,56 B1 + 0,06 B2 (Eq.2)
Where: [A1] = Carbopol 940 at low level ; [A2] = Carbopol 940 at high level ; [B1] = Tween 80:PEG
400 (6:5) at low level ; [B2] = Tween 80:PEG 400 (6:5) at high level .
From the Eq. 2, it can be seen that the Carbopol 940 at the low level increased the
spreadability and vice versa. This is because the small amount of Carbopol 940 causes the
degree of swelling of the gel to be low so that the viscosity is low. The spreadability is
opposite with viscosity, so the lower viscosity the higher spreadability. The relationship
between spreadability and Carbopol 940 at the low, medium and high levels of Tween
80:PEG 400 (6:5) is shown in figure 3.

Fig. 3. The Influence of Carbopol 940 on spreadability at the low, medium and high levels of
Tween 80:PEG 400 (6:5)

Figure 4. The Influence of Carbopol 940 on Dispersability at low-level Tween 80:PEG 400 (6:5) (20%)
The coefficient B1 and B2 in equation 2 were each positive, but the value of B1 was greater
than B2. This showed the effect of increasing the spreadability caused by Tween 80: PEG
400 (6: 5) at the low level was greater than that caused by Tween 80: PEG 400 (6: 5) at the
high level.
The presence of surfactant and cosurfactant causes a decrease in the contact angle between
nanoemulgel and the glass surface used for spreadability test so that the spread of
nanoemulgel becomes wider. At low surfactant-cosurfactant levels, increased spreadability
by surfactant-cosurfactant is supported by low viscosity so that the spread of nanoemulgel
becomes even wider. However, at high levels, the increase in spreadability by surfactants and
cosurfactants is held back by the high viscosity of nanoemulgel, so the effect of increasing
spreadability is not as high as at the low level. The relationship between spreadability and
Tween 80:PEG 400 (6:5) at the low, medium and high levels of Carbopol 940 is showed in
figure 4.
 Fig. 4. The Influence of Tween 80:PEG 400 (6:5) on spreadability at the low, medium and
high levels of Carbopol 940

Optimization of the formula that compromises the best viscosity and spreadability was shown
by the value of desirability. The closer to 1, the greater the chance of the optimum formula to
achieve the target response value predicted by the software. An optimum formulation
solution was obtained with a desirability value of 1. The optimum formulation composition
consists of 10% of nutmeg seed oil, 0.5% of Carbopol 940 and 10.9 % of Tween 80:PEG 400
(6:5).
Optimum Formula Verification
The optimum formula solution from the software needs to be proven by testing the optimum
formula responses experimentally. The optimum formulation response result was tested using
one-sample T-test with a confidence value of 95 %. If the responses generated from the
experiment do not differ significantly from the response predicted by the software, then the
model used (main effect) can be said to be valid. The comparison of the response of
experimental results with the prediction of software can be seen in table 4.
Based on table 4, the optimum formula from the experiment was not significantly different
from the optimum formula from Design Expert 7.0.0 software. Hence, the main effect model
was valid to optimize the formulation of nutmeg seed oil nanoemulgel in this study.
Cycling Test Result of Optimum Formulation
The cycling test result can be seen in table 5. The physical stability of the optimum formula
was evaluated from organoleptic, pH, viscosity, and dispersability test results. White colour
was obtained from the optimum composition formula of Carbopol 940 and Tween 80 and
PEG 400, and there was no colour change during 6 cycles storage. The nutmeg seed oil
aroma during the 6 cycles storage was getting stronger. It was because of the diffusion
process of a substance penetrating the oil layer as the permeable membrane or because of the
depletion of the oil layer that causes nothing’s inhibiting the active substance of the product
(Ainurrofiq, 2006).
The pH of each cycle at table 5 met the parameter for skin, i.e 4.5-6.5. The pH data normality
evaluated using Shapiro-Wilk showed that the data was normally distributed because the
significance value of its cycle was >0.05 in each cycle. Furthermore, from the one way
ANOVA analysis, there was a significant increase in pH during the 6 cycles observation.
The result of the viscosity test can be seen in table 5. The viscosity of optimum formulation
in 6 cycles still in the range of 20.000-40.000 which is the ideal viscosity for semisolid
preparation (Garg et al., 2002). There was a significant decrease in viscosity during storage
(p<0.05). This decrease in viscosity is common in gels during storage, which is caused by the
occurrence of syneresis, the phenomenon of the release of solvent molecules from the
polymer chain network. The longer the storage, the more free solvent molecules, so the
viscosity decreases.
The dispersability of the optimum formula increased. This was related to a decrease in
viscosity during storage. However, the increase was not significantly different (p>0.05).
Conclusion
Optimum formulation consist of Carbopol 940 of 0.5 %, Tween 80 of 10.9 %, PEG 400 of
9.1 %, TEA of 2 %, nutmeg oil of 10 % and aquadest 67.5%. The General Factorial Design
method can produce a valid model by seeing the significance value of the optimum formula
(>0.05) in the T-Test.
.
References
Agustinisari, I., Purwani, E.Y., Harimurti, N., Yuliani, S., 2014. Antimicrobial Activity of
Nutmeg Seed Oil Nanoemulsion (in Indonesian). J Pascapanen, 1 1.
Garg, A., Aggarwal, D., Garg, S., and Singla, A. 2002. Spreading of Semisolid Formulation.
Pharmaceutical Technology, pp. 84-102.
Huang, C., Lee, C., Chen, L., Wang, C., 2010. Antimicrobial herbs against Staphylococcus
aureus and Propionibacterium acnes. Planta Medica:76, 500.
Mulyawanti, I., Budijanto, S., Yasni, S. 2016. Optimization of Formula and Microscopic
Structure of Gluten-Free Paste Made from Pure Purple Sweet Potatoes and Green
Bean Flour (in Indonesian). Jurnal Agritech 36,15.
https://doi.org./10.22146/agritech.10678
Panggabean, A.K., 2016. Utilization of Nutmeg Extract (Myristica fragrans) as Antimicrobial
in Sausage Tempe Products (in Indonesian). Thesis. Universitas Medan : Sumatera
Utara.
Salih, Zainab, T., Fatima, Al-Gawhari., Nawal, A. Rajab. 2017. Preparation, Release,
Rheology And Stability Of Piroxicam Nanoemulgel. International Journal of
Applied Pharmaceutics http://dx.doi.org/10.22159/ijap.2018v10i1.20234
Tables:
Table 1. General formula of the nutmeg seed oil emulgel
Ingredient Percentage (%)
Nutmeg seed oil 10
Carbopol 940 0.25-0.75
Tween 80:PEG 400( (6:5) 20-40
Triethanolamine (TEA) 2
Aquadest Ad 100

Table 2. The experimental design and the response values

Factor 2 Response
Factor 1
Std Run B: Tween 80:PEG 400 Viscosity Dispersability
A : Carbopol (%)
(6:5) (%) (cps) (cm)
6 1 0.75 30 56,900 4.174
1 2 0.25 20 2,120 8.15
3 3 0.75 20 52,580 4.47
7 4 0.25 40 5,640 6.03
2 5 0.5 20 29,320 5.15
8 6 0.5 40 44,560 4.44
5 7 0.5 30 35,420 4.59
4 8 0.25 30 3,600 7.52
9 9 0.75 40 57,520 3.74

Table 3. Statistics of the Main Effect Model of the Response using Design-Expert

ANOVA Fit statistic summary

Response P-Value Significance Adj R- Pred R- Adeq

R-Squared
Squared Squared Precision

Viscosity 0.0003 Significant 0.9895 0.9790 0.9468 23.969

Dispersability 0.0049 Significant 0.9588 0.9176 0.7915 12.941

Table 4. Optimum formulation Responses from experiment and Design Expert software
Response Experimental Data Design Expert Data Significance
Viscosity 32,640±572 32,478 0.672
Dispersability 5.13±0.18 5.28 0.272

Table 5. Cycling test result

 Mean of Response in Cycle
Response
0 1 2 3 4 5 6
pH 5.49±0.02 5.76±0.14 5.84±0.11 5.35±0.41 6.04±0.12 6.03±0.08 6.03±0.10

Viscosity 35,420±434 34,380±180 33,700±489 32,640±572 30,000±1352 25,622±1745 23,500±544

Dispersability 5.00±0.10 5.18±0.09 5.47±0.38 5.33±0.21 5.23±0.15 5.21±0.35 5.33±0.25