Int J Pharm Pharm Sci, Vol 6, Issue 9, 485-489Original Article

PREPARATION AND CHARACTERIZATION OF NIGELLA SATIVA MICROEMULSIONS

NURUL HAFIZAH MOHD NOR1, MOHD AFFENDI MOHD SHAFRI2, 3, FARAHIDAH MOHAMED1, 3, 4*

1Department of Pharmaceutical Technology, Kulliyyah of Pharmacy, International Islamic University Malaysia (IIUM), 25200 Kuantan, Malaysia, 2Department of BioMedical Science, Kulliyyah of Allied Health Sciences, International Islamic University Malaysia (IIUM), 25200 Kuantan, Malaysia, 3International Institute of Halal Research & Training (INHART), Ground Floor, Block E0, Kulliyyah of Engineering, IIUM, P. O. Box10, 50728, Kuala Lumpur, Malaysia, 4IKOP Sdn Bhd, Kulliyyah of Pharmacy, International Islamic University Malaysia (IIUM), 25200 Kuantan, Malaysia.
Email: farahidah@iium.edu.my

Received: 03 Aug 2014 Revised and Accepted: 04 Sep 2014

ABSTRACT

The aims of this study were to develop and characterize an oil-in-water (o/w) Nigella sativa (N. sativa) microemulsion. The microemulsions were prepared by drop-wise titration of N. sativa oil into mixtures of surfactant blends (Span 20, Span 80, Tween 20, Tween 80, Tween 85) and water. All transparent ternary mixtures were characterized for their viscosity and droplet size. The stability of the microemulsion was evaluated by subjecting them to stressful conditions, namely centrifugation (2000 g for 20 minutes) and heating in a drying oven (60 °C to 105 °C for 5 hours) and the droplet size was determined following one month storage at room temperature (25 °C) thereafter. Based on the results, a phase diagram was constructed from corresponding volumes of those 3 components. N. sativa mixtures (ranging from 7.4% to 10.7%) prepared at HLB 16 of surfactant blends (Tween 20: Tween 80; 6:4) with water (ranging from 17.9% to 18.5%) yielded transparent liquids. The constructed phase diagram displayed regions of a few types of microemulsion and emulsion. Interestingly, droplet size of freshly prepared mixtures was wider in range (5 to 15.6 nm) than the size following stressful condition (11.3 to 12.4 nm). It was concluded that N. sativa oil could be formulated into microemulsion at specific HLB value of surfactant blends. Such system was envisaged to enable routine rapid in vitro test on neuron cell lines loaded with N. sativa oil or possibly other lipophilic materials whenever viewing of neurite extension is required.

Keywords: Nigella sativa, Microemulsions, Sorbitan-based surfactants, Phase diagram

INTRODUCTION

Nigella sativa L. (N. sativa) from Ranunculaceae family is a plant that is usually found in the Mediterranean area, the desert of the Middle East, the South Asia, and the Far East. The oil has been used to treat human illnesses since Pharaoh’s era and is often mentioned in some of the oldest religious and medical manuscripts. N. sativa was selected as the model lipophilic agent in this study to evaluate its potential as neuro-regenerative agent. It was reported earlier that N. sativa carries numerous therapeutic effects including as antioxidant, anti-diabetic and anti-inflammatory [1]. Active constituents that thought to be responsible for its multiple therapeutic properties include several fatty acids, nigellicine, arvacrol, thymoquinone, tannins, essential amino acids, and oxy-coumarin [2]. Studies also had been done on the main active constituent of N. sativa, thymoquinone and it showed potential pharmacological effect on pathologies implicating neuro-degeneration such as cerebral ischemia [2]. In a study done by Kanter [3] on sciatic nerves in experimental diabetic neuropathy, it showed that there were fewer morphologic alterations had occurred to the drug-induced diabetic rats after treatment with thymoquinone and especially N. sativa. Myelin breakdown had significantly reduced and ultra structural features of axon had remarkably improved. In another separate study, it was also found that thymoquinone was effective in preventing hippocampal neuro-degenerative in rats exposed to toluene [4].

However, it is not feasible to directly test the N. sativa oil on cell line due to its immiscibility with the aqueous media. This makes rapid screening for therapeutic activity impossible. Although an emulsion system of the oily agent of interest can be formulated to improve its miscibility with the aqueous media, its high turbidity makes viewing under the microscope difficult. Formulation of a clear and transparent liquid form of N. sativa is required particularly in the study of its ability to induce neurite extension on the neuron cell line. A microemulsion was proposed to be the best form of the oily agent for the purpose.

Microemulsions have been extensively used to improve solubility of poorly water-soluble materials [5]. It is defined as the dispersion of oil and water that requires stabilization by surfactants to impart a thermodynamically stable system [6]. The microemulsions can be spontaneously formed, but at a narrow range of concentration of the three components. The size of microemulsion must be below 100 nm, which is significantly smaller than emulsions and must be accompanied by a transparent characteristic [7]. Microemulsion can be further categorized into three types; Type I (oil-in-water microemulsion); Type II (water-in-oil microemulsion) and Type III (bicontinuous microemulsion) [8]. All these can be distinguished based on the nature of the dispersed phase and the dispersion medium of the microemulsion. A co-surfactant free of oil-in-water (o/w) microemulsion could be employed using a mixture of nonionic surfactants [9], in which the hydrophilic surfactants (Tween 80, 60, 40 and 20) were combined to the hydrophobic surfactants (Span 80, 60, 40 and 20). In this study, sorbitan-based surfactants with different combination were tested in the preparation of sorbitan-based N. sativa microemulsions.

MATERIALS AND METHODS

N. sativa oil, imported from Karachi, Pakistan was supplied by Al-Mustafa Enterprises (Brand Name: Hemani). Surfactants namely Span 20 (HLB = 8.6), Span 80 (HLB = 4.3), Tween 20 (HLB = 16.7), Tween 80 (HLB = 15), Tween 85 (HLB = 11) were purchased from Merck (Germany). Water of deionized grade was used.

Preparation of N. sativa microemulsions

Briefly, surfactants were prepared by blending Span 80, Tween 80, Span 20, Tween 85 and Tween 20 at pre-determined HLB values (Figure 1). Spontaneous emulsification was then applied by mixing deionized water with appropriate surfactant blends at different ratios (Table 1) using magnetic stirrer for 10 minutes at 640 rpm. After that, the N. sativa oil was titrated drop-wise until a transparent solution was formed. Only formulation that appeared transparent was further characterized and subjected to stress-testing.

Determination of Droplet Size

The size analysis was conducted for fresh microemulsion and following storage at either stressful or at normal condition (25°C). The droplet size of the microemulsions was analyzed by Zetasizer Nano series ((Malvern Instrument Ltd., United Kingdom)) by briefly loading 1.5 x 10-3 L of individual microemulsion into a polystyrene cuvette. The reading was obtained at 173° detection angle. The droplet size was expressed as the volume mean diameter of triplicates.

Stress-testing and Characterization of the Microemulsions

Method previously employed by Cho et al. [9] was adopted. Briefly, the microemulsions were subjected to extreme conditions by centrifuging them at 2000g for 20 minutes and heated at 60°C to 105°C for 5 hours in a drying oven (Memmert, Germany). The microemulsions were observed by naked eyes for any phase separation following the stressful conditions. Apart from particle sizing, the zeta potential of the microemulsion was also measured. The measurement of the zeta potential was done using Malvern ZetaSizer Nanoseries Z (Malvern Instrument Ltd., United Kingdom), in which 1.5 x 10-3 L microemulsions were loaded into a clear disposable zeta cell and the reading was reported as mean ± SD.

Construction of Phase Diagram

The ternary diagram was plotted for evaluation by Triangular Diagram plotting Spreadsheet (TRI-PLOT, United Kingdom).

Statistical Analysis

Statistical analysis was performed where all data were represented as the mean value of triplicate samples. One-Way ANOVA (Minitab 16, USA) was used to determine the significant difference (p < 0.05) between the mean values.

RESULTS AND DISCUSSION

Formulation of N. sativa Microemulsions

Microemulsion needs a proper HLB value because it will indicate how the surfactant will behave in the solution. Hence, to manipulate the HLB value, combination of two surfactants is used. In order to ensure the presence of adequate number of surfactant molecules to stabilize the nanodroplets in the system, an optimum concentration of surfactants are used [10]. This is important to promote the formation of microemulsions within the range of 100 nm. Also, microemulsions will form under the influence of two factors; interfacial layer and interfacial tension [11].

Additionally, in the free dispersion energy, the droplet size of microemulsion systems is usually minimally equilibrated at 100 – 1000Å of change [7]. For this reason, an optimum surfactant concentration is required in accommodating and in consideration of these interfacial areas. The most important outcome in this study is in the production of flexible layer and a very low tension at the interface of water/oil of the microemulsions.

Fig. 1: A flowchart to describe preparation of sorbitan-based N. sativa microemulsions


Table 1: Preparations of 16 formulations made up of various surfactant types, blends and different HLB. Abbreviation used: F = Formulation; W = water; O = oil, S = surfactant; S80 = Span80; S20 = Span20; T20 = Tween20; T80 = Tween80; T85 = Tween85

F Percentage (%) Surfactant Blends HLB
W: S (W: S): O W S O
1 - - 66.7 33.3 - -
2 - - 33.3 66.7 - -
3 - - 8.3 16.7 75.0 83% S80 / 17% T85 5.5
4 - - 16.1 64.5 19.4 68% S80 / 15% S20 / 17% T20 7
5 18.2:81.8 25:75 - - - 68% S80 / 15% S20 / 17% T20 7
6 16.7:83.3 25:75 - - - 28% S80 / 72% T85 9
7 - - 14.3 71.4 14.3 28% S80 / 72% T85 9
8 - - 4.8 23.8 71.4 60% T20 / 40% T85 14
9 - - 27.8 55.5 16.7 60% T20 / 40% T85 14
10 - - 12.7 75.9 11.4 60% T20 / 40% T85 14
11 - - 25.0 60.0 15.0 60% T20 / 40% T85 14
12 - - 17.2 69.0 13.8 60% T20 / 40% T80 16
13 - - 22.0 65.0 13.0 60% T20 / 40% T80 16
14 - - 17.9 71.4 10.7 60% T20 / 40% T80 16
15 - - 18.2 72.7 9.1 60% T20 / 40% T80 16
16 - - 18.5 74.1 7.4 60% T20 / 40% T80 16

Referring to (Table 2), formulation 1 and 2 were used as the controls in this experiment. This was because; to form a microemulsion, a sufficient amount of surfactants were needed in the formulation. As formulation 1 and 2 lack in the surfactant, no microemulsions should form in the formulae.

Type II microemulsions were prepared using surfactant blends with HLB values of 5.5 and 7, resulted in formulation 3 and 4. However, these formulations failed to produce microemulsions as they appeared turbid. This implied that N. sativa oil was incompatible with the surfactant blends’ chain length [12]. The results suggested that fatty acid compositions in N. sativa oil interacted differently in a ternary phase constituted of surfactants, water and oil.

Formulation 5 (HLB 7) and 6 (HLB 9), a modified formulation adopted from a study by Rukmini et al. [13] were also fabricated. They were of Type II and Type I microemulsion systems, respectively. However, these also did not produce microemulsions, probably because the oil type and surfactant blends used in this study was different than that of Rukhmini's.

As the theoretical HLB values of 8 to 16 from the pseudoternary graph were indicative of a suitable oil-in-water microemulsion system, hence the formulations 6 to 16 were prepared. Type I of microemulsion system was prioritized in this project, as the aqueous phase must be created in order to give a whole homogenized system when loading into the cell lines. These medium HLB values also indicated that a more stable oil-in-water microemulsion might be formed [14]. Therefore, at the very first place in this experiment, formulation 12 using combined surfactants of HLB 16 (60% Tween 20 and 40% Tween 80) successfully produced transparent solution. Following this result, the formulation 13 (HLB 16), 14 (HLB 16), 15 (HLB 16) and 16 (HLB 16) were then generated with little modification in terms of the oil concentrations and the volume of surfactant blends. However, of all the transparent solutions produced, only formulation 14, 15 and 16 exhibited nano range size (Figure 2).

Table 2: HLB values of sorbitan-based N. sativa micro emulsions. Abbreviation used: F = Formulation; W = water; O = oil, S = surfactant; S80 = Span80; S20 = Span20; T20 = Tween20; T80 = Tween80; T85 = Tween85

F Surfactant Blends HLB Value Observation
1 - - Cloudy
2 - - Cloudy
3 83% S80 / 17% T85 5.5 Cloudy
4 68% S80 / 15% S20 / 17% T20 7 Cloudy
5 68% S80 / 15% S20 / 17% T20 7 Slightly cloudy
6 28% S80 / 72% T85 9 Cloudy
7 28% S80 / 72% T85 9 Cloudy
8 60% T20 / 40% T85 14 Cloudy
9 60% T20 / 40% T85 14 Slightly cloudy
10 60% T20 / 40% T85 14 Slightly cloudy
11 60% T20 / 40% T85 14 Translucent
12 60% T20 / 40% T80 16 Transparent
13 60% T20 / 40% T80 16 Transparent
14 60% T20 / 40% T80 16 Transparent
15 60% T20 / 40% T80 16 Transparent
16 60% T20 / 40% T80 16 Transparent

Fig. 2: Physical appearance of successful sorbitan-based N. sativa microemulsions

In this experiment, different concentrations of the N. sativa oil have been formulated using different HLB value of the surfactant blends. It was shown that at HLB 16, a high concentration of the N. sativa oil would produce a cloudy solution. Hence, the microemulsion systems were achieved when oil concentrations were reduced in corresponding to the decrement in the surfactant blends concentration to the lowest concentration possible.

Droplet Size of N. sativa Microemulsions

In measuring the particle size of the microemulsions, measurement was done only to trial preparations with transparent appearance as this characteristic gave a high possibility of the microemulsion production. Therefore, only formulations 11 to 16 were tested. The result showed that only formulation 14, 15 and 16 could be defined as microemulsion (Table 3) as they exhibited less than 100 nm range of particle size. The result One-Way ANOVA posthoc Tukey’s test multiple comparison also showed that these three formulations were significantly different (p < 0.05) having 97.50% of individual confidence level.

Table 3: Physical characteristic of sorbitan-based N. sativa microemulsions

Formulation Percentage (%) Mean Particle Size before Storage ± SD (nm) Mean Particle Size after
Storage ± SD (nm)
Water Surfactant Oil
14 17.9 71.4 10.7 4.99 ± 0.89 12.39 ± 0.17
15 18.2 72.7 9.1 8.06 ± 1.84 11.43 ± 0.19
16 18.5 74.1 7.4 15.62 ± 2.3 11.31 ± 0.08

Abbreviation used: F = Formulation; SD = Standard deviation

These formulations remained transparent with no turbidity after being subjected to vortex mixer. In contrast, formulations 11, 12 and 13 were not termed microemulsions despite their translucent/ transparent appearance by naked eyes observations as their droplets size was not in the nanodiameter region.

Stress-testing and Characterization of the Microemulsions

Some of the characteristics of a stable microemulsion are (a) it does not give phase separation, (b) it forms spontaneously at room temperature and (c) it requires very little energy as compared to conventional emulsions. Hence, to check for the thermodynamic stability of N. sativa formulations, the following stress-testings were conducted (Table 4).

For stability study of the microemulsion systems, the zeta potential measurements were taken, in which for formulation 14, the reading obtained was -35.4 ± 1.20 mV (Figure 3), followed by formulation 15 having -32.27 ± 1.20 mV (Figure 4) and formulation 16 with -32.03 ± 2.00 mV (Figure 5). The high value of these readings indicated that microemulsions have excellent dispersion stability. Also, this showed that the systems remained stable upon storage, which was later confirmed by turbidity observation following storage for a month at room temperature.

Table 4: The stress-testing observation for phase separation of sorbitan-based N. sativa microemulsions

Formulation Centrifugation Heating
14 No separation No separation
15 No separation No separation
16 No separation No separation

Fig. 3: The triplicate measurement of the zeta potential of formulation 14


Fig. 4: The triplicate measurement of the zeta potential of formulation 15


Fig. 5: The triplicate measurement of the zeta potential of formulation 16

The stability of the microemulsions can be evaluated via the turbidity changes [2]. Turbidity is parallel to the mean diameter of the particles, thus giving it the most common method in measuring microemulsions stability. The turbidity of formulation 14, 15 and 16 was negative, indicating that the microemulsions remained stable following exposure to stressful conditions. The microemulsions were also monitored for one month storage at room temperature.

There was no significant change in the appearance of the microemulsion, and turbidity result was negative, even after the stored microemulsions were heated and centrifuged (Table 5). This might happen due to the behaviour of the polysorbate chain [10], in which any change in the turbidity of the microemulsion system was probably affected by the changes in the size of the head-group of the surfactant systems [15].

Table 5: The stability study of sorbitan-based N. sativa microemulsions

Formulation Storage Centrifugation after Storage Heating after Storage
14 Clear Clear / No separation Clear / No separation
15 Clear Clear / No separation Clear / No separation
16 Clear Clear / No separation Clear / No separation

Phase behavior of N. sativa microemulsions

The components of microemulsion structure can be explained through a pseudoternary phase diagram. In high concentration of oil, the water molecules solubilized-reverse micelles can be produced by the surfactant in its hydrophilic portion [10]. Lack of water will result in the change from a clear to turbid emulsions. In contrast, continuous addition of water produces water-in-oil microemulsions in which the interfacial layer of the combined surfactants will contain stable water droplets. More addition of water will result in a translucent region in which the water is shoved between double layers of surfactants. However, if this continues, the lamellar structure will break down, thus forming oil-in-water microemulsions, where surfactants stabilized-oil droplets become the continuous phase in the system.

Fig. 6: Pseudoternary diagram of sorbitan-based N. sativa microemulsions

In order to determine the phase behaviour of the microemulsions, Triangular Diagram plotting Spreadsheet (TRI-PLOT, United Kingdom) was used to plot pseudoternary diagram using formulation 3 to 16. Formation of these microemulsion systems were explained in a pseudoternary phase diagram and their regions were observed (Figure 6). Formulation 3, 5, 6 and 8 were located at the lower right of the diagram, indicated that they were of the macroemulsion systems.

On the other hand, formulation 4, 7, 9, 10, 11, 12 and 13 were scattered at the upper middle of the diagram, suggesting the formation of the liquid crystal percolate or the bicontinuous structure of the solution systems [7]. In addition, formulation 14, 15 and 16 were closely spotted near to the upper left of the phase region, indicating that these formulations were oil-in-water microemulsions and corresponding to the hypothetical phase pseudoternary diagram [7].

In order to retain the good solubilizing effect of microemulsion, it must therefore be formulated accordingly. The most important one is the prudent selection of surfactants to obtain an ultralow interfacial tension (< 10-3mN/m) at the oil/water interface [7]. By monitoring parameters like concentration, structure and temperature, the surfactant could act as the building block to form aggregates of molecules. The chemical structure of surfactants must be considered as the microemulsion formation was highly influenced by the compatibility of the oil and surfactant chain length [12, 16]. Commonly, microemulsions contain high quantity of surfactants and hence have the propensity to be high in toxicity. As far as the safety usage of the surfactants is concerned, polysorbate is known to be safe for the human usage, as these surfactants are of the nonionic type. According to U. S. Food and Drug Administration (FDA); 10mg/kg body weight per day was considered as the acceptable daily intake (ADI) for 4 known surfactants Tweens; Tween 20, Tween 60, Tween 65 and Tween 80. For this reason, these surfactants are applied widely in the pharmaceutical industry, food as well as in cosmetics. They are inexpensive and most importantly, minimally toxic to living organisms [17]. In addition, there have been no occurrences of genotoxicity and carcinogenicity as reported by Japan Food Additives Commission [18]. Study also showed that taking 2g of Tween 20 for 3 times per day in one week gave no adverse effect of the human body system [18]. The highest amount of surfactant used in this study only 2.4g in total, fell within allowable safe limit and therefore, these microemulsions could be concluded as safe and non-toxic for human purposes. In this study, the method of using several surfactants in combination was employed, as combined use of surfactants has a few advantages. Among others, this can increase the stability properties, improve the loading of the bioactive compound and allow use of smaller size particle with good self-emulsifying properties [19]. Some co-surfactants such as phenol and ethanol are not used due to their toxicity and irritating potential, caused by the short and medium length chains of the alcohols [15]. They are also avoided as they can result in the partition in the continuous phase [20] and hence disrupt microemulsion formation.

CONCLUSION

Stable sorbitan-based N. Sativa oil-in-water microemulsions had been successfully produced by appropriate blending of surfactant types with N. sativa oil and water. The findings also suggested that range of HLB ratio for similar surfactant groups could be utilized as guideline to find correct ratio between the ternary components. The correct formulation of N. sativa microemulsion is very important to allow use in vitro study where it may influence the direction of studies involving N. sativa.

ACKNOWLEDGEMENT

This research was funded by the Ministry of Science, Technology and Innovation Malaysia (MOSTI) under grant number SF11-006-0035.

REFERENCES

  1. Najaran ZT, Sadeghnia HR, Asghari M, Mousavi SH. Neuroprotective effect of Nigella sativa hydro alcoholic extract on serum/glucose deprivation induced PC12 cells death. Physiology and Pharmacology 2009;13:263–70.
  2. Al-Majed A, Al-Omar F, Nagi M. Neuroprotective effects of Thymoquinone against transient forebrain ischemia in the rat hippocampus. Eur J Pharmacol 2006;543 Suppl 1-3:40-7.
  3. Kanter M. Effects of Nigella sativa and Its Major Constituent, Thymoquinone on Sciatic Nerves in Experimental Diabetic Neuropathy. Neurochem Res 2008;33 Suppl 1:87-96.
  4. Kanter M. Nigella sativa and derived thymoquinone prevents hippocampal neurodegeneration after chronic toluene exposure in rats. Neurochem Res 2008;33 Suppl 3:579-88.
  5. He CX, He ZG, Gao JQ. Microemulsions as drug delivery systems to improve the solubility and the bioavailability of poorly water-soluble drugs. Expert Opin Drug Delivery 2010;7:445-60.
  6. Nagarajan R, Ruckenstein E. Molecular Theory of Microemulsions. Langmuir; 2000. p. 6400-15.
  7. Bagwe RP, Kanicky JR, Palla J, Patanjali PK, Shah DO. Improved drug delivery using microemulsion: rationale, recent progress, and new horizons. Crit Rev Ther Drug Carrier Syst 2001;18(1):77-140.
  8. Shiau BJ, Sabatini DA, Harwell JH. Solubilization and microemulsification of chlorinated solvents using direct food additive (edible) surfactants. Ground Water 1994;32 Suppl 4:561-9.
  9. Cho YH, Kim S, Bae EK, Mok CK, Park J. Formulation of a cosurfactant-free o/w microemulsion using nonionic surfactant mixtures. J Food Sci 2008;73 Suppl 3:115-21.
  10. Patel MR, Patel RB, Parikh JR, Bhatt KK, Kundawala AJ. Microemulsion: as novel drug delivery vehicle. Pharm Info net [Internet] 2007 [cited 22 May 2012]. Available from: http: //www.pharmainfo.net/reviews/microemulsions-novel-drug-delivery-vehicle.
  11. Parekh K. Preparation, characterization, and in vitro protein release studies in pharmaceutically relevant lecithin microemulsions. The University of Toledo; 2011.
  12. Bayrak Y, Iscan M. Studies on the phase behavior of the system non-ionic surfactant/alcohol/alkane/H2O. Colloids and Surfaces A: Physicochemical and Engineering Aspects 2005;268 Suppl 1-3:99-103.
  13. Rukmini A, Raharjo S, Hastuti P, Supriyadi S. Formulation and stability of water-in-virgin coconut oil microemulsion using ternary food grade nonionic surfactants. Int Food Res J 2012;19 Suppl 1:259-64.
  14. Yuwanti S, Raharjo S, Hastuti, Pudji, Supriyadi. Stable o/w microemulsion formulation using combination of three nonionic surfactants with low, high and med. J Agritech Fakultas Teknologi Pertanian 2012;31 Suppl 1.
  15. Flanagan J, Singh H. Microemulsions: a potential delivery system for bioactives in food. Crit Rev Food Sci Nutr 2006;46:221-37.
  16. Fanun M. Properties of microemulsions based on mixed nonionic surfactants and mixed oils. J Molecular Liquids 2009;150 Suppl 1-3:5-32.
  17. Yaghmur A, Aserin A, Garti N. Phase behaviour of microemulsions based on food-grade nonionic surfactants: effect of polyols and short-chain alcohols. Colloids and Surfaces A: Physicochemical and Engineering Aspects 2002;209:71-81.
  18. Food Safety Commission (Japan). Evaluation report of food additives. Polysorbates (Polysorbates 20, 60, 65 and 80);2007.
  19. Li P, Ghosh A, Wagner RF, Krill S, Joshi YM, Serajuddin ATM. Effect of combined use of nonionic surfactant on formation of oil-in-water microemulsions. Int J Pharm 2005;288:27-34.
  20. Warisnoicharoen W, Lansley AB, Lawrence MJ. Nonionic oil-in-water microemulsions: the effect of oil type on phase behaviour. Int J Pharm 2000;198:7-27.