1Department of Animal Nutrition and Feed Science, Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, Indonesia, 2Department of Animal Production, Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, Indonesia, 3Department of Pharmaceutics, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia
Email: [email protected]
Received: 03 Oct 2018, Revised and Accepted: 19 Nov 2018
Objective: Focus of this study was to optimize and to characterize the self-Nano emulsifying drug delivery system using lemongrass (Cymbopogon citratus) essential oil.
Methods: The optimum formulas were analyzed using a D-Optimal mixture experimental design and performed using a Design Expert® Ver. 7.1.5. Formulation variables which include in the design were: oil component X1 (a mixture of Cymbopogon citratus essential oil and virgin coconut oil/VCO), surfactant X2 (Tween 80), and co-surfactant (PEG 400), while emulsification time in a sec (Y1) and transmittance in percent (Y2) as responses.
Results: The optimum formula for SNEDDS in the current study were: Cymbopogon citratus essential oil (7.147%), VCO (7.147%), Tween 80 (71.417%), and PEG 400 (14.290%). From the optimizing formula can be shown that the mean of droplet size, polydispersity-index, zeta potential, and viscosity were: 13.17±0.06 nm, 0.17±0.05,-20.90±1.47 mV, 200±0mPa. s (n=3), respectively. Furthermore, the optimized formula has passed the thermodynamic stability test; meanwhile, transmission electron microscopy displayed spherical shape.
Conclusion: The optimized SNEDDS formula was improving solubility of poorly soluble Cymbopogon citratus essential oil.
Keywords: Cymbopogon citratus, D-optimal mixture design, optimization, self-nanoemulsifying drug delivery system (SNEDDS)
© 2019 The Authors.Published by Innovare Academic Sciences Pvt Ltd. This is an open-access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
The productivity of broiler chickens can be optimal when the health condition of the gastrointestinal tract is in good condition . The balanced population between pathogenic and beneficial microflora in the gastrointestinal tract plays an important role in nutrient digestion and absorption [2-4]. Escherichia coli, Salmonella typhimurium, and Clostridium perfringens are the three major pathogens in the gut of poultry . Since the high public concerns on antibiotic resistance, the use of antibiotics as growth promoters in the diets of poultry has been banned. There is an urgent need to explore the alternative for antibiotic growth promoters .
Essential oils are potential alternatives for an in-feed antibiotic as they have antibacterial properties . Essential oils are classified as phytobiotics that have pharmacological effects . As shown in lemongrass (Cymbopogon citratus) essential oils, this phytobiotic have been found to be effective in inhibiting pathogenic bacteria, such as: Salmonella typhimurium , S. enterica , Escherichia coli, Staphylococcus aureus, Listeria monocytogenes , Klebsiella pneumonia [12, 13], and also antifungal activity against Candida albicans .
Essential oils are volatile substances, liquid, soluble in lipid and organic solvents , and hydrophobic . Essential oils have antibacterial activity but low aqueous solubility . Self-nanoemulsifying drug delivery system (SNEDDS) is one of the effective strategies to improve oral bioavailability , solubility  and it can control the drug release . The purpose of the formula optimization study is to determine the variable level from which a strong product with high-quality characteristics can be produced .
The best formula to combine oils in water is needed to be generated. The present experiments were carried out in order to find the optimum formula of SNEDDS of Cymbopogon citratus essential oil followed by characterization.
Preparation of SNEDDS
Firstly, surfactant (Tween 80, Kao Indonesia Chemical, Bekasi, Indonesia), co-surfactant (PEG 400, idCHEM Co., Ltd., Kyunggi, South Korea), Cymbopogon citratus essential oil (Lansida Group, Yogyakarta, Indonesia) and virgin coconut oil/VCO (Healthy Co, Yogyakarta, Indonesia) were mixed using a magnetic stirrer (IKA® C-MAG HS 7, IKA WORKS Inc., Wilmington, NC, USA). Secondly, ultrasonicator (J. P Selecta, Barcelona, Spain) was utilized to make the SNEDDS formula uniformly disperse. Thirdly, SNEDDS formula was placed into a water bath (Memmert GmbH and Co. KG, Schwabach, Germany) at 45˚C for 15 min .
SNEDDS formulations were added with a ratio of 1:50 with distilled water and mixed using vortex for 30 min. The percent transmittance was measured using UV-vis spectrophotometer (Genesys 10, Thermo Scientific Inc, Waltham, MA, USA) at 650 nm [21, 22].
The SNEDDS formula was added drop wise to 500 ml of artificial gastric fluid at 37˚C±2˚C. The artificial gastric fluid was composed of hydrochloric acid 37%, NaCl (Merck, Germany), and distilled water. Gentle agitation was provided by a magnetic stirrer (Stuart CB162 Hotplate and Magnetic Stirrer, New Jersey, USA) rotating at 100 rpm. The assessment was conducted visually and the time for emulsification was taken .
Optimization of SNEDDS
Along with the formula generated in our previous study , SNEDDS were optimized using a D-Optimal mixture experimental designed and performed using a Design Expert® Ver. 7.1.5. software (Stat-Ease Inc., Minneapolis, USA)  by selecting the percentage of the oil component X1 (a mixture of VCO and Cymbopogon citratus essential oil), the surfactant X2 (Tween 80), and the co-surfactant (PEG 400) as independent variables, while emulsification time in sec (Y1) and transmittance in percent (Y2) as responses. Data were analyzed by one-way ANOVA at 0.05 levels .
Characterization of SNEDDS
Measurement of droplet size and zeta potential
The droplet size and zeta potential of the optimum formula were determined by dynamic light scattering techniques using a Zeta potential/Particle-sizer (Horiba Scientific SZ-100, Horiba, Kyoto, Japan). Liquid SNEDDS (1 ml) was diluted to 100 ml with distilled water and performed in triplicate .
The viscosity of optimum formula was measured by Brookfield viscometer (Brookfield engineering laboratories, Stoughton, MA, USA) at 25±0.5˚C, and at 12 rpm. The viscosity of each SNEDDS was performed in triplicate .
Thermodynamic stability study
According to the method of , the study of the thermodynamic stability was implemented for optimum formula in three steps:
Heating-cooling cycle: The optimum formula was stored at 4˚C and 45˚C for 48 h at each temperature using constant climate chamber bath, it was repeated for 6 cycles in triplicate. The formulation that withstands the heating-cooling cycle was continued to the centrifugation test.
Centrifugation test: The optimum formula passed heating-cooling cycle were centrifuged at 5000 rpm for 30 min, observed for any sign of creaming, cracking, or separation phase.
Freeze-thaw cycle: The optimum formula passed centrifugation test were exposed at-21˚C and 21˚C for 24 h at each temperature, centrifuged at 3000 rpm 5 min, and continued visual observation.
Transmission electron microscopy (TEM)
The micro-morphology of the optimum formula was observed by TEM Joel JEM-100 CX (Joel, Tokyo, Japan). The SNEDDS was diluted with water (1:1000), a sample drop was stained with 2% phosphotungstic acid solution, and placed on a copper grid for 30 s .
Table 1: Level of the factors analyzed using D-optimal mixture experimental design
|X1= Quantity of Oil (%)||14.29||20.00|
|X2= Quantity of Tween 80 (%)||60.00||71.43|
|X3= Quantity of PEG 400 (%)||14.29||20.00|
Table 2: Percentage of oil, tween 80, and PEG 400 and observed responses
|Oil (%)||Tween 80 (%)||PEG 400 (%)||Transmittance (%)||Emulsification time (s)|
The optimum formula was replicated three times and verified with a single sample t-test with OpenStat® (Industrial Technology Department, Iowa State University, USA). The optimum formula was selected for characterization.
Optimization Formula of SNEDDS
Table 3: The statistical parameter of each response using an experimental design
|Y = Emulsification time|
|Lack of fit||4.66||0.0535||Not significant|
|Y = Transmittance|
|Lack of fit||0.24||0.9652||Not significant|
The probability of the model was less than 0.05, and the lack of fit was more than 0.05 (table 3). This result indicated that the chosen model can describe the relationship between the variables. Fig. 1 showed that the residual is normally distributed around the line and nothing stands out. The relationship between variables was illustrated using a special model cubic (fig. 2).
Fig. 1: The normal probability plot residual of emulsification time
Fig. 2: Special model cubic of emulsification time of the mixture study result
Emulsification time used as a response is a way to determine the ability of surfactant and co-surfactant components to emulsify oil components . Selection of the optimum formula based upon “trading off” from the minimization of emulsification time. Emulsification time is one of the important keys to estimating emulsification efficiency .
Fig. 3: The normal probability plot residual of transmittance
The probability of the model and the lack of fit were both more than 0.05 (table 3). The data indicated the chosen model can describe the relationship between the variables. Fig. 3 showed that the residual was normally distributed around the line and no one stands out. The relationship between variables was illustrated using a special model cubic (fig. 4).
Fig. 4: Special model cubic of transmittance of the mixture study result
The optimization formula aimed to find products that have high-quality characteristics that will be produced . Transmittance must be maximized to produce good quality products. The transmittance value of all treatments is above 99 percent, indicating the efficiency of self-nanoemulsion . A good nanoemulsion has a transmittance value close to 100 percent that indicated a clear nanoemulsion .
Fig. 5: Desirability
The optimized formula that selected was the one that have a desirability value of close to 1 . Fig. 5 showed the desirability which used to predict the optimum formula. The first solution found through numerical optimization and desirability value was 0.861. The composition of optimum formula consists of an oil component (14.293%), Tween 80 (71.417%), and PEG 400 (14.290%).
Table 4: Predicted value and actual data of the optimized SNEDDS
|Response||Actual data±SD||Predicted value||P-value|
*Actual data are mean±SD, n=3 [SD: Standard deviation], the actual data and predicted value are shown in table 4. The P-value of emulsification time and transmittance was higher than 0.05, that indicated no significant difference between actual data and the predicted value.
Characterization of SNEDDS
Table 5: Droplet size, PI, zeta potential, and viscosity of the optimum formulation
|Replication||Droplet size (nm)||Polydispersity index (PI)||Zeta potential (mV)||Viscosity (mPa. s)|
* SD: Standard deviation, n=3
The droplet size of nanoemulsion was found to be 13.17±0.06 with polydispersity-index (PI) 0.17±0.05 (table 5). Droplet size is a crucial factor to evaluate the system of nanoemulsion . Smaller particle size causes better absorption at gastrointestinal tract . The optimum formula shows a PI value lower than 0.5 which indicate uniformity droplet size distribution .
The zeta potential of the optimum formulation was 20.90±1.47 mV as shown in table 5. The fatty acid content in the formula may cause the negative charge of the zeta potential .
The viscosity of the optimum formulation is 200±0 mPa. s (table 5). The concentration of surfactant and carrier oil used in SNEDDS is related to the value of viscosity . The viscosity is directly proportional to the oil concentration .
The optimum SNEDDS formula did not change visible appearance which showed good stability at various storage conditions (table 6). Nanoemulsion is more stable than an emulsion, SNEDDS that will form nanoemulsion must be thermodynamically stable with the characteristic of not experiencing precipitation, cracking, or creaming .
Table 6: Results for thermodynamic stability studies
|Replication||Heating-cooling cycle||Centrifugation test||Freeze-thaw cycle|
Fig. 6: TEM of optimized SNEDDS formulation (80,000×; dilution 1000-fold with water)
Transmission electron microscopy (TEM)
The TEM of the optimum formula is presented in fig. 6. After dilution, all droplets demonstrated spherical shape and almost same size, represented a successful formation of SNEDDS. The TEM of the optimized formulation shows no coalescence signs indicating that the selected formula has good quality .
The optimization of Cymbopogon citratus SNEDDS using D-Optimal mixture experimental design was performed. The optimum formula for SNEDDS was a mixture of Tween 80, PEG 400, VCO, and Cymbopogon citratus essential oil at a ratio of 71.417: 14.290: 7.147: 7.147 (%). From the optimizing formula can be shown that the mean of droplet size, PI, zeta potential, and viscosity were: 13.17±0.06 nm, 0.17±0.05,-20.90±1.47 mV, 200±0 mPa. s, respectively. Furthermore, the optimized formula was passed the thermodynamic stability test, meanwhile team displayed spherical shape. The optimized SNEDDS formula was improving solubility of poorly soluble Cymbopogon citratus essential oil.
The authors are thankful to Biopharmaceutics Laboratory, Department of Pharmaceutics, Faculty of Pharmacy, Universitas Gadjah Mada Yogyakarta for an availing facility for these optimization studies. The author expresses gratitude towards DIKTI Ministry of Research, Technology, and Higher Education Indonesia for funding the project under the scheme of PMDSU No. 2050/ UN1/DITLIT/DIT-LIT/lT/2018.
All the authors have contributed equally
All the authors declared none
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