FORMULATION AND EVALUATION OF MICROEMULSION CONTAINING NEEM SEED OIL

Objective: The objective of the present study was to formulate Microemulsion containing seed oil. Neem seed (Azadirachtaindica) oil was extracted from its seeds by the soxhlet apparatus. Acetone is used as a solvent. PEG 400 and Carbopol 940p was select as surfactant, co-surfactant and hydrogel thickening agent. Microemulsions were characterized for pH, viscosity, spreadability, in vitro drug transport study and in vivo antibacterial activity and shows satisfactory results. Antibacterial activity of formulation against E. coli Shows at a concentration of 3%. The neem seed oil microemulsion has the potential for antibacterial activity. Methods: A ratio of surfactant and cosurfactant i.e, S/CoSchoosen and corresponding mixture was made. The mixture was mixed with oil. Each mixture was mixed thoroughly using magnetic stirrer until homogenous dispersion/solution was obtained. Double distilled water was used in this formulations as to prevent the incorporation of surface active impurities. The mixture was titrated with water and ambient temperature with constant stirring at the endpoint where the mixture become clouded, the quantity of aquous phase added. The percentage of three different pseudo-phases incorporated were calculated. Results: Solubility studies in various solvents reveals that the oil is insoluble in distilled water and ethanol. Soluble in methanol. Conclusion: It was observed that the microemulsion having multilamellar nature. Batches with carbopol shows better homogenous distribution. The stability of microemulsion prepared with carbopol 71 was gretter than with xanthan gum. The in vitro study of microemulsion was performed and Batch (F7) is optimized batch which shows highest drug release.


INTRODUCTION
Microemulsions are thermodynamically stable isotropically clear dispersion of two immiscible liquids, like oil and water, stabilized by an interfacial film of surfactant molecules, with a size range of 5-200 nm and have very low interfacial tension [1]. Microemulsions could be an alternative carrier in topical drug delivery and as it has high Solubilization capability and nanometer size, it is believed that microemulsion will be a better candidate in delivering drug topically. They are composed of surfactant, water, and oil having cosurfactants provide better therapeutic action when compared to the traditional cream and lotions [2].
It is a topical delivery in skin, which makes the drug delivery difficult. This factor is consideration of preparation of microemulsions which have low skin irritation, high drug loading capacity, It reduce the diffusion barrier of corneum by dissolving the lipids in the Stratum corneum and enhancing the permeation of drug [3].
Traditionally neem seed oil and leaves used as insect repellent and as pesticides. Neem oil containing Azadirachatin could be used in hair care formulation due to their antiheadche, antidandruff, and antifungal properties [4]. Almost all parts of neem have been used as traditional Ayurvedic, in unani, and siddha medicine in india. Neem oil is used to control various skin infections [5].
The aim of the present study is to formulate topical microemulsion using Neem seed oil, tween 80, PEG 400, xanthan gum, carbopol 940. Tween 80 is a non-ionic, non-toxic surfactant that is not affected by change in pH. The antibacterial efficacy of the formulated microemulsion is additionally investigated.

Extraction of neem seed oil [6]
Neem seeds were washed three times thoroughly till no dust or other impurities left. Neem seeds were heated in a temp of 50 °C for one hour so they would dry out so kernels can be separated from wooden parts of seeds. The kernels were grounded using grinder. Soxhlet apparatus used for the extraction of neem seed oil. The kernel powder was placed into the soxhlet apparatus. Acetone is used as solvent in the ratio of 1:5 (W/V). The solvent was hated at 50 °C for eight hours so no oil left in the neem kernel. Then the mixture of oil and solvent was kept in room temperature for 48 h so the solvent evaporates completely and eventually oil is left.

Determination of specific gravity
The specific gravity of the oil was determined using specific gravity bottle.

Determination of refractive index
This is physical attribute of triglycerine measures by the angle through which a beam of light is bent when passing through a thin film of melted fat few drops of oil were placed on one face of glass prism of a refractometer and it was gently spread, closed and tightened. Ample time is allowed for the prism and oil to achieve steady temperature. The refractive index was then read from demarcation line.

Determination of pH
The pH of oil was determined by using digital pH meter.

Preparation of standard calibration curve of neem oil [8]
Different concentrations of Neem oil were prepared with different solvents and scanned in the UV range. The wavelength 220 nm was selected for neem oil, where it show maximum absorbance. Lower concentrations does not show any absorbance, hence concentration (above 10 mcg) were taken for calibration. The calibration curves were linear over the concentration range of 30-70 ug/ml of neem oil. Absorbance versus concentration plotted

Formulation of microemulsion [6]
A ratio of surfactant and cosurfactanti. e,S/CoSchoosen and corresponding mixture was made. The mixture was mixed with oil. Each mixture was mixed thoroughly using magnetic stirrer until homogenous dispersion/solution was obtained. Double distilled water was used in this formulations as to prevent the incorporation of surface active impurities. The mixture was titrated with water and ambient temperature with constant stirring. At the end point where the mixture become clouded, the quantity of aquous phase added. The percentage of three different pseudo-phases incorporated were calculated. Same procedure was followed for all S/Co ratios. Phase diagrams were prepared after calculating the percentage of each phase required to form microemulsions. After preparing the pseudo ternary phse diagram the medicated microemulsions were formulated.

Evaluation of microemulsion [9]
Physical appearance Prepared microemuisions were observed for color, homogeneity, consistency, texture, and pH.

Spreadability measurement
The spreadability of the microemulsion was determined by placing 0.5 g micoemulsion within a pre-marked circle of 1 cm diameter on a glass plate over which a second glass plate was placed. A weight of 5 g was allowed to rest on the upper glass plate for five min. The increase in the diameter due to spreading of the microemulsion was noted, and the mean diameter was taken.

Globule size determination
The globule size of the microemulsion was determined by optical microscope.

Viscosity
It was determined at 25±1 °C by means of Brook field viscometer.

In vitro skin permeation [10]
In vitro skin permeation study was performed by using Franz diffusion cells with an effective diffusion area of 2.8 cm 2 . The excised skin samples of rat were clamped in between donar and receptor compartment of Franz diffusion cell.1g of sample was added to the donar compartment. The receptor compartment was filled with PBS pH 7.4 and maintained at 37 °c with stirring at 100 rpm. At predetermined time of interval 3 ml of sample was withdraw and fresh same quantity of sample was added. Samples were filtered through whatman filter paper and analysed by UV spectrophotometer at 220 nm. The amount of drug permeted per unit surface area (µg/cm 2 ) was plotted versus time (h) and the flux (µg/cm 2

Antimicrobial study [11]
) was calculated from slope of the line.
Antimicrobial test was performed on previously prepared agar plates. On which E. coli saline solution was spread with the help of glass spreader. Wells were prepared with the help of borer, samples were withdrawn with the help of micropipette (100ug/ml) and poured into the prepaped wells. Then plates were incubated at 37 °c for 24h. And zone of inhibition was measured with the help of scale after 24h.

Determination of solubility study
Solubility studies in various solvents reveals that the oil is insoluble in distilled water and ethanol. Soluble in methanol.

Calibration curve for neem oil
Calibration curve for Neem seed oil by UV spectrophotometer. The equation found was Y=mx+c and R 2 value was 0.9985. The concentration of drug was in the range of 30-70µg/ml.

Antimicrobial activity of microemulsion
Antimicrobial activity of oil was performed on E. coli. The inhibitory action was showed at concentration of 3%. The seed oil at concentration of 0.3% on agar plate was found to be active against Staphylococcus Aureus and at 0.4% was active against Salmonella typhosa. The Neem seed oil was found to be inactive against Pseudomonas aeruginosa.
The bacteria (E. coli) was tested against marketed formulation, as a placebo and with oil.

CONCLUSION
Neem seed oil microemulsion was prepared in order to enhance skin penetration. Microemulsion were prepared by phase titration method using tween 80 surfactant or PEG 400 as cosurfactant and thickening agent. Neem seed oil as antibacterial agent. It was observed that the microemulsion having multilamellar nature. Batches with carbopol shows better homogenous distribution. It is also depends on stirring time. Batches with carbopol 71 having better viscosity than batches with xantham gum. The stability of microemulsion prepared with carbopol 71 was gretter than with xanthan gum. The in vitro study of microemulsion was performed and Batch (F7) is optimized batch which shows highest drug release. The formulation tested against bacteria (E. coli) and have antimicrobial activity. The bacteria tested against marketed formulation, neem seed oil microemulsion, and as a placebo. Highest zone of inhibition was observed by Neem seed oil microemulsion.

ACKNOLEDGEMENT
The auther's are thankful to D. S. T. S. Mandals college of Pharmacy Solapur for providing raw materials and necessary facility to carry out this research work successfully. The auther's are also thankful to Dr. Yogesh S. Thoratfor providing timely guidance and assistance.