Formulation and Characterization of SNEDDS of Dayak Onion Extract with Comparative Variation of Surfactants, Co-Surfactants, and Palm Oil

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Introduction
The development of formulation technology has attracted many researchers as an effort to produce new drugs with ideal properties by considering molecular ion balance, hydrophiliclipophilic equilibrium, biopharmaceutical processes, metabolism and biodegradation, drugreceptor affinity, physiological considerations, and the biocompatibility of the system as the main factors influencing the development of formulation technology. Commonly done in research is about nanotechnology (Martien et al., 2012). Nanoemulsion is a thermodynamically stable preparation, transparent dispersion of oil and water stabilized by the interfacial film of surfactant and co-surfactant molecules and has a droplet size of less than 100 nm (Shafiq-un-nabi et al., 2007).

PROCEEDING 1 st International Pharmacy Ulul Albab Conference and Seminar (PLANAR)
Self-Nanoemulsifiying Drug Delivery System (SNEEDS) is one of the developments of nanoemulsion delivery systems that can penetrate cell tissue by considering the physicochemical properties of the active ingredients and additives in the formulation so that they affect the resulting nanoemulsion preparations, such as droplet size, size distribution, and emulsification time. (Date et al., 2010). The components of SNEEDS are influenced by the oil phase, surfactant, and co-surfactant (Huda et al., 2016).
The oil component in this preparation is the primary carrier of the active substance. It is a determinant of the droplet size of the emulsion formed. The oil used is palm oil (Elaeisguineensis Jacq). Palm oil is a food oil with dominant long-chain fatty acids, which are essential to reduce unsaturation, prevent oxidative degradation, and affect drug solubility in water (Marpaung, 2014).
The next component, namely surfactants, reduces the size of droplets or emulsion droplets and stabilizes the active substance at the absorption site. There is no deposition in the gastrointestinal tract. The surfactants used were Transcutol, Span 20, Tween 80, and Tween 20. Co-surfactants function to assist surfactants in finding the surface tension of water and oil, increasing dissolution, and improving active substances' absorption (Marpaung, 2014). The cosurfactant used is PEG with stable properties, easily soluble in warm water, non-toxic.
The number of comparisons between oil, surfactant, and co-surfactant in this study used three ratios of variation between the three constituent components, namely 1:8:1, 1:7:2, 2:7:1. It is also influenced by the HLB value to get the most stable SNEDDS. SNEDDS with HLB between 11-15 is a stable vulnerability in the manufacture of SNEDDS systems (Winartiet al., 2016).
The natural extract preparations that have been developed produce therapeutic effectiveness with large enough doses, low solubility, and less than optimal oral bioavailability. SNEDDS is used to increase the absorption and bioavailability of drugs in the body, especially for low solubility in water (Nasr et al., 2016). The utilization of natural materials in this research uses dayak onion extract with naphthoquinone secondary metabolite compounds that have bioactivity as anticancer. The purpose of this research is expected to be an innovation for the development of drug delivery systems with extracts of natural ingredients using various concentrations of surfactants-co-surfactants with the oil used to improve the bioavailability of active substances in the body.

SNEDDS Preparation
The preparation of SNEDDS, namely hydrophilic and lipophilic surfactants, was stirred at 300 rpm for 10 minutes. Co-surfactant PEG 400 was added and stirred for 10 minutes, finally added oil little by little and stirred for 10 minutes. SNEDDS were stored for 24 hours and observed for phase separation. The most stable preparation with the lowest surfactant composition, the highest oil component, and the highest HLB was chosen as the SNEDDS formula for dayak onion extract (Winarti et al., 2016).

Preparation of SNEDDS Bawang Dayak Extract
The design of the optimized SNEDDS formula consisting of oil, surfactant, and cosurfactant added 50 mg of dayak onion extract, then mixed until homogeneous with a magnetic stirrer LAB MS-H (Heidolph, Germany) for 10 minutes and stored at 25 o C for further characterization. SNEDDS Characteristic Test of dayak onion extract.

Transmittan Percent Test
Measurement of percent (%) transmittance of SNEDDS was carried out using a UV-Vis 1800 spectrophotometer (Shimadzu, Germany). Measurement by taking 100 L of each formula then diluted with distilled water to 100 mL. The mixture was homogenized with a magnetic stirrer at 200 rpm. SNEDDS was measured at a wavelength of 650 nm to determine the percent transmittance (Nasr et al., 2016).

Emulsification Time Test
The SNEDDS formula was evaluated visually to determine the emulsification time using a magnetic stirrer. A total of 100 L of SNEDDS was dropped into a beaker containing 100 mL of simulated gastric fluid (SGF) without enzymes pH 1.2 ± 0.05 and simulated intestinal fluid (SIF) at pH 6.8 ± 0.05 without enzyme (Ren et al., 2009), temperature 37 o C with stirring 200 rpm. The time for emulsification was determined as the SNEDDS time to form a homogeneous mixture after mixing (Basaliusetal., 2010).

Particle Size Measurement
SNEDDSparticles were measured using the Microtrac Nanotrac wave II Particle Size Analyzer (PSA). Take 100 L of SNEDDS, then put it into a cuvette. The cuvette used must be free of foam and grease. The cuvette that has been filled with the sample is inserted into the sample holder. The tool is turned on, and the particle size menu is selected.

pH measurement
The pH measurement of each formula was carried out using a calibrated digital pH meter pH-700. Take 5 mL of SNEDDS, the electrode is inserted into the SNEDDS, and the number indicated by the pH meter is recorded (Annisaet al., 2016).

Viscosity Measurement
Viscosity measurements were carried out to see the viscosity of SNEDDS produced due to the influence of the addition of other ingredients such as surfactants and the power of manufacturing techniques. Viscosity measure meant using a Brookfield cone and plate viscosimeter. A stationary plate forms the bottom of the movable sample cup and is filled with 0.5 mL-2.0 mL SNEDDS. The system is accurate within ± 1.0% of the fulls clearance. Reproducibility ± 0.2%. The tool works in a temperature range of 0-100oC (Zhao et al., 2015).

Dilution with Various Media
The stability of the dayak onion extract in nanoemulsion after dilution with water, SGF, and SIF was examined by monitoring the concentration of whole dayak onion extract during incubation at room temperature. SNEDDS were added to 100 mL of distilled water, artificial intestinal fluid (SIF), and artificial gastric fluid (SGF). The mixture was then homogenized with a vortex for 2 minutes (Ren et al., 2009) (Astutiet al., 2018). 7. ThermodynamicStability

Heating-cooling Cycle
The test was carried out by taking 2 mL of SNEDDS diluted with 10 ml of aquadest and stored at 4°C and 45°C for 48 hours. The temperature was exchanged for each preparation. Physical damage to the SNEDDS preparation was observed (Winartiet al., 2016).

Freeze-Thaw Cycle
The test was carried out by taking 2mL of SNEDDS, diluting with 10ml of aquadest, and stored at -20°C and 25°C for 48 hours. The temperature was exchanged for each preparation. Physical damage to the SNEDDS preparation was observed (Winartiet al., 2016).

Centrifugation
Thermodynamic testing of 10 mL SNEDDS was carried out using a Hettich Rotofix 32 centrifuge at 3500 rpm for 30 minutes. Then the SNEDDS were stored at -20ºC and the others at 25ºC. Stability observations were carried out after 24 hours of storage (Winartiet al., 2016).

Result and Discussion
Optimization of the composition of the SNEDDS material was carried out by mixing the ratio of surfactants (Tween 80, Tween 20, Span 20, and Transcutol) and co-surfactants (Transcutol) with palm oil as a carrier oil with an HLB range of 11-15 which is a stable HLB in SNEDDS (Syukri et al., 2019). Palm oil is an extended chain oil group that has the advantage of increasing drug transport through lymphatics, thereby reducing first-pass metabolism, but its ability to emulsify compared to medium-chain triglycerides, diglycerides, or fatty acid esters (Sapraet al., 2012). The use of HLB ranges from 11-15 because this value shows O/W droplets, and the higher the HLB value, the more hydrophilic nanoemulsion preparations are obtained. Furthermore, the ratios of 1:8:1, 1:7:2, and 2:7:1 to determine the stable formulation of SNEDDS. The comparison between the amount of surfactant by mixing hydrophilic and hydrophobic surfactants to form nanoemulsions with better characteristics and affect the surface tension of the preparation (Debnath et al., 2011).
The composition of the ingredients was mixed until homogeneous and not observed for 24 hours to determine the formula for the HLB value and a stable ratio indicated by the absence of phase separation. Preparations with a clear physical appearance and no phase separation will be selected next to be tested for physical characteristics of SNEDDS preparations. The resulting formula is 60 formulas using three ratio ratios of SNEDDS components. A formula with a clear appearance and no phase separation indicated a stable preparation in the formation of SNEDDS.  F32, F33, F34). Of the 15 formulas, the ratios are 1:8:1 and 1:7:2, which can produce stable compositions when diluted, with good droplet sizes and meet the stability test requirements (Syukriet al., 2019).
The 15 formulas were then tested for %transmittance to determine the level of clarity of the preparation using UV-VIS Instruments at a wavelength of 650nm. F13 and F34 were obtained at HLB 13 and 14, respectively, with a ratio of 1:8:1 and 1:7:2, which had %transmittance values >90% (Wirnartiet al., 2018). Furthermore, both formulas were tested for emulsification time with dilution in SIF and SGF liquids. Both formed homogeneity with a value of <2 minutes (Winartiet al., 2016). Then proceed to the particle size test on the preparation by diluting SGF and SIF. The range of values obtained is between 10-200 nm (Syukriet al., 2016).
The preparation was carried out by mixing the composition of the SNEDDS material with a predetermined ratio in a stable formula, then homogenized and observed for 24 hours at room temperature. In this case, the formula that is made is a formula that is stable at the time of optimization of SNEDDS without dayak onion extract, namely F13 and F34.
The selection of this formula will then be tested for the physical characteristics of the SNEDDS preparation by adding dayak onion extract to improve the bioavailability of the active substance in the body.

Transmittance Percent Test
The data from the percent transmittance test are in Table 2. This result shows a value that is far from the range, namely >90% (Sahumena, 2014). This result contradicts the research, which stated that the emulsion with transparent and clear preparation conditions had a transmittance value close to aquadest. It could be concluded that the emulsion droplet size value was 10-200 nm (Syukriet al., 2016). SNEDDS, which has a low transmittance value, shows a larger particle size. A macroemulsion is formed to look cloudy because its solubility with water is very low (Syukriet al., 2018).
The lack of clarity of SNEDDS, which can be indicated, is that the oil globules are not dispersed with the active ingredients and other components in a homogeneous and nano-sized manner . It can also be indicated that dayak onion extract, which has lipophilic properties, also affects the instability of the oil because of the amount of oil in the preparation increases. Increasing the amount of oil in the preparation can reduce the stability of the preparation if it is not accompanied by an increase in the amount of surfactant, which functions as a decrease in surface tension and can produce smaller globules . Both formulas are still being continued for other characteristic tests.

Particle Size Test
Droplet size characterization was carried out to determine the nanoemulsion droplet size. This particle size affects a larger interfacial surface area for drug absorption. The size of the nanoemulsion has a droplet size of less than 200 nm (Syukri et al., 2019). The droplet size can be known through the appearance of the preparation. The more cloudy the preparation is, the more likely it is to have a large droplet size (Winarti et al., 2016). Table 3 in F13 shows that the particle size value when diluted follows the parameter value range, which is 10-200nm. However, at F34, the particle size value is below the parameter range or smaller than the parameter. The Polydispersity Index (PDI) value is also obtained in Table 4 if the value <1 indicates the uniformity of particle size is well-formed and uniform.
The size of the dispersed phase dramatically affects the appearance of the emulsion to be transparent or cloudy, and this is due to the size of the oil droplets dispersed in water. Suppose light passes through an emulsion system with very small droplet sizes. In that case, the light beam will be transmitted so that the color of the solution looks transparent and the resulting transmittance is more excellent (Sahumena, 2014).

Emulsion Time Test
The data from the emulsification time test in Table 5 aims to determine the SNEDDS preparation formed when peristalsis occurs in the gastrointestinal tract by diluting it with simulated intestinal and gastric fluids. In formulas F13 and F34, homogeneous preparations were included when diluted and stirred for >2 minutes. The best results are shown if the practice shows an emulsion time >2 minutes (Wirnarti et al., 2018).  Table 6 states that the pH test results of SNEDDS preparations can penetrate well at pH 6-9, which is the pH of the intestine (Zhao et al., 2015). The formula with a pH of 6-8 nanoemulsion W/A will produce a large negative charge and prevent droplets from approaching each other and aggregating to form a stable nanoemulsion preparation (Komaiko and McClements, 2015).

Viscosity Test
The purpose of the viscosity test is to determine the level of available viscosity of SNEDDS due to the influence of other materials such as surfactants and preparation techniques. The results of the viscosity test are listed in Table 7. The formulas F13 and F34 showed an increase in the viscosity of the formula with an increase in the proportion of surfactant in the formulation to achieve an optimal formulation ranging from 7.0 ± 0.1 to 42.0 ± 0.2 centipoises (Syukriet al., 2019).

Dilution Test with Various Media
The fluid used is a simulated fluid with pH in the gastrointestinal tract, namely the stomach and intestines and distilled water. The pH values in the intestines ranged from 6-9, and the pH in the stomach was 1.2. The results in Table 8 are stable preparations and provide values according to the pH parameters in each gastrointestinal tract. The physiological environment has a pH range varying from pH 1.2 (pH in the stomach) to 7.4 and greater (pH of blood and intestines) (Syukri et al., 2019). The pH value of the resulting nanoemulsion is safe to use as a drug base because it follows the pH of the small intestine (7-7.24) as the main organ of drug absorption (Jusnita et al., 2019).

Thermodynamic Stability Test
This test was carried out with three cycles with one cycle at a temperature of -20̊ and 25̊ C with a storage time of 48 hours per cycle. The results showed that in the preparations F13 and F34, there was a separation of the clear phase and the cloudy phase on the upper surface of the preparation.
A freeze-thaw test was carried out with three cycles at -20̊ and 25̊ C, with a storage time of 48 hours for each process. The effect of this temperature is to observe the instability of preparations such as cracking, creaming. The results on F13 and F34 preparations were unstable due to physical changes in the practices and cracking in each trial.
Centrifugation test on SNEDDS F13 and F34 preparations to determine the presence of deposits after screening at a certain speed and time, namely at a speed of 3500 rpm for 30 minutes (Syukriet al., 2018). The results that appear after screening are preparations that occur separation of the clear yellowish phase and the dark red phase. The practice was then placed at a temperature of -20̊ C and 25̊ C and allowed to stand for 24 hours. The results were observed and showed that both formulas froze and indicated that the preparation was unstable.
The instability of the preparation is due to the surfactant being unable to reduce the interfacial free energy and providing a mechanical barrier for coalescence to occur, resulting in a less spontaneous thermodynamic dispersion (Pratiwi, 2018).

Conclusion
The SNEDDS formula using the ratio of palm oil, surfactant, and co-surfactant used in this study was able to form SNEDDS, but after the addition of the active ingredient dayak onion extract as the active ingredient showed less stable results on the stability of the physical preparation of SNEDDS with marked cracking and creaming in practice. When testing the characteristics of the SNEDDS preparation.