Publication Ref No.

: IJPRD/2011/PUB/ARTI/VOV-2/ISSUE-12/FEB/003

ISSN 0974 9446

DEVELOPMENT OF ANTIFUNGAL EMULSION BASED GEL FOR TOPICAL FUNGAL INFECTION(S). Ankur Jain1*, Piyusha Deveda1, Naveen Vyas1, Jitendra Chauhan1, Hemant Khambete1, Dr.Sanjay Jain1
1

Ankur Jain

Smriti College of Pharmaceutical Education, 4/1 Pipliya Kumar Kakkad, Mayakheri road Indore-452010, India E-mail: ankurjain200@gmail.com

ABSTRACT Emulsion in gel have emerged as one of the most interesting topical drug delivery system as it has dual release control system i.e. emulsion and gel. Also the stability of emulsion is increased when it is incorporated in gel. Miconazole nitrate, a fungicidal, is effective for the local as well as Systemic fungal infections. The objective of this project was to develop an emulsion based gel for control delivery for miconazole to treat local as well as systemic fungal infections. In present work we prepare emulsion and then incorporated in Carbopol gel. The prepared formulation was evaluated on basis of pH, spreadability, Viscosity, drug content, in vitro release and Stability Studies. The result of studies reveled that the optimized batch shows 94.80% release in 24 hours and stable for around three months. The microbial assay was also performed using fungal strain and compared with marketed product, the result shows 39.7% inhibition of optimized batch where as marketed preparation shows only 28.3% inhibition. Hence it can be concluded that emulsion based system is more effective system for controlled delivery of antifungal agent(s).

Key Words: Emulsion, Gel, Emulgel, Miconazole nitrate, Topical Drug Delivery. INTRODUCTION Topical drug administration is a localized drug delivery system anywhere in the body through ophthalmic, rectal, vaginal and skin as topical routes. Skin is one of the most readily accessible organs on human body for topical administration and is main route of topical drug delivery system1.Topical drug delivery systems have been used for centuries for the treatment of local skin disorders, one side the topical applications of the drug offer the potential advantages of delivering the drug directly to the site of action and delivering the drug for extended period of time at the effected site that mainly acts at the related regions2. On the other hand, topical delivery system increases the

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contact time and mean resident time of drug at the applied site leading to an increase in local drug concentration while the pharmacological activity of Emulgel formulations may not change as rapidly as the solution form3. When applied to diseased skin, topical drug products induce one or more therapeutic responses, where onset, duration, and magnitude depend on the relative efficiency of three sequential processes, namely, (1) the release of the drug from the dosage form, (2) Penetration of the drug through the skin barrier, and (3) generation of the desired pharmacological effect. Because topical products deliver the drug directly to or near the intended site of action, measurement of the drug uptake into and drug elimination from the stratum corneum For the most part topical preparations are used for the localized effects at the site of their application by virtue of drug penetration into the underlying layers of skin or mucous membranes4. Although some unintended drug absorption may occur, it is sub therapeutics quantities and generally of minor concern5. Emulsified gel is stable one and better vehicle for hydrophobic or water insoluble drugs6. Emulsified gels are based on the two formulation emulsion and gel. An emulsion may be defined as a dispersion of two or more mutually insoluble liquids, one in the other7. The liquids are typically water and oil. The dispersion may either be that of oil in water (oil-in-water) or vice versa (water-in-oil) 8. A gel is an apparently solid, jelly-like material formed from a colloidal solution. By weight, gels are mostly liquid, yet they behave like solids due to the addition of a gelling agent. It is an emulsion either of the oil-in-water or water in oil type, which are gelled by mixing with a gelling agent 9. Oil-in-water emulsions are most useful as water washable drug bases and for general cosmetic purposes, while water-in-oil emulsions are employed more

widely for the treatment of dry skin and emollient applications 10.Emulsions tend to have a cloudy appearance, because the many phase interfaces (the boundary between the phases is called the interface) scatter light that passes through the emulsion11-13.A stable emulsion consisting of two pure liquids cannot be prepared; to achieve stability, a third component, an emulsifying agent must be present. Generally, the introduction of an emulsifying agent will lower the interfacial tension of the two phases14. Gels for dermatological use have several favorable properties such as being thixotropic, greaseless, easily spreadable, easily removable, emollient, nonstaining, compatible with several excipients, and water-soluble or miscible15-16. MATERIALS AND METHODS Materials: Miconazole nitrate gifted from Gufic Bioscience Ltd Mumbai, Carbopol 934; Carbopol 940; Tween 20; Span 20; Propylene glycol; Methyl paraben; Propyl paraben were purchased from loba chemie , Mumbai. Light liquid paraffin purchased from Rankam Mumbai. Ethanol; were purchased from c.y.company china. All chemicals were pharmaceutical grade and used without further modification. Analytical method The estimation of drug Miconazole nitrate in the samples collected from in vitro release studies was performed by a UV spectrophotometric method. In order to generate calibration curve, an accurately weighed amount of Miconazole nitrate was solubilize in the methanol to obtain a primary standards in the concentration range of 2-20 g/mL and the calibration curve was obtained by measuring their absorbance at predetermined max of 220 nm with a Simadzu1700 UV/visible

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Publication Ref No.: IJPRD/2011/PUB/ARTI/VOV-2/ISSUE-12/FEB/003

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spectrophotometer,. The concentration of Miconazole nitrate in test samples was calculated using the linear regression equation of the calibration curve (Absorbance = 0.0637 Concentration- 0.0262, r2 = 0.9921). The high value of correlation coefficient (r2) indicates the linearity of the calibration curve and the curve did not deviate significantly from the origin as indicated by its low value of intercept. The method was validated for accuracy and precision14. Preparation of formulation Emulsion based gel containing Miconazole nitrate was prepared. The Gel in formulations were prepared by dispersing Carbopol 934 in purified water with constant stirring at a moderate speed and Carbopol 940 in purified water with constant stirring at a moderate speed then the pH are adjusted to 6 to 6.5 using Tri Ethanol Amine (TEA). The oil phase of the emulsion were prepared by dissolving Span 20 in light liquid paraffin while the aqueous phase was prepared by dissolving Tween 20 in purified water. Methyl and Propyl paraben was dissolved in propylene glycol whereas drug (Miconazole nitrate) was dissolved in ethanol and both solutions were mixed with the aqueous phase. Both the oily and aqueous phases were separately heated to 70 to 80 then the oily C; phase were added to the aqueous phase with continuous stirring until cooled to room temperature. And add Glutaraldehyde in during of mixing of gel and emulsion in 1:1 and to obtain the emulsified gel4. Optimization Experimental design: Eight Miconazole nitrate emulsified gel formulations (Table 1) were prepared according to a 23 factorial design employing the qualitative factors and levels show in table 215. (Refer Table No. 01 & 02)

Characterization of Emulsion Based Gel Physical appearance The prepared emulsified gel formulations containing Miconazole nitrate were inspected visually for their color, homogeneity, consistency and phase separation. Measurement of pH The pH of various emulsified gel formulations was determined by using digital pH meter. One gram of gel was dissolved in 100 ml distilled water and stored for two hours. The measurement of pH of each formulation was done in triplicate and average values are calculated. Spreadability One of the criteria for an emulsified gel to meet the ideal quantities is that it should possess good spreadability. It is term expressed to denote the extent of area to which gel readily spread on application to skin or affected part. The therapeutic efficacy of a formulation also depends upon its spreading value. Spreadability is expressed in terms of time in seconds taken by two slides to slip off from emulsified gel and placed in between the slides under the direction of certain load. Lesser the time taken for separation of two slides, better the spreadability. It is calculated by using the formula. S = M. L / T Where M = wt. tied to upper slide L = length of glass slides T = time taken to separate the slides Rheological Study The viscosity of different emulsified gel formulation was determined at 370C using a brook field viscometer (Brookfield DV-E viscometer) with spindle 61. The recorded viscosities are collected in figure 2.

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Drug Content Determination Drug concentration in emulsified gel was measured by spectrophotometer. Miconazole nitrate content in emulsified gel was measured by dissolving Known quantity of emulsified gel in solvent (methanol) by Sonication. Absorbance was measured after suitable dilution at 220 nm in UV/VIS spectrophotometer (UV-1700 CE, Shimadzu Corporation, Japan). Microbiological assay Ditch plate technique was used. It is a technique used for evaluation of bacteriostatic or fungistatic activity of a compound. It is mainly applied for semisolid formulations. Previously prepared Sabourauds agar dried plates were used. Three grams of the emulsified gel are placed in a ditch cut in the plate. Freshly prepared culture loops are streaked across the agar at a right angle from the ditch to the edge of the plate. After incubation for 18 to 24 hours at 25C, the fungal growth was observed and the percentage inhibition was measured as follows : % inhibition = L2 / L1 100 Where L1 = total length of the streaked culture, and L2 =length of inhibition. In Vitro Release Study Franz diffusion cell (with effective diffusion area 3.14 cm2 and 15.5 ml cell volume) was used for the drug release studies. Emulsified gel (200 mg) was applied onto the surface of egg membrane evenly. The egg membrane was clamped between the donor and the receptor chamber of diffusion cell. The receptor chamber was filled with freshly prepared PBS (pH 5.5) solution to solubilize the drug. The receptor chamber was stirred by magnetic stirrer. The samples (1.0 ml aliquots)

were collected at suitable time interval. Samples were analyzed for drug content by UV visible spectrophotometer at 220 nm after appropriate dilutions. Cumulative corrections were made to obtain the total amount of drug release at each time interval .The cumulative amount of drug released across the egg membrane was determined as a function of time16. Stability studies The prepared emulsified gel formulations containing Miconazole nitrate were stored away from light in collapsible tube at 25 C, 40 and 4 for 3 months. After storage, the C C samples are tested for their physical appearance, pH, rheological behavior, drug release, skin irritation test and microbiological assay. RESULT AND DISCUSSION Physical examination The prepared Miconazole nitrate emulsified gel formulations were white viscous creamy preparation with a smooth and homogeneous appearance. Measurement of pH The pH values of all prepared formulation ranged from 5.5 to 5.8, which are considered acceptable to avoid the risk of irritation upon application to the skin because adult skin pH is 5.5. Spreadability The spreadability of various emulsified gel formulations are given below:

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Publication Ref No.: IJPRD/2011/PUB/ARTI/VOV-2/ISSUE-12/FEB/003

25 S p r e a d a b i l i ty g . c m . / s e c . 20 15 10 5 0 F1 F2 F3 F4 F5 F6 F7 F8 Formulation

ISSN 0974 9446

Fig 2. Viscosity of Miconazole nitrate Emulgel (Mean S.D) Drug content determination: 1 g of the prepared emulsified gel was mixed with 100 ml of suitable solvent (methanol). Aliquots of different concentration were prepared by suitable dilution after Sonication and filtering the stock solution and absorbance was measured. Drug content was calculated using the equation, which was obtained by linear regression analysis of calibration curve. The drug content of all emulsified gel formulation given below (fig.3);
100 90 80 % D r u g c o n te n t 70 60 50 40 30 20 10 0 F1 F2 F3 F4 F5 F6 F7 F8 Formulation

Fig 1. Spreadability of the various Emulgel formulations (Mean S.D) Rheological studies: The measurement of viscosity of the prepared emulsified gel was done with Brookfield viscometer (Brookfield DV-E viscometer). The emulsified gel were rotated at 10 (min.) and 100 (max.) rotation per minute with spindle 61. At each speed, the corresponding dial reading was noted. The viscosity of the emulsified gel was obtained (fig.2).
6000 5000 V i s c o s i ty c p s 4000 3000 2000 1000 0 F1 F2 F3 F4 F5 F6 F7 F8 Formulations maximum minimum

Fig 3. Comparing of drug content of various formulation of Miconazole Nitrate Emulgel (Mean S.D) Microbiological assay: The use of control plates showed that the plain emulsified gel bases were microbiologically inert toward the tested candida albicans strains. The antifungal activity of Miconazole nitrate in its different emulsified gel formulation as well as in its commercially available cream form. Percentage inhibition was taken as a measure of the drug antifungal activity. The emulsified gel formulations were found to have the same rank order in their antifungal activities as in

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the in vitro release studies. Thus, the greatest activity was observed with formula F5 and F6, where the percentage inhibition reached up to 39.7% and 38.5% respectively, while the lowest activity was found with F4 and F8, where the percentage inhibition was 29.9% and 30.9 % respectively. (Refer Table No. 03) In vitro Drug Release The in vitro release profiles of Miconazole nitrate from its various emulsified gel formulations are represented in Figure 4. It was observed that all the formulation had become liquefied and diluted at the end of the experiments, indicating water diffusion through the membrane. In general, it can be observed from figures that the better release of the drug from all emulsified gel formulation. The release of the drugs from its emulsified gel formulation can be ranked in the following descending order: F5 > F6 > F1 > F3 > F8 > F7 > F4 > F2, Where the amounts of the drug release of the drug released after 24 hours were 94.8%, 85.5%, 67.94%, 61.14%, 60.7%, 54.06%, 50.06% and 34.19%, respectively. Thus the higher drug release was observed with formulations F5 and F6. This finding may be due to presence of liquid paraffin in its low level/ high level and the emulsifying agent in its low level respectively, which lead to an increase in the hydrophilicity of the emulsified gel, which, in turn, facilitates penetration of the release medium into the emulsified gel and diffusion of the drug from the emulsified gel. Add 0.1% of gluteraldehide to give the retard the release of drug from emulsified gel formulation. Its proved that the presence of liquid paraffin led to retardation of Miconazole nitrate release from its emulsified gel formulation. Opposing to F5 and F6 formulation, F4 and F2 showed the lowest drug release. In formulation F4 and F2, liquid paraffin in its high level, while the emulsifying agent in its high level / low level respectively. This finding indicated that the lowering effects

of liquid paraffin on the release was more pronounced than the higher effect of emulsifying agents on the drug release. Although F5 is Carbopol 940-based, it showed a greater drug release than F1, which is Carbopol 934-based. The same is true for F2 and F3. This finding proved that the effect of liquid paraffin in increasing the drug release from the emulsified gel was more predominant than the decreasing effect of Carbopol 940 on drug release. Thus the 3 studied factors can be arranged according to their effect on the drug release from the emulsified gel formulation as follows: the liquid paraffin concentration > emulsifying agent concentration > the gelling agent type.
120 100 % D ru g R e le a s e 80 60 40 20 0 0 5 10 15 Time (min.) 20 25 30 F1 F2 F3 F4 F5 F6 F7 F8

Fig 4. Release profiles of Miconazole nitrate from its Emulgel formulations at 24 hours (Mean S.D) Stability studies All the prepared Miconazole nitrate emulsified gel were found to be stable upon storage for 3 months, where no change was observed in their physical appearance, pH, rheological properties and drug release.

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ACKNOWLEDGEMENTS: Authors are Thankful to Gufic Bioscience Ltd, (Mumbai, India) for providing free gift sample of miconazole nitrate and also thankful to Smriti college of pharmaceutical education, indore for work in the research article. REFERENCES 1. Kshirsagar A. Drug Delivery Systems. Indian Journal of Pharmacology 2000; 32:S54-S61. 2. Rashmi M. Topical gel: A review august vol. 2008;1-6. available from http:// www.pharmainfo.net. 3. Sharma S. Topical preparations are used for the localized effects at the site of their application by virtue of drug penetration into the underlying layers of skin or mucous membranes. Pharma Review 2008; 6:1-10. 4. Magdy I M. Optimization of Chlorphenesin emulgel formulation. American Association of Pharmaceutical Scientist 2004; 6: 1-7. 5. Siamak P and Mohammad N S. In-vitro release of Diclofenac Diethyl ammonium from lipid-based formulation. International Journal of Pharmaceutics 2002; 241: 185190. 6. Gondaliya D P and pundarikakshudu K. Studies on preparation, characterization and Transdermal permeation of nimesulide from aqueous and emulgel. Indian drugs 2002; 39: 465-473. 7. Julia W and Schreze I. Polysaccharide gel with multiple emulsions. Food hydrocolloids. 2005; 19:605-615. 8. Guido S, Fred V, Martien A, Cohen S, George A. Oil droplet release from emulsion filled gels in relation to sensory perception. Food hydrocolloids 2007; 21:977-985.

9. Hideaki T and Yuya K. Preparation of poly (N-isopropylacrylamide) emulsion gels and their drug release behaviors. Colloidals and surfaces B.Biointerfaces 2008; 67:9298. 10. Luana P, Cinzia P, Stefania M, Carlo R, Claudio N. Rheological and functional characterization of new anti-inflammatory delivery system designed for buccal administration. International Journal of Pharmaceutics 2008; 356:19-28. 11. Forster Th, Jackwerth B, Pitterman W, Rybinski W, Schmitt M. Properties of emulsions. Cosmetics and Toiletries 1997; 1:1-6. 12. Rieger M M, Lachman L, Lieberman Ha, Kanig Jl. Emulsions. The Theory and Practice of Industrial Pharmacy. Edn 3, Philadelphia, PA: Lea and Febiger; 1986, 502-533. 13. Shingel KI, Roberge C, Zabeida O, Robert M, Klemberg-Sapieha JE. Solid emulsion gel as a novel construct for topical applications: synthesis, morphology and mechanical properties. Journal of Material
Science Mater Med. 2008; 2:1-5.

14. Sheikh S, Faiyaz S, Talegaonkar S, Ali J, Baboota S, Alka A and Khar RK. Formulation development and optimization using nanoemulsion technique: A technical note. AAPS Pharm Sci Tech 2007; 8: E1E6. 15. Bolton S. Pharmaceutical statistics.inc; 3rd Ed., New York NY; 1997.p. 326-354. 16. Nathalie F, Valery G, Marie J S. The release of caffeine from hydrogenated and fluorinated gel emulsion and cubic phases. Colloidal and surfaces A. 2004; 243:117125.

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TABLES & FIGURES Table 1. Factor and level for the 23 factorial designs Factors (A) Gelling agent type (B) Liquid paraffin concentration (C) Emulsifying agent concentration

Levels +10 % -5% +7.5% -5% +2.5% - 1.5%

Table 2. Composition of miconazole nitrate emulsified gel formulation Composition Composition Formulation A B C + F1 + F2 + + F3 + + + F4 + F5 F6 + + F7 + F8 + paraffin concentration, C, emulsifying agent concentration, Factor at

(1) A B AB C AC BC ABC A, Gelling Agent type, B, liquid low level, +, factor at high level.

Table 3. Percentage inhibition as a criterion for the antifungal activity of miconazole nitrate in its different emulgel formulations

Formulation F1 F2 F3 F4 F5 F6 F7 F8 Marketed product

% Inhibition 31.6% 30.9% 31.6% 27.56% 39.7% 38.5% 32.6% 29.9% 28.3%

End
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