Optimization and Physicochemical Characterization of a Self-nanoemulsifying Drug Delivery System (SNEDDS) for Citronella Essential Oil ( Cymbopogon nardus )

Abstract

Indonesia produces abundant citronella essential oil, which contains diverse bioactive compounds with promising potential as natural feed additives, yet scientific evidence supporting its application in poultry nutrition remains limited. This study aimed to select the optimal and safest material composition for a self-nanoemulsifying drug delivery system (SNEDDS) of citronella essential oil using Design Expert Ver. 13.0.1.0, a D-optimal mixture, and to characterize the resulting formulation as a prospective poultry feed additive. Component selection, composition ratios, and oil phase proportions were determined using an exploratory approach: homogenization at 250 rpm for 15 minutes, followed by ultrasonication. The prepared SNEDDS formulations were evaluated through comprehensive physicochemical characterization, including organoleptic properties, percentage transmittance, emulsification time, dynamic light scattering (DLS) analysis, stability testing, and morphological observation. The composition ingredients of citronella SNEDDS consisted of virgin coconut oil (VCO) as the carrier oil, Tween 80 as the surfactant, and PEG 400 as the cosurfactant. The optimum formula consisted of 17.86% oil phase (citronella essential oil and VCO with a ratio of 50:50 [%]), 62.791% surfactant, and 19.169% cosurfactant. It demonstrated high transmittance (99.338% ± 0.366%), rapid emulsification (32.107 ± 0.454 s), small particle size (14.4 ± 0.141 nm), low polydispersity index (PDI) (0.088 ± 0.025), moderate zeta potential (–17.00 ± 2.980 mV), and appropriate viscosity (376.00 ± 3.637 mPa•s).

Keywords

feed additive morphology nanoemulsions physicochemical characterization selection

Introduction

Broiler chickens are one of the most affordable sources of animal protein. Poultry production can be improved by using antibiotics to combat pathogenic microbial infections and enhance growth performance.^1,2 However, several countries have banned the use of antibiotics as feed additives due to concerns about bacterial resistance, resulting in residues in the meat produced, decreased immunity, and increased environmental pollution.^3,4 The European Union has prohibited most antibiotics as feed additives since 1999, with a full ban taking effect as of January 1, 2006 (Regulation (EC) No. 1831/2003).⁵ Indonesia also enacted a ban on the use of antibiotics in poultry in January 2018, based on a regulation issued by the Indonesian Minister of Agriculture, Number 14/PERMENTAN/PK.350/5/2017, concerning the Classification of Veterinary Drugs.⁶

The prohibition of antibiotic use has encouraged efforts to develop antibiotic alternatives to optimize livestock growth performance. Phytobiotics are one type of feed additive that originates entirely from plants, making them a natural option from the environment that has the potential to replace chemical substances recognized as generally recognized as safe (GRAS). Phytobiotics encompass a range of substances, including herbs, spices, essential oils, and plant extracts.⁷ The major constituents of citronella essential oil are monoterpenes (89.93%), which include citronellal (27.34%), geraniol (23.21%), geranial (13.37%), citronellol (12.49%), and neral (10.31%)⁸ that are beneficial for improving the productivity of broiler chickens. However, essential oils are hydrophobic, have low bioavailability, and are prone to oxidation.⁹ The application of self-nanoemulsifying drug delivery systems (SNEDDS) technology is implemented to increase the surface area with droplet sizes ranging from 10 to 100 nm, enhance water solubility and stability, protect the essential oil from oxidation, and reduce evaporation.¹⁰ Additionally, the application of SNEDDS technology is easy to produce at a low cost and minimizes physical changes.

Therefore, this study aims to develop a nanoemulsion of citronella essential oil using materials that are safe for livestock and effective for enhancing the health and productivity of animals. Thus, the use of a small amount can provide positive effects, and its application is much more efficient because the resulting formulation is in liquid form that can be mixed directly into drinking water, making it more practical for application to livestock.

Materials and Methods

Materials for Citronella Essential Oil SNEDDS

Citronella essential oil, as the primary material for this study, was obtained from a local supplier, namely Lansida, Yogyakarta, Indonesia. The essential oil of citronella was extracted using the steam distillation method from the leaves. The ingredients used in the SNEDDS composition are chosen based on their acceptability. The carrier oil is derived from vegetable sources, as it does not produce odors like animal-based oils and is more cost-effective than single fatty acids, such as oleic acid.¹¹ Surfactants and cosurfactants are selected for their suitability for oral administration.¹² The materials used for the selection of the composition of SNEDDS include virgin coconut oil (VCO) (PT. Naturlife Karya Indonesia, Bogor, Indonesia), olive oil (PT. Naturlife Karya Indonesia, Bogor, Indonesia), soybean oil (Mazola, Selangor Darul Ehsan, Malaysia), canola oil (Mazola, Selangor Darul Ehsan, Malaysia), Tween 20 (Elotant TM, South Korea), Tween 80 (Elotant TM, South Korea), polyethylene glycol 400 (Petronas, Kuala Lumpur, Malaysia), propylene glycol (Dow Chemical Pacific, Singapore), hydrochloric acid 37% (Merck, Germany), and NaCl (Merck, Germany).

Selection of SNEDDS Composition Ingredients

The composition of SNEDDS for citronella essential oil was prepared using a modified method.¹³ The composition of the nanoemulsion ingredients consists of oil, surfactant, and cosurfactant mixed in a ratio of 20:60:20 (%).^14–16 The ratio of the oil phase combination, which consists of citronella essential oil and carrier oil (canola, olive, soybean, and VCO), is 50:50 (%). Each SNEDDS solution was made with three repetitions to optimize the data obtained. The SNEDDS solutions were coded for more straightforward data interpretation, as shown in Table 1.

Table 1.

Selection Code for the Composition of Citronella Essential Oil SNEDDS Ingredients.

Surfactant	Cosurfactant	Carrier Oil
Surfactant	Cosurfactant	Canola	Olive	Soybean	VCO
Tween 20	propylene glycol	1	2	3	4
Tween 20	polyethylene glycol 400	5	6	7	8
Tween 80	propylene glycol	9	10	11	12
Tween 80	polyethylene glycol 400	13	14	15	16

Citronella Essential Oil SNEDDS Preparation

The citronella essential oil was stirred for 2 minutes, then mixed with the carrier oil and stirred for 3 minutes. After that, the surfactant and cosurfactant were added and stirred with a magnetic stirrer for 10 minutes at 37°C at a speed of 250 rpm. The homogeneous solution was conditioned using an ultrasonic processor at 40ºC for 10 minutes. The well-mixed solution was then evaluated organoleptically based on criteria such as odor, homogeneity, and clarity¹⁷ as shown in Table 2. The mixture that has a characteristic aroma of citronella essential oil is homogeneous, transparent, with a transmittance of >90%, and an emulsification time of <60 seconds, and will be selected as the composition for the nanoemulsion formulation.

Table 2.

Organoleptic Test Parameters for Citronella Essential Oil SNEDDS.

Parameters	Result
Odor	Distinctively citronella aroma, slightly citronella aroma, odorless
Clarity	Clear, slightly cloudy, cloudy
Homogeneity	Homogeneous, less homogeneous, separating

Organoleptic Test

Organoleptic testing was conducted by 20 randomly selected panelists who completed a questionnaire containing organoleptic test parameters to evaluate the sensory properties of mixtures made with various nanoemulsion composition ingredients. The criteria for the panelists included males and females aged 19–25 years, a willingness to participate as panelists, and being physically and mentally healthy (i.e., not color blind, without physical disabilities, and without mental illnesses).¹⁸

Percentage Transmittance Measurement

The transmittance percentage is measured using a UV-Vis Spectrophotometer at a wavelength of 650 nm, with distilled water as the blank. The homogenized solution is placed in a cuvette, and the transmittance percentage is measured on the prepared UV-Vis Spectrophotometer. The transmittance percentage is used as an index to evaluate the solubility between oil and emulsifiers.¹⁹

Emulsification Time Measurement

A total of 0.2 mL of the solution was added to 50 mL of artificial gastric fluid with a pH of 2–3, using a magnetic stirrer at a speed of 100 rpm and a temperature of 37°C. The artificial gastric fluid consists of 37% HCl, NaCl, and distilled water. The emulsification time was determined by recording the time required for the dispersed mixture to be perfectly mixed with the artificial gastric fluid, indicated by a clear visual appearance.¹³

Citronella Essential Oil SNEDDS Optimization

In the subsequent phase, a three-component mixture design was employed, consisting of citronella essential oil and carrier oil, 50:50 (%) as the oil phase (X₁), surfactant (X₂), and cosurfactant (X₃). The response variables evaluated were percent transmittance (Y₁) and emulsification time (Y₂). The design was chosen based on its suitability for mixture optimization, and data analysis was conducted using Design-Expert software (version 13.0.1.0), D-optimal mixture, which is widely used for such experimental setups. All experiments were performed in triplicate, and statistical analysis was carried out using a one-sample t-test.¹³

DLS Analysis

The measurement of particle size, polydispersity index (PDI), and zeta potential was carried out with utmost thoroughness using a nanoparticle analyzer (HORIBA nanoPartica SZ-100, Fukuoka, Japan) based on the principle of dynamic light scattering (DLS) at a scattering angle of 90° and an instrument holder temperature of 25°C. The measurement was conducted by dissolving 0.1 mL of SNEDDS in 10 mL of distilled water, then 5 mL of the formed nanoemulsion was placed in a cuvette and positioned in the instrument holder to measure particle size, PDI, and zeta potential. The measurements were conducted with three repetitions, ensuring the validity and reliability of the results.²⁰

Viscosity Measurement

The viscosity of the nanoemulsion was measured using a digital viscometer (Brookfield AMETEK DV2T, Instruments Engineer Labs, USA). A total of 100 mL of citronella essential oil nanoemulsion was placed into a beaker. The viscometer was then set to a speed of 50 rpm for three rotations, using spindle number 2, for 15 seconds at a room temperature of 25°C, with three repetitions.²¹

Thermodynamic Stability Measurement

The thermodynamic stability test is conducted in three stages with three repetitions.²² In the first stage, heating-cooling is performed by storing SNEDDS at 45°C in a water bath for 48 hours, followed by cooling at 4°C in a refrigerator for an additional 48 hours. The first stage is performed over a total of six cycles. The second stage is centrifugation testing. SNEDDS is centrifuged at a speed of 3,500 rpm for 30 minutes. The third stage is the freeze-thaw cycle, which involves storing SNEDDS at a temperature of –18°C for 24 hours, followed by thawing at 25°C for 24 hours. The stability of the nanoemulsion is assessed by monitoring for any changes before and after the stability tests, including separation, sedimentation, cream formation, homogeneity, and clarity, through visual observation during each cycle.²³

Morphology Observation

The morphology of the SNEDDS for citronella essential oil was observed using a transmission electron microscope (TEM HT7700) with a maximum accelerating voltage of 120 kV. The nanoemulsion was diluted in distilled water, and then 15 μL was applied to a TEM grid and allowed to dry for 60 minutes.²⁴

Results and Discussion

The Selection of SNEDDS Composition Ingredients

The selection of SNEDDS composition ingredients is shown in Table 3. The SNEDDS solution, specifically formula 16, comprising citronella essential oil, VCO, Tween 80, and polyethylene glycol 400, yielded a homogeneous solution with a distinctive citronella scent, a clear and homogeneous appearance, the highest transmittance value of 99.74%, and the shortest emulsification time of 29.33 seconds. Therefore, the combination of ingredients selected for the citronella SNEDDS composition is VCO as the carrier oil, Tween 80 as the surfactant, and polyethylene glycol 400 as the cosurfactant. VCO contains Medium-Chain Triglycerides (MCTs) with a carbon chain length of 12, while others contain long-chain triglycerides (LCTs) with a carbon chain length of ≥16.¹¹ Fatty acids with shorter chains are more soluble in water, resulting in smaller nanoemulsion droplets. Therefore, higher transmittance values are associated with smaller droplet sizes, which result in a clearer nanoemulsion.²⁵

Table 3.

Character Data for the Selection of Ingredients, Citronella Essential Oil SNEDDS.

Formula	Measurement Result
Formula	Transmittance (%)	Emulsification Time(s)	Odor	Clarity	Homogeneity
1	4.43 ± 0.65	34.33 ± 2.05	D	CO	S
2	18.13 ± 3.84	48.33 ± 1.89	D	SO	S
3	3.97 ± 1.20	42.67 ± 1.70	D	SO	S
4	95.36 ± 0.92	37.33 ± 0.47	D	CO	S
5	94.86 ± 2.08	42.33 ± 0.47	D	CL	LH
6	73.73 ± 4.36	36.67 ± 0.47	D	SO	LH
7	82.50 ± 3.07	34.67 ± 1.70	D	SO	LH
8	8.82 ± 1.21	33.33 ± 0.47	D	CO	S
9	12.03 ± 4.10	36.00 ± 0.82	D	CO	S
10	18.13 ± 3.38	37.33 ± 2.05	SC	CO	LH
11	25.40 ± 6.59	34.33 ± 1.25	SC	CO	LH
12	98.68 ± 1.15	34.33 ± 1.89	SC	CL	H
13	91.81 ± 2.13	30.67 ± 1.70	D	SO	H
14	97.64 ± 0.37	39.33 ± 3.09	D	CL	H
15	86.03 ± 2.38	39.00 ± 1.41	D	SO	H
16	99.74 ± 0.18	29.33 ± 1.25	SC	CL	H

Note: D = Distinctively citronella aroma; SC = Slight citronella aroma; CL = Clear; CO = Cloudy; SO = Slightly cloudy; S = Separating; H = Homogeneous; LH = Less homogeneous.

Transmittance percentage is one of the parameters used to select the composition of the citronella SNEDDS formula. A transmittance percentage of 90% to nearly 100% is expected to produce emulsion droplets reaching nanometre size.²⁶ SNEDDS system solutions that produce a transmittance percentage of more than 90% will produce a clear visual appearance and become a qualitative indicator that the system solution can disperse spontaneously. The combination of citronella essential oil with surfactants, cosurfactants, and carrier oils that produced the highest transmittance percentage and shortest emulsification time was selected as the composition for the SNEDDS formula.

Emulsification time is one of the important parameters in determining the composition of SNEDDS ingredients. Emulsification time refers to the stability of the system. It indicates the formulation’s ability to emulsify rapidly upon contact with broiler chicken gastric fluid, due to peristaltic movement in the digestive tract.²⁷ The faster the emulsification time required, the better the water solubility of the resulting formula, due to the presence of surfactants and cosurfactants that form an oil-water interface layer.²⁸

The results of organoleptic testing of odor, clarity, and homogeneity using several SNEDDS composition ingredients, with a clear appearance, homogeneity, and a distinctively citronella aroma. A straightforward solution will yield a high transmittance percentage, indicating that the produced particle size will likely be in the nanoscale range, and a homogeneous solution can be expected to have each constituent ingredient mixed evenly and stably at various temperatures, thereby effectively increasing stability and reducing drug delivery irritation.²⁹ These results indicate that the presence of SNEDDS ingredients can maintain the characteristics of the main ingredient, which is a source of bioactive compounds. The application of SNEDDS can minimize the volatility and degradation of bioactive components in essential oils.¹⁷

The Selection of Oil Phase:Surfactant: Cosurfactant Ratios

The selection of SNEDDS composition ratios is shown in Table 4. The ratio of citronella essential oil SNEDDS composition ingredients had an effect on the transmittance percentage and emulsification time. The 20:60:20 (%) ratio achieved a transmittance percentage close to 100% with the lowest surfactant use, and the emulsification time required was classified as fast, as all mixed solutions did not exceed 60 seconds.

Table 4.

Composition Ratio of Oil Phase:Surfactant:Cosurfactant.

No.	Oil Phase (%)	Surfactant (%)	Cosurfactant (%)	Transmittance (%)	Emulsification Time(s)
1	33.33	33.33	33.33	35.88 ± 2.45	31.29 ± 1.62
2	25	50	25	94.10 ± 6.58	30.10 ± 0.49
3	20	60	20	99.49 ± 0.40	30.81 ± 2.15
4	16.67	66.67	16.67	99.27 ± 0.56	29.91 ± 3.35
5	14.29	71.43	14.29	98.65 ± 0.39	33.04 ± 0.31
6	12.5	75	12.5	99.12 ± 0.06	34.91 ± 3.21
7	11.11	77.78	11.11	100.41 ± 0.19	35.48 ± 2.17
8	10	80	10	100.34 ± 0.09	40.15 ± 1.26

The composition of SNEDDS with various ratios produces different characteristics. Ratios with surfactants ranging from 33.33 to 50 (%) produce transmittance percentages far from 100%, while surfactant amounts of 60% and above produce transmittance percentages close to 100%. This value indicates that an increase in the amount of surfactant is directly proportional to an increase in the percentage of transmittance produced. The ratio with the lowest surfactant content was selected, as it was deemed sufficient to achieve the desired emulsification, thereby optimizing the amount of oil phase as a source of bioactive compounds. A high transmittance percentage indicates that the resulting SNEDDS is highly transparent.²² The solution that produced the highest transmittance percentage, the fastest emulsification time, and a clear visual appearance was considered the best formula using the lowest amount of Tween 80 and the highest amount of essential oil.³⁰ In addition, in order to reduce costs and lower the irritation level of the resulting SNEDDS.

The Oil Phase Ratios

The oil phase ratios are shown in Table 5. The oil phase ratio, specifically the combination of citronella essential oil and VCO, had a highly significant effect on both the transmittance percentage and emulsification time. The 50:50 (%) ratio obtained a transmittance percentage close to 100%, namely 99.92%, with the fastest emulsification time of 31.21 seconds. SNEDDS solutions with various oil phase ratios affect the percentage transmittance and emulsification time produced. A formula comprising 20–70% citronella essential oil exhibited elevated transmittance values (surpassing 96%) and comparatively expeditious emulsification times (less than 40 seconds). However, elevating the citronella oil concentration to 80–90% generally resulted in longer emulsification times, with the formula at 90% exceeding 60 seconds. The utilization of elevated concentrations of citronella oil (80–90%) has the potential to augment the predominance of the hydrophobic phase, which can result in a reduction in emulsification efficiency and an increase in emulsification time. These results align with the findings of,³⁰ which indicate that a combination of essential oil and VCO in a 0.5:0.5 ratio is the optimal formula. This value suggests this ratio is the optimal ratio for developing a citronella essential oil SNEDDS formula.

Table 5.

Citronella Essential Oil SNEDDS Phase Ratio.

No	Citronella Essential Oil (%)	VCO (%)	Transmittance (%)	Emulsification Time (s)
1	10	90	20.90 ± 8.05	36.55 ± 1.41
2	20	80	98.84 ± 1.11	36.38 ± 1.05
3	30	70	98.48 ± 2.48	37.31 ± 1.00
4	40	60	96.01 ± 5.21	41.09 ± 1.39
5	50	50	99.92 ± 0.70	31.21 ± 0.26
6	60	40	97.80 ± 1.97	32.70 ± 1.96
7	70	30	98.14 ± 1.32	33.08 ± 1.61
8	80	20	95.92 ± 1.73	46.51 ± 3.77
9	90	10	96.55 ± 2.22	64.15 ± 1.46

Citronella Essential Oil SNEDDS Formula Optimization

Formula optimization was performed to determine the optimal ratios of each ingredient that yield the best formula with high precision. Based on the results of the composition and oil phase ratio selection, the ideal ratio was formula 3 (Table 4), consisting of oil phase:surfactant:cosurfactant 20:60:20 (%), with an oil phase ratio of 50:50 (%). The ratio from formulas 2 and 4 was used as the upper and lower limit of the optimal ratio. Therefore, the upper limit of 25:66.67:25 (%) and a lower limit of 16.67:50:16.67 (%) (Table 6). These limits are used as the threshold ratio, which will be analyzed using a D-optimal mixture design to obtain the optimal formula with Design-Expert software. Optimization by means of D-optimal mixture to find the optimal formula on various formulas used three independent variables, namely the amount of oil, surfactant, and cosurfactant, against two dependent variables, the percentage of transmittance and emulsification time. The formula was based on a “trading off” between increasing transmittance and decreasing emulsification time, yielding 16 formulas (Table 7).

Table 6.

The Components of the Threshold Were Analyzed Using a D-optimal Mix Design.

Independent Variables (Factors)	Range
Independent Variables (Factors)	Low	High
X₁ = Quantity of oil	16.67	25
X₂ = Quantity of surfactant	50	66.67
X₃ = Quantity of cosurfactant	16.67	25

Table 7.

Formula Using D-optimal Mix Design by Design Expert Ver. 13.0.1.0.

Run	Oil (%)	Surfactant (%)	Cosurfactant (%)	Transmittance (%)	Emulsification Time (s)
1	16.67	58.33	25	99.85	35.75
2	17.49	63.56	18.95	98.20	31.35
3	16.67	58.33	25	99.26	36.17
4	25	53.67	21.33	97.68	39.72
5	24.84	58.49	16.67	99.29	36.45
6	25	53.67	21.33	81.68	40.47
7	21.86	55.94	22.20	99.91	36.64
8	24.21	50.79	25	99.39	40.88
9	24.84	58.49	16.67	99.44	36.65
10	17.47	60.70	21.83	99.04	31.63
11	21.27	62.06	16.67	99.19	37.43
12	24.21	50.79	25	81.18	46.86
13	16.67	66.66	16.67	99.14	32.16
14	20.66	58.95	20.39	99.08	31.68
15	19.44	55.56	25	98.35	37.14
16	16.67	66.66	16.67	99.25	31.76

The results in Table 7 show that the transmittance ranges from 81.18% to 99.91% and the emulsification time is 31.35 to 46.86 seconds, obtained from variations in the composition of the oil phase, surfactant, and cosurfactant within the limits set in Table 6. Formulations with lower surfactant proportions tend to exhibit lower transmittance and longer emulsification times, indicating that the surfactant amount is insufficient to reduce interfacial tension and form small droplets. Therefore, the composition with a higher surfactant and a moderate oil phase was chosen as the optimum formula because it is estimated to produce nanoemulsions with high transmittance and faster emulsification.

Analysis of the Dependent Variable

The optimum formula consisted of 17.86% oil phase (50:50 (%) for the combination of citronella essential oil and VCO), 62.791% surfactant, and 19.169% cosurfactant. The results of the expert design analysis show that the transmittance percentage is closer to red, while the emulsification time is closer to red. This spectrum shows that the values obtained are in accordance with expectations: maximizing transmittance and minimizing emulsification time. The residual standard probability plots of transmittance percentage (Figure 1a) and emulsification time (Figure 1b) show straight lines with no outliers. The residual vs. run results for transmittance percentage (Figure 2a) and emulsification time (Figure 2b) are centered on the zero line, with no prominent differences. This indicates that the residuals are normally distributed.¹³ Figure 3a and 3b show the linear model of the relationships among oil, surfactant, and cosurfactant, yielding a predicted transmittance percentage of 99.338% ± 0.366% for the optimum formula and an emulsification time of 32.107 ± 0.454 seconds. A high transmittance value indicates that the resulting nanoemulsion is clear, exhibits minimal particle aggregation, and is highly stable.¹⁷ The addition of surfactants and cosurfactants can reduce the interfacial tension between oil and water, thereby reducing the emulsification time.²⁸

Figure 1.

The Residual Standard Probability Plots.

Figure 2.

The Residual Versus Run Results.

Figure 3.

The Linear Model of the Relationships Graphic Values.

Analysis of variance and lack-of-fit tests are presented in Table 8. The linear model showed no significant relationship ( p > .05) between the amounts of oil, surfactant, and cosurfactant and the actual transmittance percentage. In contrast, the emulsification time showed a significant relationship ( p < .05) with the amounts of oil, surfactant, and cosurfactant. This indicates that the ratio of oil, surfactant, and cosurfactant affects the emulsification time but has no significant effect on the transmittance percentage.³¹ Although the transmittance model was not statistically significant, transmittance was still used as an indicator of nanoemulsion clarity and dispersion quality, as consistently high values suggest good dispersion characteristics.

Table 8.

Analysis of the Linear Model.

Response	F Value	Probability > F
Y₁ = Transmittance
Model	2.20	0.150 nonsignificant
Lack of fit	0.248	0.960 nonsignificant
Y₂ = Emulsification Time
Model	17.64	0.0002 significant
Lack of fit	1.85	0.259 nonsignificant

The lack-of-fit analysis for both transmittance percentage and emulsification time showed no significant results (p > .05), indicating a high agreement between the predicted values and the actual data. Therefore, the optimum formula results were verified by a single-sample t-test using SPSS Statistics 23 (Table 9). Both transmittance percentage and emulsification time showed p values (p > .05), indicating that there was no significant difference between the predicted values and the actual data.³²

Table 9.

Verification of the Predicted Optimal Formulation Through Single-sample t-test Analysis.

Response	Predicted Value	Actual Data ± SD	p Value
Transmittance	99.879	99.338 ± 0.366	.125
Emulsification time	32.848	32.107 ± 0.454	.105

DLS Analysis

DLS analysis consists of droplet size, PDI, and zeta potential. Droplet size of 14.4 ± 0.141 nm (Figure 4) indicates a range of 10–100 nm with a PDI of 0.088 ± 0.025. Zeta potential of –17.00 ± 2.980 mV (Figure 5) indicates a range of −30 to 30 mV.

Figure 4.

Droplet Size Distribution Graph.

Figure 5.

Zeta Potential Distribution Graph.

Characterization of citronella essential oil SNEDDS with optimum formula using the DLS method yielded a droplet size of 14.4 ± 0.141 nm. This size is less than 200 nm, which may enhance the absorption and bioavailability of bioactive compounds by improving the stability and homogeneity of the emulsion system.^33,34 In this study, the nanoemulsion of essential oil from C. nardus showed an average droplet size of 14.4 nm, which was smaller than the nanoemulsion from C. citratus with a size of 20.7 nm.³⁰ This result indicates that the emulsification system in C. nardus essential oil has a higher nanoemulsion formation efficiency, although both are still within the optimal nanoemulsion range. Therefore, the use of nanoemulsions, which are very small in size, as feed additives will effectively enhance the absorption of bioactive compounds.

The PDI is a quantitative parameter that describes the variation in droplet size distribution obtained by comparing the standard deviation and the average droplet size of SNEDDS. A PDI of less than 0.5 indicates that the particle size distribution produced is a homogeneous and stable system.³⁵ Emulsions with a high degree of homogeneity and good stability are optimally obtained at a PDI value of less than 0.2.³⁶ The lower the PDI value obtained, the higher the uniformity of the SNEDDS produced.³⁷ Therefore, the PDI value can indicate whether the SNEDDS produced is homogeneous or not.

The zeta potential is used to measure the stability of colloids by determining the surface charge of particles, which affects interactions and dispersion stability. The formulation system is stabilized with the nonionic surfactant Tween 80 and the cosurfactant PEG 400. This suggests that steric stabilization plays a significant role. The overlapping of their polymeric layers generates repulsive forces due to osmotic pressure and the decrease in conformational entropy. Nonionic surfactants with polyoxyethylene chains form a hydrated interfacial layer around oil droplets. Generally, nonionic surfactants exhibit zeta potential values that are neutral or close to zero.³⁸ A zeta potential near zero indicates that the surface charges do not attract one another.³⁹ A negative zeta potential value indicates that the SNEDDS formulation contains fatty acids. This is attributed to the use of VCO as the carrier oil, which contains fatty acid components, including lauric acid, palmitic acid, caprylic acid, oleic acid, capric acid, and stearic acid.⁴⁰

Viscosity

The viscosity of citronella SNEDDS was found to be 376.00 ± 3.637 mPa.s. The low viscosity value indicates that the particle size falls into the small category. Viscosity indicates the resistance to flow of SNEDDS due to intermolecular friction, thus describing the viscosity level of a nanoemulsion. The droplet size in this research was small, resulting in a low viscosity.⁴¹ Viscosity in the range of 100–500 mPa•s is considered stable, making it easy to apply as a feed additive, especially in drinking water. Lower viscosity and interfacial tension within the liquid lipid phase enhance the emulsification process, resulting in the formation of ultrafine particles.⁴² The low viscosity within the nanoemulsion promotes efficient diffusion of the active compound, enabling prolonged and controlled drug delivery,⁴³ which in turn optimizes the absorption of bioactive compounds by livestock through controlled drug release.

Thermodynamic Stability

Thermodynamic stability is shown in Table 10. The results of the stability test of citronella essential oil SNEDDS showed no separation or precipitation. This indicates that citronella essential oil SNEDDS remains stable and resistant to extreme temperatures. The results of thermodynamic stability tests conducted using heating-cooling cycles, centrifugation, and freeze-thaw cycles showed that SNEDDS remained stable, with no separation or precipitation. Thermodynamic stability testing highlights SNEDDS’s capacity to maintain stability across different storage temperatures, as temperature plays a crucial role in nanoemulsion stability.⁴⁴

Table 10.

Thermodynamic Stability Test.

Stability Test	Replication 1	Replication 2	Replication 3
Heating-cooling cycle	Stable	Stable	Stable
Centrifugation	Stable	Stable	Stable
Freeze-thaw cycle	Stable	Stable	Stable

Citronella Essential Oil SNEDDS Morphology

Citronella essential oil SNEDDS morphology (Figure 6) reveals that TEM observations of citronella essential oil SNEDDS produce uniform spherical globules, indicating that citronella essential oil SNEDDS particles are homogeneously dispersed and physically stable. The TEM image of citronella oil SNEDDS shows that the particles are uniformly spherical, smooth, and do not aggregate. This image indicates that the droplets are evenly dispersed and stable, and can maintain their size without forming larger particle clusters. TEM was employed to evaluate the morphology of SNEDDS, as it provides a robust characterization for analyzing the internal structure, particle size distribution, and surface morphology of nanoemulsions in detail.⁴⁵

Figure 6.

Transmission Electron Micrograph of SNEDDS with Magnification 10 K.

TEM image citronella essential oil SNEDDS has a circular shape surrounded by a thicker line forming a ring. This image shows that SNEDDS consists of an oil phase encapsulated by surfactants, allowing the citronella essential oil to emulsify into water. The layer between the oil phase encapsulated by surfactants demonstrates the successful formation of an oil-in-water nanoemulsion.⁴⁶

Conclusion

This study successfully optimized citronella essential oil SNEDDS with 17.86% oil phase (50:50 (%) for the combination of citronella essential oil and VCO), 62.791% surfactant, and 19.169% cosurfactant with high transmittance 99.338% ± 0.366%, rapid emulsification of 32.107 ± 0.454 seconds, small particle size 14.4 ± 0.141 nm, low PDI 0.088 ± 0.025, moderate zeta potential –17.00 ± 2.980 mV, and appropriate viscosity 376.00 ± 3.637 mPa•s. It produces a uniform spherical globule visual, indicating that the citronella essential oil SNEDDS particles are homogeneously dispersed and physically stable.

Future Directions

Further investigations incorporating biological validation are warranted to support the potential efficacy of the citronella essential oil SNEDDS formulation as a feed additive and to determine how nanoemulsions behave in complex biological environments, release of active compounds, potential toxic effects from surfactants, and stability and bioavailability. Further evaluation of the long-term storage stability of nanoemulsions by conducting storage stability studies at various temperatures over a specific period, while monitoring changes in droplet size, PDI, zeta potential, and any phase separation. In addition, the chemical stability of the active compound can be analyzed using Gas Chromatography–Mass Spectrometry to detect potential degradation during storage.

Footnotes

Acknowledgements

The authors would like to acknowledge the Laboratorium Elektron Mikroskop PPNN ITB, Laboratorium Biologi Ternak Universitas Andalas, and Laboratorium STIFARM Padang, as well as all technicians for their involvement in this study.

Authors’ Contribution

All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be authors as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines.

Consent to Participate

Twenty panelists were selected at random and participated voluntarily, meeting the inclusion criteria without coercion.

Consent for Publication

The utilization and publication of anonymized data were endorsed by all participants.

Data Availability Statement

All data generated and analyzed are included in this research article.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval

Ethical approval was not required for this study because only non-invasive organoleptic evaluation of odor, clarity, and homogeneity was performed with adult volunteers.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by a 2025 PMDSU grant from the Ministry of Research, Technology, and Higher Education, under contract No. 060/C3/DT.05.00/PL/2025. We gratefully acknowledge the financial support received.

Informed Consent

The participants were made aware of the objectives and procedures of the study. The samples were not ingested, and the nanoemulsion was used at safe concentrations. Prior to participation, written consent was obtained.

Publisher’s Note

All statements and conclusions in this article are solely those of the authors and do not necessarily represent the views of the publishers, editors, or reviewers. The publisher maintains neutrality regarding jurisdictional claims about the institutional affiliations published.

Use of Artificial Intelligence-assisted Tools

The authors declare that they have not used artificial intelligence (AI)-tools for writing and editing of the manuscript, and no images were manipulated using AI. All content was critically reviewed, revised, and validated by the authors to ensure accuracy, scientific rigor, and originality.

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