Self-Nano Emulsifying Drug Delivery System (SNEDDS) of Curcuma mangga Val. Essential Oil and The Stability Study

Email: retno.sudibyo@ugm.ac.id Essential oil of Curcuma mangga Val. has been reported to have cytotoxic effect against cancer cell lines. But this oil is unstable in dispensing so that a self nano-emulsifying drug delivery system (SNEDDS) of the oil was coducted to solve the problem and improve its potency. In the study, optimization, verification, characterization, and stability test of the SNEDDS formula were carried out respectively by simplex lattice design (SLD) on Design Expert ver. 10 software, droplet size and Zeta potential determinations using particle size analyzer (PSA) instrument, as well as heating-cooling and freeze-thaw metods. The best SNEDDS formula resulted was Miglyol : Tween 80 : PEG 400 = 16.034% : 68.380% : 15.586%; with transmittance of 84.47+ 1.05%, droplet size of 15.75 nm, zeta potential of -8.54 mV, polydispersity index (PDI) of 0.188, emulsifying time of 49.67+1.7 seconds in distilled water, 24.33+4.19 seconds in artificial gastric fluid and 21.33+2.87 seconds in artificial intestine fluid. After a freeze-thaw test there was no change on the emulsion’s clarity, color, smell, as well as no separation, which means that the formula was stable thermodynamically. The optimum SNEDDS formula resulted has small particle size, better emulsifying time in artificial gastric and intestine fluids, as well as better thermodynamic stability, which in turn will improve the cytotoxic activity of the Curcuma mangga Val. rhizome oil toward cancer cells.


INTRODUCTION
The essential oil of Curcuma mangga Val. rhizomes has shown a cytotoxic effect toward some cancer cell-lines (Khasanah, 2002, Nurrokhman, 2004, Verlianara, 2004, Rumiyati, et al., 2007, and Astuti, et al., 2014. But the oil is not stable. According to Billia et al. (2014), some essential oils have some disadvantage properties such as easy to evaporate, easily decompose against heat, humidity, light and oxygen. These disadvantage properties could cause problems whenever the oil will be administrated/used as is. An appropriate formula which can protect the oil from degradation in order to improve the stability as well the activity and effectiveness is needed. This study is to find out the best nano-emulsion formula for the essential oil of C. mangga Val. in order to improve its stability and potency. The self nano-emulsifying drug delivery system (SNEDDS) formula which has components of: carrier oil, surfactant and cosurfactant was adopted to construct a nano emulsion of the C. mangga essential oil.

C. mangga Val essential oil.
The essential oil of C. mangga Val. was isolated by water steamed distillation of sliced clean rhizome in 6 hours. The oil resulted was dried by shaking it with anhydrate CaCl2 and filtered it into a clean bottle, which then stored in a freezer until it was used in the study.

SNEDDS formulation and optimization.
SNEDDS formula was prepared using Tween 80 as the surfactant, PEG 400 as cosurfactant, and a carrier oil. Different oils of Miglyol (synthetic oil), 239 virgin coconut oil (VCO) or olive oil were selected as the carrier oil. Best formula was selected from five formulas which were constructed based on the SNEDDS component ratio. The criteria for the selection were least amount of surfactant and best emulsion homogeneity or stability according to visual observation. The best formula obtained was then optimized by Simplex Lattice Design method using Design Expert Ver.10 software.
To find out the optimum formula, two parameters of transmittance and emulsifying time were measured, as well as stability tests of heating-cooling and freeze-thaw tests were conducted on the emulsions. Three different emulsion media of distilled water, artificial gastric fluid (AGF) and artificial intestine fluid (AIF) (Astuti et al., 2018) were used for making the emulsions. To determine the optimum formula the two parameters of the three different nano-emulsions resulted were used as the goal limits.

SNEDDS verification and characterization.
Verification was carried out by measuring the transmittance and emulsifying time of the emulsions. The measurements were then compared to those of the suggested values. The emulsion was also characterized through its organoleptic, pH, % transmittance, droplet size and Zeta potential. The pH was determined using pH meter. The % transmittance was measured by UV Spectrophotometer at 650nm using solution of 100µL SNEDDS diluted in distilled water up to 10mL. The droplet size and Zeta potential were determined triplicate from the SNEDDS dilution using particle size analyzer (PSA).

SNEDDS emulsifying time.
Emulsifying time test of SNEDDS formula was conducted in three different fluids of CO2 free distilled water, artificial gastric fluid (AGF) with pH 1.2 and artificial intestine fluid (AIF) with pH 7. On each fluid, a 0.5mL SNEDDS was dispersed in a 250 ml flask, at 37±2°C on a magnetic stirrer with 50 rpm rotation. The emulsifying time was started as the SNEDDS contact the fluid, and was stopped when nano-emulsion was completely formed (Astuti et al., 2018;Beandrade, 2018;Khan et al., 2015).

RESULT AND DISCUSSION
Five tentative compositions of SNEDDS formulas (Table I.A), while the formula stability using three different oils (Table I.B). Based on formula stability observation (Table I.B.), Miglyol was the best carrier oil. So that Miglyol was selected as the C. mangga oil carrier. Combination 1:2:1 had already resulted isotropic mixture, therefore the SNEDDS formula optimization could be conducted within combinations of 1:2:1 (Table I.B.). The lower and upper limits of Miglyol, Tween 80 and PEG 400 were respectively 15-25, 60-70, and 15-25, which were optimized by Simplex Lattice Design (SLD) method using Design Expert Ver.10 software.
The SLD method resulted in 14 SNEDDS combinations to be prepared for measuring the transmittance and emulsifying time (Table II.A). These measurement values were then uploaded back to the program to find the optimum formula. The formula was then determined statistically using ANOVA upon the transmittance and emulsifying time data, and resulted equation models (Table II.B).
All the responses showed p values of <0.05 (Table II.B.); which means that any proportion change will significantly change the responses. The higher transmittance response value the better the SNEDDS formula. Positive value of the coefficient in the equation means positive synergy of the component in the emulsion in giving responses; while negative value coefficient means the component giving negative synergy (Huang et al, 2004). The increasing transmittance response of the SNEDDS was supported by Tween 80 (B) and PEG 400 (C); while the Miglyol (A) reduced the transmittance response (Table II.B). This is in line with the fact that increasing oil concentration will decrease the transmittance due to its larger droplet size (Wulandari et al, 2016). Different from the transmittance response; the lesser the value of emulsifying time the faster the time for emulsifying, which means the better the SNEDDS formula. Other responses (out of emulsifying response) were due to the component interactions. Interaction between Miglyol and PEG 400 (AC) gave negative values and causing decreasing emulsifying time and faster impact to those three-emulsifying responses (Table II.B). However, the interaction between Tween 80-PEG 400 (BC) gave negative coefficients (faster emulsifying time) only in AGF and AIF media, but not in distilled water.
The relationship between the transmittance and surfactant (Tween 80) -co-surfactant (PEG 400) of the SNEDDS formula is shown as a contour plot (Figure 1.A). The higher of % transmittance of the formula the yellowbrighter was shown in the figure. It was showed that the higher the surfactant and co-surfactant, the higher the transmittance (Figure 1.A).

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Volume 31 Issue 4 (2020) This because the surfactant and co-surfactant reduced the water surface-tension and causing it more flexible and causing the system produced much smaller (nano size) oil droplets; so that the UV beam could easily went through the solution and increased its transmittance.
The relationship between the emulsifying time and surfactant (Tween 80) -co-surfactant (PEG 400) of the formula is shown as contour plots (Figure 1. B, C and D). The faster the formula producing nano-emulsion, the dark-bluish was shown in the figure. The yellow-reddish color expressed the slowest time of emulsifying ( Figure  1. B, C and D). The faster the emulsifying time, the easier the SNEDDS forming oil in water (O/W) nano-emulsion. In this case the same reason was occurred (Figure 1.A).
To find out the optimum SNEDDS formula, transmittance limit of 86.1% and emulsifying time of 60 seconds were assigned to the SLD program as the goal limits for all the three different nanoemulsions. There were 4 formulas suggested by the SLD program (Table 3.A), which one of them would be selected as the optimum SNEDDS formula. Formula 1 with the best desirability value of 0.904 was selected as the optimum SNEDDS formula with composition of Miglyol: Tween 80:PEG 400 = 16.034%:68.380%:15.586% (Table 3.A).

Optimum SNEDDS verification, characterization, and stability
The optimum SNEDDS verification of transmittance and emulsifying time were shown in Table 3.B. It was found that the observation values were much better than the prediction values, either the transmittance or emulsifying time in all fluids. The optimum SNEDDS formula characterizations are shown in Figure 2. The droplet size was 15.75 nm (Figure 2A), which was considered as small size droplet, because the nano-emulsion must have droplet size 20 -200 nm (Aboofazeli, 2010). The droplet size is an important factor in drug absorption and delivery (Senapati et al., 2017). This droplet size is influenced by shear and surfactant effects, as well as the relative viscosity between oil (dispersed phase) and water (continuous phase) (Singh et al., 2017).
The droplet uniformity is measured by polydispersity index (PDI). The PDI value of optimum SNEDDS was 0.188 (Figure 2A),  which means uniform and monodisperse as it was <0.2 (Singh et al., 2017). Zeta potential value showing the charge difference between the medium and the particle carrier, as well as the repulsion strength between the medium and the dispersed particle (Chabib et al., 2016). The higher the zeta potential the stronger the repulsion and the better emulsion stability because it decreases the possibility of the particles to aggregate, flocculate, coalesce and coagulate (Khan et al., 2014, Chabib et al., 2016, Ke et al., 2016. The optimum SNEDDS zeta potential was -8.54 mV which is stable but not in a long time since the value of 8.54 < 30 (no matter of positive or negative values) (Singh et al., 2017).
There was no change on the emulsion's clarity, color, smell, as well as no separation after the heating-cooling and freeze-thaw tests. This showed that the optimum SNEDDS formula was stable thermodynamically.