Research Article |
Corresponding author: Nining Sugihartini ( nining.sugihartini@pharm.uad.ac.id ) Academic editor: Milen Dimitrov
© 2023 Naelaz Zukhruf Wakhidatul Kiromah, Nining Sugihartini, Laela Hayu Nurani.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Kiromah NZW, Sugihartini N, Nurani LH (2023) Development and characterization of clove oil microemulsion. Pharmacia 70(1): 233-241. https://doi.org/10.3897/pharmacia.70.e98096
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Clove oil is one type of essential oil from clove flower buds (Syzygium aromaticum) which contains eugenol compounds. Clove oil is volatile in nature and highly affected by heat, thus incorporating it in a microemulsion system can increase its shelf life of the oils. This study aimed to formulate and characterize microemulsion preparations with clove flower essential oil. The microemulsion existence region was determined by constructing a pseudo-ternary phase diagrams and prepared with four components, i.e. isopropyl myristate, tween-80 as a surfactant, polyethylene glycol 400 (PEG 400) as a co-surfactant, and water as the aqueous phase. The optimized clove oil microemulsion formulation was subjected to an evaluation of various parameters, such as organoleptic properties, %transmittance, pH, viscosity, stability, particle size, zeta potential, and polydispersity index (IP). Based on the results of the study, the highest compound component in clove oil was eugenol with a % area of 63.79%. The results revealed that the construction of a phase diagram and the use of the phase titration method constituted a suitable technique for the preparation of microemulsions as most of the formulations were transparent. It was found that the tween-80:PEG 400 ratio of 2:1 with an oil:S-mix ratio of 1:9 generated an optimum results. The clove oil microemulsion had a globule size of 17.69±0.025nm, a polydispersity index value of 0.057±0.0043, zeta potential of-5.36±0.23mV, and a pH value of 7.3±0.1, a viscosity value of 466.7±9.06., and %transmittance of 99.9±0.1. According to these findings, the microemulsion formulation might serve as a suitable drug delivery system.
microemulsion, clove oil, formulation, characterization
The clove (Syzygium aromaticum) is included in the Myrtaceae family, which is widely produced in Indonesia. The Clove is primarily produced in Indonesia, accounting for roughly 70% of total global clove production yearly (
The potency of clove leaf essential oil still needs to be improved. It is immiscible with water, which minimizes contact with polar ingredients (
Microemulsions are thermodynamically stable, transparent, and homogeneous (
A microemulsion is a bicontinuous system containing water and oil, separated by a surfactant and a cosurfactant. Microemulsions have low interfacial tension. It will be challenging to achieve the required interface area if only a single surfactant is used; thus, a co-surfactabt is needed (
This study aimed to make a microemulsion preparation containing clove flower essential oil with various concentrations of tween 80 as a surfactant and PEG 400 as a co-surfactant. The results of the formulation of the microemulsion preparation will be characterized to obtain a stable and high-quality clove flower essential oil microemulsion.
The main ingredients used in this research was clove flower essential oil, which was obtained from the Center of Essential Oil Studies (CEOS) of the Indonesian Islamic University, Yogyakarta. The additives used in the formulation of microemulsion gel preparations, including isopropyl myristate, tween-80, PEG 400, and aquadest, were of pharmaceutical grade and obtained from CV Nurul Jaya Medicallabsains, Banyumas.
Instruments in this study were: Iwaki Pyrex glassware, GC-MS instrument, magnetic stirrer, digital pH meter (Pico+ Labindia, Mumbai, India), V-Visible spectrophotometer (UV, 1700, Shimadzu, Japan), Brookfield viscometer (DV- II+Pro Brookfield, USA), and Zetasizer (Malvern instrument ltd ZEN3600, UK).
GC-MS analysis was performed using an Agilent 7890 gas chromatograph connected to an Agilent 5975C Mass Spectrometry detector. The column used was HP-5MS UI (cross-linked 5% methyl phenyl silox) capillary column (30 m × 0.25 mm, film thickness 0.25 m). The oven temperature is raised from 40 °C to 200 °C at a rate of 6 °C/min, and further from 200 °C to 280 °C at a rate of 30 °C/min. Post-run was then conducted for 10 minutes at 280 °C. The carrier gas was Helium with a flow rate of 1 mL/min. The injector and detector temperatures was 250 °C (
The pseudo ternary phase diagrams of surfactant and co-surfactant mixture (S-mix), oil and doubled distilled water were plotted by the water titration method. The surfactant used was tween-80, and the co-surfactant used was PEG 400. The ratio of surfactant (S) to co-surfactant (CoS) was fixed at different ratios of 1:1, 2:1, and 1:2 on the weight basis for each phase diagram. The oil phase was mixed with the surfactant and co-surfactant mixture at the ratios (volume basis) of 1:9, 1:8, 1:7, 2:12, 2:10, 2:8, 2:7, 2:6, 3:7, 3:6, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1(w/w) (
The oil-S-mix mixture at each ratio was slowly titrated with distilled water. The addition of distilled water was carried out with a constant stirring speed, and the final mixture was stirred with a constant stirring speed for 15 minutes at room temperature until a microemulsion (ME) was formed, i.e., a transparent liquid with low viscosity (
The most stable microemulsion base was achieved. The manufacture of clove oil microemulsions was carried out by weighing all ingredients using a 10% clove oil concentration. The surfactant and co-surfactants were mixed in a beaker glass, to produce a surfactant mixture by stirring at 500 rpm using magnetic stirrer for 5 minutes until homogeneity was achieved. Oil was added by continuously stirring with the same stirring speed using a magnetic stirrer for 5 minutes until homogeneity was achieved. Weighed clove oil was added to the mixture by continuously stirring with the same stirring speed using a magnetic stirrer for 5 minutes until homogeneity was achieved. Distilled water was also added little by little to the mixture until all the distilled water was added. Stirring was carried out continuously using a magnetic stirrer for 15 minutes until homogeneity was achieved.
An organoleptic test was carried out by observing the shape, color, and smell of the clove oil microemulsion. Observations were made visually and using the five senses (Fitriani et al. 2016).
The apparent pH of the prepared formulations was measured (in triplicate) by using a calibrated digital pH meter (Pico+ Labindia, Mumbai, India) at ambient temperature with a glass electrode at 25±1 °C.
Transparency of the microemulsion was determined by measuring the percentage of transmittance at 650 nm against distilled water as the blank using a UV-Visible spectrophotometer (UV, 1700, Shimadzu, Japan).
The viscosity of the samples was measured at 25 °C with a Brookfield viscometer (DV-II+Pro Brookfield, USA) using a spindle no 5 with a shear rate 6 rpm. Each measurement was performed in triplicate.
Particle size measurements
The average droplet size of the samples was measured at 25 °C by SCATTER SCOPE 1 QUIDIX (South Korea), and their polydispersity index (IP) was also calculated.
Zeta potential determination
The zeta potential of the samples was measured by Zetasizer (Malvern instrument ltd ZEN3600, UK). The samples were placed in clear disposable zeta cells and the results were recorded.
Centrifugation
Formulations passing from the heating and cooling cycle were centrifuged at 3500 rpm for 30 min. All formulations that did not show any phase separation were taken for the heating and cooling stress test.
Six cycles between refrigerator temperatures of 4 °C and 45 °C with storage at each temperature for no less than 48 h were studied. Those formulations which were stable at these temperatures were subjected to a centrifugation test.
All the experiments were repeated three times, and data were expressed as the mean values ± SDs. Statistical data were analyzed by a one-way analysis of variance (ANOVA), and P<0.05 at a 95% confidence interval was considered to be significant.
In this study, the analysis of the chemical components of clove flower essential oil was carried out using gas chromatography (GC-MS). Gas chromatography is able to read compounds with the lowest concentrations; thus, secondary metabolites in plants can be identified with results in the form of chromatograms and mass spectra (
The results of the analysis showed that there were 57 peaks can be seen in Fig.
No | Peak | Name of Component | Molecular structure | Weight | Rt(minutes) | % area |
---|---|---|---|---|---|---|
1. | 18 | Eugenol | C10H12O2 | 164 | 19.17 | 63.79 |
2. | 22 | Caryophyllene | C15H24 | 204 | 20.58 | 10.43 |
3. | 20 | Phenol, 2-methoxy-3-(2-propenyl)- | C10H12O2 | 164 | 19.62 | 8.57 |
4. | 32 | Phenol, 2-methoxy-4-(2-propenyl)-, acetate | C12H14O3 | 206 | 23.21 | 8.48 |
5. | 19 | Eugenol | C10H12O2 | 164 | 19.60 | 2.08 |
For the microemulsion base, this study used the surfactant tween-80 and the co-surfactant PEG 400 because previous studies have proven that the combination of both is the right choice to produce microemulsion preparations with good physical characteristics and stability. The ratio of oil, surfactant, and co-surfactant in the microemulsion region was determined using cosurfactant using a pseudo-ternary phase diagram. Pseudo-ternary diagrams were created to obtain the maximum ratio that would precisely describe the phase formation limit (
If the ratio of surfactant to co-surfactant changes, where the former continuously increases, the interfacial tension will become better and more optimal, producing a good microemulsion. However, if the amount of co-surfactant is elevated beyond the surfactant, a reduction in emulsification will occur, therefore, it is better to use more surfactant than co-surfactant (
Fig.
The composition of the selected microemulsion base is shown in Table
Formulation | S/C | %Oil | %(S+C) | %water |
---|---|---|---|---|
ME-1 | 1:9 | 7 | 60 | 33 |
ME-2 | 1:8 | 8 | 68 | 24 |
ME-3 | 1:7 | 9 | 64 | 27 |
M3-4 | 2:10 | 13 | 64 | 23 |
ME-5 | 2:8 | 15 | 59 | 26 |
The first evaluation carried out was the organoleptic evaluation. This evaluation was carried out by storing the microemulsions at room temperature (25 °C) for 30 days. In all formulas, the microemulsion was clear, transparent in appearance, and with no precipitate. The reason for this was the presence of both hydrophilic and hydrophobic chains in eugenol, which resembled the structure of a surfactant and, therefore, could align themselves along with the original surfactant, i.e., tween-80, at the interfacial film. The results of the organoleptic evaluation can be seen in Fig.
A pH measurement is important to determine the suitability of the pH of the preparation with the pH of the skin. The results of the pH measurements in this study are shown in Fig.
The results showed that the base microemulsion formulations were stable under acidic conditions. This was due to the use of non-ionic surfactants in the microemulsion manufacture. Non-ionic surfactants are uncharged emulsifiers; thus, they are not affected by the concentration of H+ , which makes microemulsions stable under acid conditions (
The percentage of transmittance was measured using a UV-Vis spectrophotometer with distilled water as the blank. The transmittance percentage can be used to indicate the level of clarity of the microemulsion preparations. If a microemulsion preparation has a % transmittance value close to 100%, it can be concluded that the microemulsion preparation is optically clear (
In Fig.
Viscosity serves to see the flow properties of a preparation, which is an important parameter in examining the physical properties and stability of a preparation (
The results of measuring base was measured for 30 days using a Brookfield viscometer at a temperature of 27 °C. The results showed that all the base microemulsion formulas tended to increase viscosity in the first week and decreased viscosity from day 7 to day 30.
The resulting viscosity was not too large, this indicates that the base microemulsion formulations contained well-dispersed particles, which results in a good flow rate (300–1000 Cps). In addition, a statistical analysis was also carried out on the storage time and its relationship with the viscosity of the base microemulsion formulations. Statistical results showed that there was a significant difference between the formulas, with p<0.05. This means that that the storage time had an effect on the viscosity value. This increase, according to the statistical analysis data of the viscosity of each base microemulsion formulations, showed a significant difference (p<0.05). This indicates that the viscosity of the base microemulsion formulations was influenced by the ratio of surfactants to co-surfactants.
The results of the globule measurements of all base microemulsion formulas (Table
Formulation | Particle size (nm) | Polydispersity | Zeta potential (mV) |
---|---|---|---|
ME-1 | 19.02±0.01 | 0.222±0.004 | 5.70±0.36 |
ME-2 | 34.24±0.94 | 0.645±0.093 | -6.43±0.72 |
ME-3 | 59.85±0.80 | 1.0±0.00 | -8.68±0.25 |
ME-4 | 138.07±2.17 | 0.243±0.005 | -7.47±0.18 |
M3-5 | 128.30±0.71 | 0.168±0.009 | -5.49±0.11 |
A zeta potential measurements was carried out to measure the surface charge of the globules, which functioned to maintain optimum distance and prevent coalescence (
The stability test consisted of two tests: the centrifugation test and the heating and cooling test, which tested the physical stability of the microemulsion base. A centrifugation test was performed to determine whether or not there was a phase separation caused by gravitational force. The principle of centrifugation was to separate particles based on their molecular density, with centrifugal force causing particles with smaller thicknesses to be on top and particles with larger densities to go down (
Based on the optimization results and observations made on each base microemulsion formula, it was determined that the 1:9 formula was chosen. After obtaining the optimal microemulsion base, the clove flower essential oil microemulsion was made at a concentration of 10%. Clove oil microemulsion preparations and evaluations were carried out. These included observations of organoleptic properties, percent transmittance, pH, viscosity, determination of globule size, polydispersity index, and zeta potential. The results can be seen in Table
Evaluation | Results | |
---|---|---|
Day 0 | Day 30 | |
Organoleptic properties | Color: yellow, clear | Color: yellow, clear |
Odor: specific | Odor: specific | |
Form: one phase | Form: one phase | |
%Transmittance | 99.9±0.1 | 98±0.0 |
pH | 6.32±0.1 | 6.57±0.1 |
Viscosity | 269±3.77 | 222.2±15.7 |
Globule size (nm) | 17.69±0.025 | |
Polydispersity index | 0.057±0.0043 | |
Zeta potential (mV) | -5.36±0.23 |
The resulting transmittance percentage met the requirements where the microemulsion system was clear with a transmittance percentage close to 100%. Likewise, the pH measurements results fell within the expected range of 4.5–8.0. Afterward, characterization of the clove oil microemulsion preparation was carried out using a PSA (Particle Size Analyzer) to determine the size of the globules. The globule size distribution is a very important factor to determine the stability of a microemulsion preparation. The stability of the microemulsion depends on the droplet size in the dispersed phase. The results of the observation of the globule size in Table
The particle size distribution is an important characteristic of microemulsion systems as it can affect drug release and the stability of a microemulsion. It is expressed in terms of polydispersity index. The polydispersity index is categorized into two, namely, monodispersity (unimodal) and polydispersity (bimodal). The formulation of the clove oil microemulsion in this study was monodisperse (Table
The zeta potential predicted the stability of the clove oil microemulsion preparation. The interaction between particles has an important role in the stability of a colloid, while the zeta potential is a measure of the repulsive strength between the particles (
A thermodynamic stability test was carried out to see the physical stability of the microemulsion, which included centrifugation and a heating and cooling cycle. Centrifugation is a mixture separation method used to separate insoluble liquids and solids based on differences in the particle size of the mixed substances. In the centrifugation test, the phase separation that occurred in the clove oil microemulsion preparation was observed. The results showed that the clove oil microemulsion did not undergo phase separation ; thus, the clove oil microemulsion was declared stable.
The heating and cooling test refers to an accelerated condition of temperature fluctuations to determine the stability of the preparation during storage. Heating and cooling were performed to see if crystallization, phase separation, loss of viscosity, aggregation and precipitation occurred and if the changes that occurred were reversible or not. This test was carried out by testing the stability of the microemulsion alternately at low and high temperatures, each for 24 hours. The test was carried out for 6 cycles. The results showed that the preparation could pass 6 cycles well. The preparation remained clear, homogeneous, and with no separation. This shows that the changes in properties that occurred when the preparation was stored at a high temperature of 40 °C or a low temperature of 4 °C were reversible.
Pseudo-ternary phase diagrams of mixtures of oil phase, surfactant, co-surfactant, and water were created. The microemulsion prepared with 7 w/t% of oil phase, 60 w/t% of surfactant, and co-surfactant mixture (S-mix ratio of 2:1), 33 w/t% of water, and 10 w/t% of clove oil was found to have the most favorable characteristic in terms organoleptic properties, % transmittance, pH, viscosity, physical stability, globule size, polydispersity index, and zeta potential. The use of this novel approach to delivering pharmacologically active natural oils in this study can indeed prepare a base research for the upcoming survey so we can see a simultaneous commercial formulation shortly.
The researchers would like to thank the Ministry of Education and Culture of the Republic of Indonesia for having funded this research with contract number 1989.10/LL5-INT/PG.02.00/2022 and to Ahmad Dahlan University and Muhammadiyah University of Gombong for facilitating and assisting this research.