22%, and for response Y3 (drug release in 12 h.) were 494.11–769.41. The fitted models could be viewed as regression equations as shown in Table 5 generated by the software (Design Expert 8.0.7.1). equation(4) Y1=324.07+57.50X1−75.12X2−62.50X3−67.50X1X2+91.50X1X3+78.25X2X3Y1=324.07+57.50X1−75.12X2−62.50X3−67.50X1X2+91.50X1X3+78.25X2X3 equation(5) Y2=95.65−0.91X1+1.765X2−0.8850X3+7.65X1X2+7.59X1X3+0.155X2X3−6.172X12−0.327X22+1.772X32

equation(6) Y3=531.75+15.88X1−5.275X2+72.35X3−52.94X1X2−3.552X1X3−14.11X2X3+14.70X12+0.589X22+113.53X32 see more The three dimensional plots were used to study the effects of two factors on the response at a time, when the third factor was kept at a constant level (Fig. 5, Fig. 6 and Fig. 7). GW-572016 supplier The drug entrapment efficiency (EE) was determined by measuring the concentration of free drug in the dispersion medium with ultrafiltration technique.12 The diluted sample was centrifuged at 5000 rpm for 10 min. The free drug from the sample was estimated by UV spectroscopic method. In vitro drug diffusion study was performed using the Diffusion cell assembly. Five hundred microliters of the sample was withdrawn at fixed time intervals and the same volume of fresh medium was added accordingly. Samples were analyzed by using UV spectroscopy method at 274 nm wavelength. All the operations were carried out in triplicate ( Table 4, Table 5 and Table

6). The optimized formulation F 5 and F9, F10 formulations which were better in the in vitro diffusion study were selected for in vivo rat skin permeability study. The permeability of the drug was quantified in terms of cumulative amount permeated per unit time and per unit area and the permeability was plotted against the time ( Table 7 and Table 8). The graph of permeability study showed the linearity in the permeation. The log amount of

drug permeated was also plotted with time and permeability coefficient and flux were determined for the optimized and other two formulations (Fig. 8 and Fig. 9). The permeability coefficient values were found to be significant and in agreement with the enhancement ratio of the formulation (Table 7 and Table 8). The primary irritancy index determined for optimized formulation, Florfenicol plain gel and vehicle were found to be 0.00, as no edema/erythema was observed. This ensures the safety of the formulation under study. In the in vivo animal study volume of inflamed paw goes on decreasing as time increases that shows drug acting on inflammation cause by Carrageenan. Optimized NLC gel showed significant reduction in paw volume as compared with the control as well as standard group. The formulation showed the reduction in the inflammation to the larger magnitude and also showed sustained action during the study period. From the graph of % inhibition rate with time in hours.