Titrating A Turbid Oil Biology Essay

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Hoar and Schulman coined the term microemulsion in 1943 to define a transparent system obtained by titrating a turbid oil - in - water emulsion with a medium - chain alcohol (Raid et al. 2008). Microemulsions are isotropic and thermodynamically stable dispersed micro-heterogeneous systems composed of water, oil and amphiphile. Their stability and unique solubilization properties have drawn attention for their use as vehicles for drug delivery. Microemulsion and other related colloidal systems have received increased attention during the past few years (Subramanian et al. 2005). Microemulsion contains nanometer-sized droplets of oil or water has several interesting characteristics namely: enhanced drug solubility, good thermodynamic stability, ease of manufacturing and enhancing effect on transdermal ability over conventional formulation. There are several permeation enhancement mechanisms of microemulsion such as an increased concentration gradient and thermodynamic activity toward skin and the permeation enhancement activity of the components of microemulsion. So far, much attention has been focused on the topical delivery of drugs (Jia et al. 2004). Microemulsions provides promising alternative for dermal and transdermal delivery of both hydrophilic and lipophillic drugs (Boltri et al., 1994, Kogan et al., 2006, Maghraby et al. 2008).

The high localized concentration of drug at the site of application that have been exploited using microemulsion systems with several advantages such as excellent for drugs with short half lives, bypasses first pass metabolism, reduced side effects, decreased dosing and it can be self administered (Raid et al. 2008). Skin provides huge area for drug application to the formulator; most drugs are difficult to be delivered into and through skin because of its barriers (Barry B.W, 1983). There has been increased interest during recent years in use of topical vehicle systems that could modify drug penetration into the skin. Many of the dermal vehicles contain chemical enhancers and strong solvents to achieve these goals (Sintov et al. 2004).

Flurbiprofen, 2-(2-flurobiphenyl-4yl) propionic acid is a non-selective non-steroidal anti-inflammatory drug (NSAID), related to ibuprofen and naproxen, that inhibits the cyclooxygenase enzyme necessary for the formation of prostaglandins and other autocoids. Flurbiprofen is used to treat rheumatoid arthritis, osteoarthritis, acute and chronic painful conditions. The gastrointestinal disturbances irritation and ulcerative effects in the stomach or intestine along with the short half-life (3-4 hours) has lead to the design and development of transdermal delivery of flurbiprofen microemulsion (Gilman et al.1990).

The purpose of this work was topical delivery of microemulsion have the potential to increase the solubility of poorly water soluble drugs, enhance the bioavailability of problematic drugs by increasing the local or systemic delivery of a drug also it can avoid the first pass metabolism and there is a potential to deliver the drug in a controlled manner and the possibility to immediate withdrawal of the treatment if necessary (Subramanian et al. 2004). Clove oil, essential oil that has the anti-inflammatory activity and anti-oxidant property because of the high concentration of eugenol (In vivo anti-inflammatory action of eugenol on lipopolysaccharide-induced lung injury. (Magalhães1 et al. 2010, Jirovetz et al. 2006). Generally clove oil used as a penetration enhancer for drugs. (Setty et al 2010).

The present aim was to prepare microemulsion using clove oil as an oil phase for improved transdermal delivery of Flurbiprofen. In this present work the microemulsions were prepared with different concentration of clove oil, Tween-20 and phosphate buffer and characterized.

Material and methods


Flurbiprofen was kindly gifted by Sun Pharma, Mumbai. Tween 20, Clove Oil, and Potassium dihydrogen orthophosphate were purchased from SD Fine Chemicals, Mumbai. Acetonitrile and Methanol (high-performance liquid chromatography [HPLC] grade) were purchased from Merck, Mumbai. All solvents used were HPLC grade and all chemicals were analytical grade.

Preparation of Flurbiprofen microemulsion

Flurbiprofen microemulsion was prepared by simple mixing of Flurbiprofen, clove oil, Tween 20 and phosphate buffer pH 7.4 by using vortex shaker. The compositions of formulations are given in Table.No.1 The rationale behind the absence of the cosurfactant in the formation of microemulsion is the use of higher HLB value, single nonionic surfactant (Tween 20) is probably well enough to reduce the interfacial tension between oil and water to form stable microemulsion which will minimizing the use of cosurfactant as well as the toxicicity of the same.

Preparation of Conventional gel

1% of drug weighed and dissolved in 10% Poly ethylene glycol 400. 1% of Carbopol was dispersed in minimum quantity of water. These two phases were mixed with vigorous stirring and prepared to 100gm with water.

Characterization of Microemulsion

Droplet size measurement and Surface morphology

The droplet size distribution, average droplet size of the Flurbiprofen microemulsions was characterized by photon correlation spectroscopy using a particle size analyzer (Microtrac Bluewave Particle Size Analyzer, USA ) with the measuring range from 0.01to 2000 microns. (Kyung et al. 1999).

Morphology and structure of the microemulsion were studied using transmission electron microscopy operating at 200kv (Philips EM 430 Transmission Electron Microscope, USA) and capable of point-to-point resolution. In order to perform transmission electron microscopy observations, a drop of the microemulsion was suitably diluted with water and applied on a carbon-coated grid, then treated with a drop of 2% phosphotungstic acid and left for 30s. The coated grid was dried under vacuum and then taken on a grid holder and observed under the transmission electron microscope (Adnan et al. 2009).

Transparency, pH & Viscosity Measurements

The transparency of the formulations was studied by visually observing the clarity of the formulations. pH values of the formulations were measured by a calibrated pH meter ( Zhu et al. 2009 and Chandra et al. 2008). The rheological property of the microemulsion was evaluated by using an Ostwald viscometer at 30°C. Experiments were performed in triplicate for each sample, and results were presented as average ± standard deviation

Skin Preparation

Skins were obtained from the abdominal region of wistar rat after removing hair carefully with a razor and then the subcutaneous fat and connective tissue were trimmed (Zhu et al. 2009). The excised skins were washed with 0.9% saline solution and examined for integrity then used for permeation studies.

In - vitro Skin Permeation Studies

Permeation of Flurbiprofen from the microemulsion across the excised skin was performed using Franz diffusion cells. The excised skin was clamped between the donor and the receptor chamber of vertical diffusion cells where the stratum corneum side faced the donor compartment, and the dermal side faced the receiver compartment with an effective diffusional area of 3.14 cm2. Phosphate buffer pH 7.4 was used as receptor medium (Haubing et al. 2004). Flurbiprofen microemulsion (1mL) was placed on donor side and the diffusion cell was maintained at 37°C by a re-circulating water bath and stirred at 600 rpm using a magnetic stirrer throughout the experiment. Aliquots were drawn from the receiver compartment and replaced immediately with an equal volume of fresh phosphate buffer pH 7.4 at predetermined time intervals and analyzed by HPLC at 248nm (Yin et al. 2008).

Dye binding studies

Full thickness hair removed rat skin was treated with a drop of microemulsion and a drop of water. Methylene blue staining on both pretreated skin was performed by 5 minutes surface application of few drops of methylene blue solution, followed by removal of unbind dye using sterile saline swabs and then with alcohol swabs. The skins were photographed using a digital camera (Canon, USA) (Chandra et al. 2008).

Histopathological examination of skin specimens

Abdominal skins of Wistar rats were treated with optimized microemulsion formulation. After 24 h, rats were sacrificed and the skin samples were taken from treated and untreated (control) area. Each specimen was stored in 10% formalin solution. The specimens were cut into section vertically. All section was dehydrated using ethanol, embedded in paraffin for fixing and stained with hematoxylin and eosin. These samples were then observed under light microscope and compared with control sample. In each skin sample, three different sites (epidermis, dermis and subcutaneous fat layer) were scanned and evaluated for mechanism of skin permeation enhancement (Faiyaz et al 2008)

Scanning Electron Microscopy

Freshly excised full thickness hair removed wistar rat skin was treated with microemulsions and fixed for scanning electron microscopy using 2.5% glutaraldehyde and washed; excess water was blotted and later skin samples were dried in vacuum. They were then mounted on a metal stub using double-sided carbon sticky tape and sputter coated using gold and then they were examined under scanning electron microscope (Zhu et al. 2009).

In-vivo anti-inflammatory activity study

The anti-inflammatory action of the optimized clove oil microemulsion (F-3) was evaluated by the carrageenan-induced hind paw edema method. Adult wister rats of either sex were divided six each per treatment group. The increase in volume (cm3) of the hind paw was measured with a LETICA digital Plethysmometer (LE 7500) before and at 20 min interval after the injection of egg albumin for a period of 2 h. Control rats received an equivalent amount of normal saline while ASA (150 mg/kg bw) served as reference. The percentage inhibition of oedema was calculated for each dose.

Result and Discussions

pH and Viscosity measurements

The pH and Viscosity of microemulsions are reported in Table 2. pH of all Flurbiprofen microemulsion formulations were around 7 and the viscosity was in range of 28 to 31 cps.

Droplet size measurement and TEM analysis

When the drug molecules are dispersed in the form of very small droplet size that offers a very large surface area for drug transfer to skin. This microstructure of the microemulsion provides the option to direct transfer of drug from the microemulsion through the stratum corneum.(Peltola et al., 2003). The droplet size range of clove oil microemulsion formulations varies from 58 nm to 60 nm (Figure 1). That indicates no change in droplet size when increasing the oil concentration of microemulsion. Hence the narrow droplet size of the clove oil microemulsions can be explainable to provide option to the direct transport of the flurbiprofen to skin.

The TEM image of clove oil Flurbiprofen microemulsion appeared dark and the surrounding surface were bright (Figure 2). It might be formed as a result of contact with water. The result shows that all the droplets were distributed uniformly.

The in-vitro skin permeation was studied and compared the drug permeation from three different formulations with conventional gel. In vitro skin permeation of Flurbiprofen microemulsions were found to be 45.60 %, 50.35 %, 93.45% at 24 hrs for F-1, F-2 and F-3 respectively. Flurbiprofen gel shows 39.08% of skin permeation at 24 hrs (Figure 3).Clove oil having the penetration enhancement property (Ref) and the use of high concentration of clove oil in the formulation (F3) results in a significant enhancement of the skin permeation when compared to other microemulsion formulations and gel.

The penetration enhancement is not only the microemulsion components. Likelihood the solubility of the drug in the skin is increased when microemulsion components can enter the skin as monomers (Kreilgaard, 2002).

Partitioning of the drug will increase into the upper layer of the skin creating high concentration gradient which is the driving force for transdermal drug delivery. This mechanism can be understandable by the resulting of enhancement of transdermal delivery of flurbiprofen from the clove oil microemulsion (F3), compared with the gel formulation.

The SEM picture (Figure 4) of microemulsion (F-3) treated skin showed that the microemulsion had a disruption in the stratum corneum (SC) while untreated skin remained undisturbed. It revealed that the enhancement effect on drug penetration was attaining by this structural change of SC which might be beneficial for topical delivery of flurbiprofen.

The Dye binding studies result showed that microemulsion treated area took up the dye whereas the rest of the skin remained impermeable. It indicates that the dye diffuses due to the formation of micro channels in stratum corneum (Figure 5).

Histopathological examinations of skin treated with formulation, control were done and the control group showed the well defined epidermal and dermal layers (Figure 6). The skin was treated with microemulsion showed significant changes in the skin morphology (Figure 6) such as drug adherence and disruption of lipid bilayer. These studies indicate the observed improvement in drug permeation might be due to the disruption of stratum corneum.

F-3 formulation was selected for the evaluation of anti-inflammatory activity based on higher drug permeation, optimum droplet size, and high viscosity. The anti-inflammatory activity of Formulation F-3 and conventional Flurbiprofen gel was evaluated in albino rats and compared. The percentage inhibition of edema after three hours of drug administration was found to be high with F3 treated groups (99.05 %) as compared with conventional gel treated groups (68.49%) and it was statistically significant at P<0.01.


The clove oil microemulsions containing Flurbiprofen (F3) was formulated and it possesses pH of 7, viscosity of 31cps, droplet size of 60 nm. Further F3 showed higher drug permeation and good in-vivo anti-inflammatory activity compared to conventional gel. The developed clove oil microemulsion might be used to provide better transdermal delivery of anti-inflammatory drug, Flurbiprofen.