Shweta Vashist. et al. / International Journal of Biopharmaceutics. 2015; 6(1): International Journal of Biopharmaceutics
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1 48 e- ISSN Print ISSN International Journal of Biopharmaceutics Journal homepage: IJB FORMULATION AND EVALUATION OF PRONIOSOMAL GEL OF DICLOFENAC SODIUM BY USING 3 2 FACTORIAL DESIGN Shweta Vashist*, Sunil K.Batra, Satish Sardana Department of Pharmaceutics, Hindu College of Pharmacy, New-Delhi, India. ABSTRACT The aim of the present study was to formulate proniosomal gel of Diclofenac sodium by using 3 2 factorial design. Proniosomes are semisolid liquid crystalline product of non-ionic surfactant easily formed on dissolving the surfactant in minimal amount of acceptable solvent and least amount of aqueous phase. The proniosomes were prepared by using different concentration of surfactant (span 60) and cholesterol. They were optimized using 3 2 factorial designs to study the effect of independent variables, i.e. concentration of span 60 (X1) and cholesterol (X2) on dependent variables like vesicle size, % entrapment efficiency and % drug release at 24 hrs. The prepared proniosomal gel was characterized for ph, vesicle size, viscosity, spreadability, entrapment efficiency and exvivo drug permeation study (by modified franz diffusion cell). The drug release profile exhibited zero order kinetics. From the regression analysis, it was observed that all two independent variables have significant effect on response variables. Formulation was optimized using contour plot, predicted v/s actual plot and response surface plot. The optimized formulation was found to be F5 containing medium concentration of span 60 (1350 mg) and medium concentration of cholesterol (250 mg). The stability study of optimized formulation was also done. Results suggest that proniosomal gel can enhance delivery of diclofenac through skin and can improve the bioavailability of drug. Key words: 3 2 factorial design, Entrapment efficiency, Exvivo drug permeation, Proniosomal gel of Diclofenac sodium. INTRODUCTION The transdermal route is very useful, but the stratum corneum acts as major barrier which is present on the top of the epidermis and behaves as a rate limiting membrane for penetration of drugs. The vesicular drug delivery system is potentially beneficial as the vesicles tend to fuse and adhere to the cell surface, thus increasing the permeability of the drug (Vyas SP and Khar RK, 2001). They are useful vehicle for drug delivery of both hydrophobic drugs, which associates with the lipid bilayer and hydrophilic drugs, which are encapsulated in the interior aqueous compartment (Aggarwal D et al., 2007). The drug delivery using colloidal particulate Corresponding Author Shweta Vashist shwetavashist55@gmail.com carriers such as liposome or noisome but these system have some chemical problem associated with degradation by hydrolysis or oxidation as well as physical problems as sedimentation, aggregation, or fusion during storage. So, a novel approach were adopted in dealing with these problem, is proniosome which are converted to niosomes upon hydration (Cevc G, 2006). Proniosomes are provesicular approach which overcomes the limitations of other vesicular drug delivery systems. Proniosome is a compact semisolid liquid crystalline product of non-ionic surfactants easily formed on dissolving the surfactant in minimal amount of acceptable solvent and the least amount of aqueous phase. This compact liquid crystalline gel can be readily converted into niosome on hydration (Ashwani S et al., 2011).
2 49 Preparation of the noisome from the proniosomes can be achieved by two ways a.) Hydration by skin: The hydration is achieved by skin itself i.e. the water in the skin is used to hydrate the proniosome formulation and conversion to noisome (Mahdi et al., 2004). b.) Hydration by solvents: Aqueous systems i.e purified water, saline solution and buffers are used to convert proniosome to noisome with or without agitation and sonication. MATERIALS AND METHODS Materials Diclofenac sodium was gift sample from combitic caplet global Pvt. Ltd., Sonepat. Span 60 was gift sample from central drug house Pvt. Ltd., New- Delhi. Cholesterol and soya-lecithin was purchased from central drug house Pvt. Ltd. Carbopol 934 was purchased from central drug house Pvt. Ltd., New-Delhi. All other ingredients used throughout the study were of analytical grade and were used as received. Design of experiment for preparation of proniosomal gel A 3 2 design was used for exploring quadratic response surfaces and constructing polynomial models with Design Expert Version The two independent variables such as surfactant (A) and cholesterol (B) were selected on the basis of preliminary studies carried out before the experimental design being implemented. The experimental design was applied to investigate the effect of different independent variables such as A, B. The interaction term (A, B) shows how the response changes when two factors are changed simultaneously. The polynomial term (A 2, B 2 ) are included to investigate non linearity. METHOD OF PREPARATION OF PRONISOMAL GEL The gel formulation of Diclofenac sodium was prepared by using vesicular approach (proniosome). First, the proniosome was prepared and then mixed with carbopol 934 gel which is used as a penetration enhancer. Proniosome was prepared by co-acervation phase separation method. Precisely weighed amount of surfactant (span 60), lecithin (carrier), cholesterol, and drug was engaged in a clean and dry wide mouthed glass vial and alcohol 2.5 ml is added to it. After warming, all the ingredients are mixed well with a glass rod; the open end of the glass bottle is enclosed with a lid to prevent the loss of solvent from it and warmed over waterbath at C for about 5 min until the surfactant mixture is dissolved completely. Then the aqueous phase (0.1% glycerol solution) is added and warmed on a water bath for about 2 min. Then mixed with 2% w/v of carbopol gel which is converted into proniosomal gel and carbopol neutralize with triethanolamine (q.s) (Gupta A et al., 2008). CHARACTERIZATION OF PRONIOSOMAL GEL Entrapment efficiency:- Accurately weighed 200 mg of gel was dispersed in ph 7.4 phosphate buffer up to 10 ml. It was ultrasonicated for 10 mins. and centrifuged at 20,000 rpm at room temperature for 30 mins. The supernatant was taken and analyzed spectrophotometerically at 276 nm (Ankur Gupta et al., 2007). The percentage of drug encapsulation was calculated by this equation-ee (%) = [1- (Unencapsulated drug/total drug)] 100. Ex-vivo permeation study The diffusion studies were done to get an idea of permeation of drug through barrier from the transdermal system. In this work, Franz diffusion cell was used. Diffusion cells generally comprise two compartments, one containing the active compartment (donor compartment) and the other containing receptor solution (receptor compartment), separate by barrier i.e. rat abdominal skin. The outlet and inlet was connected with latex tube so the jacket had stagnant water inside and heat was provided by hot plate. The Teflon coated bead was used to stir the receptor solution using magnetic stirrer. The rat abdominal skin was placed on receptor compartment and both compartments held tight by clamps (Camelo Puglia and Francesco Bonina, 2008). RESULTS AND DISCUSSION ph The ph of all the nine formulations were in the range of 5.78 to 7.05 that suits the skin ph, indicating skin compatibility. Homogeneity All developed gels showed good homogeneity with absence of lumps. Vesicle size and shape The vesicle size of all the nine formulation ranged from 4.98 to µm. Vesicle size depends on cholesterol concentration. On increasing the concentration of cholesterol the hydrophobicity increases and surface energy decreases which subsequently reduces vesicle size. The vesicles are round in shape. Viscosity The viscosity of all the nine formulations ranged from to cps. Gels with high viscosity do not easily extrude from the tube whereas, low viscous gels may flow quickly and hence suitable viscosity is required to extrude a gel. Viscosity of F5 (72380 cps) formulation was found to be excellent when compared to other formulations.
3 50 Spreadability The spreadability of formulations ranged from to (g.cm/sec). The values of spreadability indicate that the gel is easily spreadable with minimal of shear. Extrudability The extrusion of the gel from the tube is an important parameter during its application. Gels with high consistency may not extrude from the tube whereas, low viscous gels may flow quickly. Hence suitable consistency is required to extrude a gel from the tube. Entrapment efficiency The entrapment efficiency of all nine formulations ranged from to 97.84%. The percentage entrapment efficiency was maximum when concentration of cholesterol was 250 mg and surfactant 1350 mg but on further increasing the concentration of cholesterol and surfactant the entrapment efficiency of drug decreased. This may be due to the reason that cholesterol molecule compete with drug for the space within the bilayer, remove the drug from the bilayer and in addition to this disrupt the vesicular membrane structure and mixed micelles formation along with the niosomal vesicles with high concentration of surfactant and this may lead to lower entrapment efficiency. Ex vivo drug permeation study The extent to which the concentration of excipients alters the drug permeation was studied by ex vivo permeation studies using rat skin as membrane barrier. Ex vivo permeation study gives the information about the behaviour of molecule in vivo. The amount of drug permeated gives the information about the amount of drug absorbed into the blood. The graphical representation of the cumulated percent of drug permeated as a function of time through rat skin is shown in Table 5.10 and Figure The percentage of drug permeation for various formulations (F1-F9) was to % respectively. Response analysis for optimization Statistical validation of polynomial equation generated by Design Expert Version and was established on the basis of ANOVA provision in the software. A total of 9 runs (F1-F9) were generated. The 3-D response surface plots were drawn using this software. The resultant experimental data of response properties were compared with that of the predicted values. Effect of cholesterol on vesicle size Vesicle size depends on cholesterol concentration. On increasing the concentration of cholesterol and surfactant the hydrophobicity increases and surface energy decreases which subsequently reduces vesicle size but further increasing it will increases the vesicle size. Effect of cholesterol and surfactant on entrapment efficiency The variation in the concentration of cholesterol significantly effect the entrapment efficiency. The observed entrapment efficiency was incresed significantly when cholesterol amount was increased from 200 mg to 250 mg, but further increase in cholesterol concentration from 250 to 300 mg decrease the entrapment efficiency. The reason behind decreased entrapment efficiency may be due to the reason that a cholesterol molecule will compete with drug for the space within the bilayer, remove the drug from the bilayer and in addition to this will disrupt the vesicular membrane structure. And the Initial increase in the concentration of span 60 from 900 mg to 1350 mg shows increase in entrapment efficiency may be due to increased in the number of niosomes formed; therefore, the volume of hydrophobic domain increases and hence increases in entrapment efficiency but further increases in concentration of span 60 shows decreases in entrapment efficiency due to the formation of mixed micelles along with the niosomal vesicles, which may lead to lower entrapment efficiency. Effect of cholesterol and surfactant on drug permeation The variation in the amount of cholesterol and surfactant showed the significant effect on the permeation of drug through the skin. The increase in drug permeation due to increase in surfactant concentration from 900 mg to 1350 mg may be due to the non-ionic surfactant present in it which modifies the stratum corneum and increase the thermodynamic activity of drug as well as skin vesicular partioning. And the cholesterol concentration did not show much singnificant effect in the permeation. Optimization data analysis Analysis of variance (ANOVA) and estimated regression coefficient were applied to evaluate response Y1, Y2, Y3. A series of experiments was carried out by considering a 3 2 full factorial design. Various statistical data (standard error of estimate, sum of squares of the errors, F statistics, and P value) were examined. Y 1 = A- 1.09B AB+ 3.47A B 2 Y 2 = A- 1.13B AB A B 2 Y 3 = A-1.37 B-1.69 AB A B 2 Stability study The optimized formulation (F5) was found to be stable for period of one month; it can be observed that the gel formulation showed no major alteration in relation to encapsulation efficiency and vesicle size.
4 51 Table 1. Correlation of actual and coded values Level Coded Value Actual value (mg) Surfactant (A) (Factor 1) Cholesterol (B) (Factor 2) Low Medium High Table 2. Formulation selected using 3 2 factorial design (coded values) S. No. Batch No. Independent variables A B 1. F F F F F F F F F Table 3. Composition of different formulations (Actual values) Formulation Drug (mg) Span-60 Carbopol Cholesterol 934(%) (mg) Soya lecithin (mg) F F F F F F F F F Table 4. The composition and observed response from randomized runs in 3 2 factorial design Batch Factor 1 Factor 2 Response Response Response No. Surfactant Cholesterol F F F F F F F F F Table 5. Stability study of optimized formulation (F5) Initial After 1 month S. No. Temp. Encapsulation Encapsulation Vesicle size efficiency efficiency Vesicle size 1. 2ºC 97.21± ± ± ± ºC 97.21± ± ± ± ºC 97.21± ± ± ±0.95
5 52 Fig 1. Preparation of proniosomal gel Fig 2. Graphical representation of ex vivo permeation study of F1 to F9 across rat skin data with different ratio of span 60 and cholesterol Fig 3. Contour curve plot and Response surface plot and linear correlation plot between predicted v/s actual plot showing the effect of surfactant (span 60) and cholesterol on response Y1.
6 53 Fig 4. Contour curve plot and Response surface plot and linear correlation plot between predictes v/s actual values showing the effect of surfactant (span 60) and cholesterol on response Y2 (Percent drug entrapped) Fig 5. Contour curve plot and Response surface plot and linear correlation plot between predictes v/s actual values showing the effect of surfactant (span 60) and cholesterol on response Y3 (Percent drug permeated)
7 54 CONCLUSION A successful attempt was made to develop proniosomal gel for transdermal delivery of diclofenac sodium by utilizing 3 2 factorial design by non-ionic surfactant(span 60): cholesterol, soya-lecithin using coacervation phase separation method. The results of exvivo skin permeation studied showed highest permeation of diclofenac sodium from formulation F5 containing span 60: cholesterol (1350: 250). The optimized formulation showed good stsbility at 30 days. REFERENCES 1. Aggarwal D, Pal D, Mitra A.K, and Kaur I.P. Study of the extent of ocular absorption of acetazolamide from a developed niosomal formulation humor. International journal of pharmaceutics. 2007; 338: Ankur Gupta, Sunil Kumar Prajapati, M Balamurugan, Mamta Singh, Daksh Bhatia. Design and development of a proniosomal transdermal drug delivery system for captopril. Tropical journal of pharmaceutical research. 2007; 6: Ashwani S., Murugesan S., Bharat K., Proniosome gel: A novel topical delivery system. International journal of recent advancement in pharmaceutical research. 2011; 3: Camelo Puglia and Francesco Bonina. Effect of polyunsaturated fatty acids and some conventional penetration enhancers on transdermal delivery of atenolol drug delivery. IJPS. 2008; 15: Cevc G. Lipid vesicles and other collids as drug carriers on the skin. Adv. Drug Deliv. Rev. 2006; 56: Gupta A, Singh M. Design and development of a proniosomal transdermal drug delivery system for captopril. Tropical journal of pharmaceutical research. 2007; Mahdi, Jufri, Effionora, Anwar. Preparation of Malodextrin DE 5-10 based ibuprofen proniosomes. Majalah Ilmu Kefarmasian. 2004; 1: Vyas SP and Khar RK. Niosomes. Targeted and controlled drug delivery novel carrier system. C.B.S Publication, 1st edition. 2001; 23:
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