FORMULATION DESIGN AND EVALUATION OF ANTI-INFLAMMATORY ACTIVITY OF COMMIPHORA MUKUL LOADED EMULGEL

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1 J Res Educ Indian Med, April-June 2014; Vol. XX (2): ISSN FORMULATION DESIGN AND EVALUATION OF ANTI-INFLAMMATORY ACTIVITY OF COMMIPHORA MUKUL LOADED EMULGEL TEJINDER KAUR MARWAHA, 1 AND KIRAN S BHISE 2 Department of Pharmaceutics, MCE Society s Allana College of Pharmacy, Azam Campus, Pune Maharashtra (India) Abstract : Background: Commiphora mukul widely known as Guggulu. An ethyl acetate extract of guggul resin, obtained from Commiphora mukul (Fam.: Burseraceae) known as guggulipid is official in Indian Pharmacopoeia 1. The active constituent of gugulipid is guggulsterone which has been found to potently inhibit the activation of nuclear factor-kappab (NF-kappaB), a critical regulator of inflammatory responses. Commiphora mukul (Guggul extract) was found to be efficacious and cheap anti-inflammatory drug with least side effects. Therefore it was selected for the present study. Objective: Considering the above justification an attempt was made to design and develop herbal anti-inflammatory topical drug delivery system wherein herbal drug in the form of emulgel was formulated and its effect was compared to marketed preparation. Method: In the study, topical emulgel of Commiphora mukul (Guggul extract) was formulated. The optimized batch was evaluated for %EE and %DR followed by in vivo studies. Result: From the in vitro studies, formulation S3 showed maximum release of % in 6 hrs. The formulation S3 was comparable with marketed tacrolimus ointment. The results indicated that the optimized batch containing drug Commiphora mukul (Guggul extract) exhibited good results. Conclusion: Thus, the results indicate that an effective biphasic herbal anti inflammatory formulation containing Commiphora mukul (Guggul extract) can be prepared in the form of emulgel. Keywords : Anti inflammatory, Commiphora mukul, Gugulipid, Emulgel Introduction Gugulipid is an ethyl acetate extract of guggul resin, obtained from Commiphora mukul (Fam.: Burseraceae) and is official in Indian Pharmacopoeia 1. The active constituent of gugulipid is guggulsterone (4,17(20)-pregnadiene- 3,16-dione), which is present in a concentration of %. The guggulsterone is present in guggulipid in the form of stereoisomers E- guggulsterone and Z-guggulsterone. Gugulipid is a potent hypolipidemic agent. Apart from its hypolipidemic activity, a large number of therapeutic activities like antimicrobial, anthelmintic, anti-inflammatory, antiarthritic and antioxidant have been reported 2-4. Guggulsterone has been found to potently inhibit the activation of nuclear factor-kappab (NF-kappaB), a critical regulator of inflammatory responses. Such repression of NF-kappaB activation by guggulsterone has been proposed as a mechanism of the antiinflammatory effect of guggulsterone 5 A wide range of therapeutic activities, low bioavailability and aqueous insolubility of guggulipid led to explore an approach, which in addition to removing its undesired pharmacological action also improves its therapeutic concentration at the site of inflammation. Tacrolimus ointment is used as a comparator marketed preparation. Tacrolimus being topical immunosuppressive agent has various side effects in ointment manufacturing as well as several applications after effects. Topical application of anti-inflammatory agents at the site of inflammation can overcome their systemic side effects and improve their therapeutic activity. Guggul has been reported to mix properties of liposome, solid lipid particles 1. M.Pharma Student 2. Principal

2 76 Tajender Kaur and Kiran Bhise and multiple emulsions etc. 6,7. Emulgel system was selected as it meets all the criterias laid down for topical drug delivery of guggul. Emulgels are particularly light weight products with a low emulsifier and lipid content. They are characterized by the fact that they can be distributed readily on the skin and impart a feeling of freshness. Emulgels have emerged as one of the most interesting topical delivery system as it has dual release control system i.e. gel and emulsion 8,9. The aim of the present investigation was to explore the possibilities of using anti inflammatory herbal drug in the topical dosage form and to design and develop biphasic drug delivery system of herbal anti-inflammatory drug in the form of emulgel. Materials Guggul extract was generously gifted by Kuber Impex Ltd, Indore (M.P). Capryol 90 and Capmul MCM C8 were obtained as gift samples from Gattefosse India Pvt. Ltd. and Abitec Corporation, Mumbai respectively. Cremophor RH 40 was gifted by SigNET AC Limited, UK. Rest of the chemicals used was procured from Loba Chem. Pvt. Ltd., Mumbai, India. 12 wistar albino rats were procured from Yash Farms, Pune, India Methods I. Preformulation Study for Drug and Polymers The samples of Guggul Extract, Oils, Surfactants and Co Surfactants were subjected to the following preformulation studies (I). Characterization of Guggul extract A. Organoleptic Properties 10 Guggul extract was analyzed for the organoleptic properties like colour, odour and appearance. B. Micromeritic Properties Powdered extract of Gum guggul was evaluated for parameters like Bulk density, Tapped density, Carr s index, Angle of repose, and Hausner ratio. The observations were recorded in triplicate. C. Solubility Profile Solubility of Guggul extract was checked visually in distilled water, methanol, ethanol, chloroform, acetone, DMSO and phosphate buffer (ph 5.4). Accurately weighed one gm of drug was transferred in a clean and dry test tube followed by addition of the solvents individually and shaken vigorously and the solubility of drug was checked visually.determination of solubility in DMSO and phosphate buffer ph 5.4 mixture for determination of ratio: Accurately weighed 20 mg of Guggul extract was added individually to ten clean and dried volumetric flasks each of 10 ml capacity. DMSO and phosphate buffer ph 5.4 solvent system was added in the ratio 1:9, 2:8, 3:7, 4:6, 5:5, 6:3, 7:3, 8:2, 9:1 and the samples were shaken for 1 hour on linear motion shaker (Model no. REMI RQ 123, Spectra Whirlmatic Lab,India). The solutions were checked visually for their clarity. D. Analytical Methodology 14 The ultraviolet absorption spectrum of a solution of Guggul extract in DMSO and (8:2) mixture of DMSO and Phosphate buffer ph 5.4 was obtained using UV/VIS spectrophotometer (V-630, Jasco Corporation Tokyo, Japan) over a wavelength range of 200 to 400 nm. Maximum absorption (ëmax) values were determined and further used for plotting calibration curve. 2. Characterization of Oil, Surfactant and Co-Surfactant for Microemulsion A. Solubility Determination in various Oil, Surfactant and Co-surfactant 15 The solubility of drug in various oils, surfactants and co-surfactants was measured and the solvents for the study were selected based on the good solublising capacity for drug. In present study the solubility of drug was investigated in different oils like Isopropyl myristate, Captex, Capryol 90 etc, surfactants and co-surfactants like Tween 80, Cremophor RH40 etc. An excess amount of drug was added

3 Anti-inflammatory activity of commiphora mukul loaded emulgel 77 into each vehicle followed by vortex mixing for 30sec (Remi mixer, Mumbai). Mixtures were shaken for 48 h at 30 0 C, followed by equilibrium for 24 hr. The equilibrated samples were then centrifuged at 1000 rpm for 10 min to remove the insoluble drug and clear supernatant liquid was decanted. An aliquot of the supernatant was diluted with DMSO and solubility of drug was estimated by UV spectroscopy at 328 nm. B. Screening of oils and surfactant The oils and surfactants were selected on the basis of their Figure tendency 7 for instant emulsification. The oils selected for this investigation were Captex, Capryol 90, IPM, Wheatgerm oil, Olive oil and Soyabean oil. The surfactants selected were Cremophor RH-40, Tween 80, Tween 20, Span 80, Span 20, Soya lecithin and Acconon MC8-2. The oils and surfactant were mixed in a ratio of 1:1. Briefly, 150 mg of the surfactants were added to 150 mg of the oily phase. Each mixture, 100 mg, was then diluted with distilled water to 100 ml in a stoppered conical flask. Ease of emulsification was judged by the number of flask inversions required to yield homogenous emulsion. Emulsions were allowed to stand for 2hr and their % transmittance was evaluated at 638 nm by UV-Visible spectrophotometer (Jasco V 650, Japan) using distilled water as a blank. Emulsions were furthermore observed visually for any turbidity or phase separation. C. Preliminary screening of co-surfactants The selected oily phase and surfactant were used for further screening of the different cosurfactants (Ethylene glycol, Propylene glycol, PEG 400, Capmul PG8 and Capmul MCM C8) for their emulsification ability. Mixtures of 200 mg of co-surfactant, 400 mg selected surfactant, and 600 mg screened oil were prepared and evaluated in a similar fashion as described in preliminary screening of surfactants. D. Pseudo-Ternary Phase iagram: 16,17 The microemulsion existence region was determined by constructing pseudo-ternary phase diagrams. Titration method was employed for its determination. On the basis of the solubility study of drug; oils, surfactants, co-surfactants and aqueous phase were used for construction of phase diagram. Surfactant and co-surfactant (Smix) in each group were mixed in different weight ratio of (1:0, 1:1, 1:2, 2:1, 1:3, 3:1, 1:4, 4:1). For each phase diagram oil and specific Smix ratio were mixed thoroughly in different weight ratio from 1:9 to 9:1 (1:9, 2:8, 3:7, 4:6, 5.0:5.0, 6:4, 7:3, 8:2, 9:1,) in different glass vials. Different combination of oils and Smix were prepared for the study to delineate the boundaries of phase precisely formed in the phase diagrams. Pseudo-ternary phase diagram was developed using aqueous titration method. Water was added dropwise with constant stirring on 6 Station magnetic stirrer (Spectralab, Whirlmatic, India) until homogeneous dispersion or solution was obtained. After each addition the system was examined for appearance and flow property. The end point of the titration was the point in which the solution becomes cloudy or turbid. The quantity of aqueous phase required to achieve turbidity point was noted. Slow titration with aqueous phase is performed to each weight ratio of oil and Smix. The physical state of the microemulsion was marked on a pseudo-threecomponent phase diagram with one axis representing aqueous phase, the other representing oil and the third representing a mixture of surfactant and co-surfactant at fixed weight ratios (Smix ratio). (II). Formulation Study A. Formulation of Emulgel The factorial Batches of microemulsion that contained Gum guggul extract was formulated by mixing oil, surfactant and co-surfactant with varying component ratio. Accurately measured 0.15 (% w/v) of Gum Guggul extract was dissolved in the mixture of oil, surfactant and co-surfactant and then an appropriate amount of water was added to the mixture dropwise with constant stirring on 6 station magnetic stirrer (Spectralab, Whirlmatic, India). Microemulsion of Gum Guggul was obtained spontaneously on stirring the mixtures at ambient temperature.

4 78 Tajender Kaur and Kiran Bhise Various gelling agents namely, Sodium CMC, Hydroxypropyl methylcellulose (Methocel K4000M), HPMC CR, Carbopol 934 and Carbopol 940 were evaluated for their ability to gel medicated microemulsion. Gelling agent was dispersed slowly in the medicated microemulsion with the help of overhead stirrer. In case of Carbopol 940 and 934, the dispersion was neutralized by using triethanolamine to obtain the gel. B. Factorial Formulations: The effect of formulation variables on the responses were statically evaluated by applying one-way ANOVA, using the commercially available software package Design-Expert version factorial design was implemented for optimization of microemulsion that contained three independent variables at two levels +1 and -1. The factorial batches are given in Table No. 1 and 2.The batches were evaluated and the effect of individual variable was studied according to the response surface methodology. C. Evaluation of the Factorial Batches Of Microemulsion Based Gel Of Guggul extract % EE Accurately weighed 1 mg of gel was mixed in 10 ml calibrated volumetric flasks containing 9 ml DMSO; it was diluted appropriately and drug content was determined spectrophotometrically in triplicate. % EE was calculated as per the formula: Actual drug content - Free drug content X 100 % EE = Actual drug content 2.% DR The in vitro drug release studies were carried out using a modified Franz diffusion (FD) cell. Accurately weighed 1gm of sample was applied on cellophane membrane which was placed between donor and receptor compartment of the FD cell. 8:2 mixture of DMSO and Table Factorial design of Gum guggul Microemulsion:Levels of variables in coded and actual form Variables for Experimental Designs Independent variable: X1= Capryol 90 X2= Cremophor RH 40 X3= Capmul MCM Dependent variable: Y1= % Entrapment efficiency Y2= %Drug release at 6 hr Levels (Code Value) X 1 (% w/v)(capryol) Actual values X 2 (% w/v)( Cremophor) X 3 (% w/v)(capmul MCM) Response Y 1 Y % Entrapment % Drug Efficiency Release Table 2. Composition of Factorial batches of Gum guggul Microemulsion Based Gel with C940 as Gelling Agent Sr. No Formulation Code C mukul (%) (w/v) Capryol (%) (v/v) Ingredients Cremophor RH 40 (%) (v/v) Cap MCM (%) (v/v) Water (%) (v/v) 1 S Upto S Upto S Upto S Upto S Upto S Upto S Upto S Upto 100

5 Anti-inflammatory activity of commiphora mukul loaded emulgel 79 Phosphate buffer ph 5.4 was used as a dissolution media. This whole assembly was kept on a magnetic stirrer and the solution was stirred continuously using a magnetic bead. Accurately weighed 1gm of drug containing gel was placed in the donor compartment. Samples (0.5ml) were withdrawn from the receptor fluid at 1 hr interval for upto 6 hrs after the application. An equal volume of the fresh phosphate buffer was immediately replenished after each sampling maintaining sink conditions. The temperature was maintained at 37 ± 0.5 C with constant rotation speed of 100 rpm. Samples were analyzed by UV-visible spectrophotometer (Jasco, V630) at 328 nm with suitable dilutions and the cumulative % drug release was calculated. D. Formulation of the Optimised Batch: The optimised batch was formulated using the predicted values of the %EE and %DR from the software. E. Evaluation of Optimised Batch Containing Guggul extract Microemulsion Based Gel: The optimized gels were evaluated for %EE and %DR. F. Skin irritation test (patch test): Three rats was used in the study. The emulgel was applied on the properly shaven skin of rat. Undesirable skin changes, i.e., change in color, change in skin morphology were checked for a period of 24 hr. (III). In Vivo Anti-Inflammatory Activity for Optimised Batches of Gum guggul, Babchi Oil and Combined Microemulsion Based Gel 19 Wistar albino rats of either sex, weighing gm were used for the experiment. Animals were housed in standard cages in lightcontrolled room at 25 ± 3 (12 hour light/ 12 hour dark cycle and 50 ± 5 % RH, were given standard pellet diet and water ad libitum. All experiments were performed after an overnight fast. All studies were conducted in accordance with protocols, reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of Allana College of Pharmacy, Pune, Maharashtra, India. The serial number of the approval protocol was Ref/ ACP/ IAEC/11-12/29-02 for in vivo anti inflammatory studies. In order to establish anti inflammatory activity edema was induced on the left hind paw of the rats by subplantar injection of 1% (w/v) carrageenan. Formulation, i.e., S3 and standard (tacrolimus ointment) were applied 30 min before carrageenan administration. The paw volume was measured at intervals of 30, 60, 90, 120 min by mercury displacement method using plethysmometer Group 1 (Control group): Carrageenan (1%). Group 2 (Standard group): Topical marketed Tacrolimus gel + Carrageenan. Group 3 (Test): Formulations S3 + Carrageenan. The % inhibition of paw edema in drug treated group was compared with carrageenan control group and calculated according to the formula: Control paw volume Test paw volume X 100 % Inhibition = Control paw volume IV. Accelerated Stability Studies 20 The stability studies were carried out according to the ICH guidelines to assess the drug and formulation stability. The optimized formulations were sealed in aluminum packaging material. Samples were kept in a stability chamber (Thermolab) maintained at 45 ºC and 75 % RH for 3 months. The samples were withdrawn at 0, 1, 2 and 3 months. Evaluation The optimized formulations were evaluated over a period of 3 months for the following parameters: Appearance, ph, %EE, in vitro Dissolution study. Results and Discussion (I). Preformulation Studies 1.Characterization of Gum guggul: A. Physical appearance: The drug was subjected to organoleptic evaluation.

6 80 Tajender Kaur and Kiran Bhise The results obtained are shown in Table 3. Table 3. Organoleptic Properties of Gum guggul Sr. No. Parameters 1. Colour Experimental Slight yellow Gum guggul Theoretical Slight yellow 2. Odour Acrid Acrid 3. Taste Bitter Bitter Organoleptic properties obtained practically matches the properties given in the literature. Thus it conforms the identity of the drug. B. Micromeritic Properties The results of evaluation of micromeritic properties of the drug are depicted in Table 4. Table 4. Micromeritic Properties of Gum guggul Sr. No. 1. Parameters Bulk density (gm/cm3) Observation Result (Mean) Tap density (gm/cm3) Hausner ratio Compressibility index 5. Angle of repose 23.58% 23.58% The results obtained from Table No.4 exhibited that the drug had Hausner ratio within , % Compressibility within the range of 23%-28% and Angle of repose within the range of with passable to poor flow property. C. Solubility Profile Solubility of powdered extract of Gum guggul is depicted in Table 5. Table 5. Solubility Profile of Gum guggul extract No. Solvent Solubility 1. Distilled Water Insoluble 2. Methanol Sparingly Soluble 3. Ethanol Sparingly Soluble 4. Chloroform Sparingly Soluble 5. Acetone Sparingly Soluble 6. DMSO Soluble 7. Phosphate Buffer (5.4 ph) Sparingly Soluble It can be depicted from Table 5 that Gum guggul is soluble in DMSO, thus DMSO was selected for further studies. D. Analytical Methodology UV-Visible Spectroscopy 261 The gama max of Gum guggul in DMSO and (8:2) mixture of DMSO: Phosphate buffer ph 5.4 was found to be 328 nm, 329nm respectively. 2. Characterization of Oil,Surfactant and Co-Surfactant A. Selection of Excipients The excipients selected needed to be pharmaceutically acceptable, nonirritating, and nonsensitizing to the skin and to fall into the GRAS (generally regarded as safe) category. Higher solubility of the drug in the oil phase was another important criterion, as it would help the microemulsion to maintain the drug in solubilized form. Safety is a major determining factor in choosing a surfactant, as a large amount of surfactants may cause skin irritation. Non-ionic surfactants are less toxic than ionic surfactants. An important criterion for selection of the surfactants is that the required hydrophilic lipophilic balance (HLB) value to form the o/w microemulsion be greater than 10. The right blend of low and high HLB surfactants leads to the formation of a stable microemulsion formulation. In this study, we selected Cremophor RH 40 as a surfactant with an HLB value of 15. Transient negative interfacial tension and fluid interfacial

7 Anti-inflammatory activity of commiphora mukul loaded emulgel 81 film are rarely achieved by the use of single surfactant; usually, addition of a cosurfactant is necessary. The presence of cosurfactant decreases the bending stress of interface and allows the interfacial film sufficient flexibility to take up different curvatures required to form microemulsions over a wide range of composition. Thus, the cosurfactant selected for the study was Capmul MCM, which has an HLB value of 5-6. Gum guggul is a highly lipophilic drug, and its pharmacological properties suggest that it has good potential for topical drug delivery. It can be depicted from the above Table No.6 that in oils, Capryol 90, in surfactants Cremophor RH 40 and in Co-Surfactants Capmul MCM has better solubility with the drug as compared to others. Therefore on the basis of solubility data polymers were selected for formulations. B. Pseudo-Ternary Phase Diagram a)ternary diagram of Ratio of Oil to Smix Oil = Captex Smix = Cremophor : Capmul MCM (2:1) Fig. 1. Ternary Diagram of Ratio of Oil (Capryol) to Smix (Cremophor : Capmul MCM (2:1)) Thus from the ternary diagram in Fig.1, it can be concluded that best ratios of Oil:Smix that gives microemulsion region are 5:5, 4:6. b) Selection of Surfactant and Co Surfactant Ternary diagram of different concentration of surfactant and co-surfactant are given in Fig.2 From the ternary diagrams it is depicted that Cremophor (surfactant) and Capmul MCM (co-surfactant) with Capryol as oil gives the maximum microemulsion region. Phase behavior investigation of this system demonstrated the suitable approach to determining the water phase, oil phase, surfactant Fig. 2. Ternary Diagram of (i) Oil Captex, Surfactant Tween, Co-surfactant Capmul PG8 (ii) Oil Capryol, Surfactant Tween, Co-surfactant Capmul MCM (iii) Oil Capryol, Surfactant Cremophor, Co-surfactant Capmul MCM (iv) Oil Olive oil, Surfactant Tween, Co-surfactant Capmul PG8

8 82 Tajender Kaur and Kiran Bhise Table 6. Solubility Determination of Gum guggul in Various Oil, Surfactant and Co-surfactant Sr. No. 1. Oil 2. Surfactant 3. Co-surfactant Solubility (µg/ml) In Solvent Gum guggul Captex 62.2 Wheatgerm Oil 25.5 Olive Oil 24.2 Capryol Soyabean Oil 32.8 IPM 3.7 Cremophor RH Tween Acconon MC Ethylene Glycol 50.2 Propylene Glycol 56.2 PEG Capmul MCM C Capmul PG (II). Formulation Study G. Factorial Batches of Gum guggul extract Microemulsion Formulated with C940 as Gelling Agent.. Fig. 3: Linear Correlation Plot between Actual and Predicted Values for % EE (Y 1 ) concentration, and cosurfactant concentration with which the transparent, 1-phase low-viscous microemulsion system was formed. The phase study revealed that the maximum proportion of oil was incorporated in microemulsion systems when the surfactant-to-cosurfactant ratio was 5:5. From pseudoternary phase diagrams, the formulation in which the amounts of oil phase completely solubilized the drug and which could accommodate the optimum quantity of Smix and distilled water was selected for the study. Fig. 4: Response surface plot showing effect of formulation variables on % EE (Y 1 ) Fig.4 showed the response surface plot, which displayed the effect of X 1, X 2 on the %EE Y 1. From the figure, at maximum level of X 2 (0.5), and minimum levels of X 1, X 3 (0.25) results in increasing the %EE of the formulation to the maximum 94. On the other hand, increasing X 1, X 2 up to (0.5) and decreasing X 3 to (0.25)

9 Anti-inflammatory activity of commiphora mukul loaded emulgel 83 results in decreasing the %EE to the minimum 91. Also, it was found that, high level of X 1 (0.5) and low levels of X 2 (0.25) resulted in high value of %EE. The %EE of the formulation will be maximized either at low 0.25 or high 0.5 of X 2, where %EE of the formulation was 93 and 94. Fig.5: Contour plot showing the effect of X 1, X 2 on % EE (Y 1 ) Contour plot represented in Fig.5 gave an idea about the exact percent of X 1, X 2 at which the %EE becomes at minimum level. Fig. 7. Response surface plot showing effect of formulation variables on % DR (Y 2 ) Fig. 7 showed the response surface plot, which displayed the effect of X 1, X 2 on the %DR Y 2. From the figure, at maximum level of X 2 (0.5), and minimum levels of X 1, X 3 (0.25) results in increasing the %DR of the formulation to the maximum 77. On the other hand, increasing X 1, X 2 up to (0.5) and decreasing X 3 to (0.25) results in decreasing the %DR to the minimum 75. Also, it was found that, high level of X 1 (0.5) and low levels of X 2 (0.25) resulted in high values of %DR. The %DR of the formulation will be maximized either at low 0.25 or high 0.5 of X 2, where %DR of the formulation was 76 and 77. Contour plot represented in Fig. 8 gave an idea about the exact percent of X 1, X 2 at which the %DR becomes at minimum level. Fig. 6 showed the linear correlation plot of predicted and actual values of %DR and these values are near by each other indicating the correctness of the model. Fig. 6. Linear Correlation Plot between Actual and Predicted Values for % DR (Y 2 ) Fig.6 showed the linear correlation plot of predicted and actual values of %EE and these values are near by each other indicating the correctness of the model. H. Evaluation of the Factorial Batches of Microemulsion that contained Gum guggul extract Based Gel From Fig.9 it can be concluded that Batch S3 showed good drug release as compared to other batches thus it was selected as an optimized batch.

10 84 Tajender Kaur and Kiran Bhise Table 7. Factorial batches that Contained Gum guggul extract Microemulsion with C940 as Gelling Agent Parameter/ Batch /Code %EE % DR at 6 hrs S ± ±0.26 S ± ±0.17 S ± ±0.21 S ± ±0.15 S ± ±0.14 S ± ±0.01 S ± ±0.03 S ± ±0.31 The evaluation results of factorial batches are depicted in Table No. 7. Batch S3 showed promising results. Fig. 9: Diffusion Graphs of Factorial batches of Gum guggul Microemulsion with C 940 as Gelling Agent I. Formulation of the Optimised Batch: The optimised batch was formulated using the predicted values of the %EE and %DR from the software and is exhibited in Table 8.. Fig.8: Contour plot showing the effect of X 1, X 2 on % DR (Y 2 ) J. Evaluation of Optimised Batch of Gum guggul extract Microemulsion Based Gel: Fig. 10 exhibits drug release of optimised batch S3. From Table 9, it can be concluded that since the experimental and predicted results obtained were found to be nearly same for batches with above mentioned composition, it Table 8. Formulation of optimised batch that Contained Gum guggul extract Microemulsion Based Gel Formu lation Code Gum guggul (%) (v/v) Ingredients Capryol(%) (v/v) Cremophor RH 40(%) (v/v) Cap MCM(%) (v/v) Water(%) (v/v) S Upto 100 Table 9. Comparison between the Experimented and Predicted Values for Probable Optimal Formulation Dependent variables Optimized formulation S3 *Experimented value Most Predicted value % Drug Release ± % Entrapment Efficiency 94.05± was subjected to stability study as per ICH guidelines The optimized batches showed similar release profile compared to factorial batches. Release shown was ±0.21 Gum guggul in 6 hrs and the best fit model was found to be Higuchi.

11 Anti-inflammatory activity of commiphora mukul loaded emulgel 85 Before Fig. 10: Diffusion Graphs of Optimised Batch K. Skin irritation test (patch test): From Fig. 11 it can be depicted that no allergic symptoms like inflammation, redness, irritation appeared on rats up to 24 h. The results indicated that the control preparation (which did not contain any drug), test gel and marketed products did not cause any skin reaction. It can be assured that drug and the excipients did not cause any skin irritation and can be used in the gel formulation. After 24 Hours (III). In Vivo Anti-Inflammatory Activity For Optimised Batches Of Gum guggul Microemulsion Based Gel Percentage increase in paw volume (inflammation) and percentage inhibition of inflammation in control group and groups treated with test and marketed products and the results are given in the Table 10 and Fig. 12. In control group which received carrageenan alone, a rapid and continuous increase in paw volume (i.e. inflammation) was observed Fig. 11: Skin Irritation test on Rats before and after 24hrs and the inflammation was sustained during the entire period of 6 hrs of study. In the groups which received test products, the percentage increase in paw volume was low when compared to the control group, indicating that the test and marketed product possess good anti-inflammatory activity. The inflammation due to carrageenan was markedly inhibited by the test and marketed products. A comparison of the percentage inhibition of inflammation (i.e., anti- A B C Fig. 12: Rat Paw Edema Test, results after 6hrs [ (a) Inhibitory Effect of Optimised Batch S3 of Gum guggul MBG ( b) Inhibitory Effect of Marketed formulation (Tacrolimus Ointment) (c)carrageenan Induced Inflammation]

12 86 Tajender Kaur and Kiran Bhise Table 10. Mean Paw Edema Volume and Percentage Inhibition of the Edema in the Albino Rats Time (in chrs) Mean Paw Edema Volume Percentage Inhibition of the Edema Control Test Standard Control Test Standard Table 11. Stability study of optimised batch S3 Formulation code S3 Parameters Initial 1 month 2 months 3 months Visual appearance No Change No Change No Change No Change ph %EE 94.05± ± ± ±1.27 Cumulative% DR ± ± ± ±0.84 inflammatory activity) of test and marketed products is made in Table 10. The % inhibition of Tacrolimus (marketed preparation) and Test formulations were found to be 74.51% and 73.22% respectively. The pharmacological activity (anti-inflammatory) of optimized batch containing the drug Gum guggul was comparable to the marketed preparation; these results indicated the rapid onset of action and higher activity during the initial periods due to their enhanced dissolution and absorption rates. (IV). Accelerated Stability Study From Table 11 it can be concluded that all the prepared emulgel formulations were found to be stable upon storage for 3 months. Conclusion Thus, the results indicate that an effective biphasic herbal formulation containing Gum guggul extract can be prepared in emulgel system and it gave promising results. Acknowledgement: Author would like to express sincere thank to Pukhraj Herbals, Indore, India for providing Commiphora mukul (Gum guggul) as a gift sample. I also thank to Abitec Corporation, Mumbai; Gattefosse India Pvt. Ltd., Mumbai and SigNET, UK Toyoma, Japan for providing Capmul MCM C8, Capryol 90 and Cremophor RH 40. References 1. Indian Pharmacopoeia, Vol. II, p. A 74, Govt. of India, Ministry of Health and Family Welfare, Controller of publication, New Delhi Chander R., Khanna A.K., Kapoor N.K.: Phytother. Res. 10, 508 (1996). 3. Beg M., Singhal K.C., Afzaal S.: Indian J. Physiol. Pharmacol. 40, 237 (1996). 4. Kimura I., Yoshikawa M., Kobayashi S.:Bioorg. Med. Chem. Lett. 11, 985 (2001). 5. Shishodia S, Aggarwal BB. Guggulsterone inhibits NF-kappaB and IkappaBalpha kinase activation, suppresses expression of anti-apoptotic gene products, and enhances apoptosis. J Biol Chem Nov 5;279(45): Epub 2004 Aug 17.

13 Anti-inflammatory activity of commiphora mukul loaded emulgel Gupta V B, Drug Delivery and allied Applications of Guggul Indian Patent, Application No. 1715/DEL/ Jain A, Gupta VB. Chemistry and Pharmacological profile of guggul- a review. Indian Journal of Traditional Knowledge:5, (2006) 8. Shekhar Verma, Anurekha Jain and V. B. Gupta. Synergistic and Sustained Anti - Inflammatory Activity of Guguul with the Ibuprofen: A P r e l i m i n a r y Study. International Journal of Pharma and Bio Sciences, Volume 1, Issue 2, 2010 (April June) 9. Khullar R, Saini S, Sethi N et al. Emulgel A surrogate approach for topically used hydrophobic drugs. Int J Pharm Biol Sci 2011; Mohamed MI. Topical emulsion gel composition comprising diclofenac sodium. AAPS J 2004; 6(3): United State Pharmacopoeia NF, volume 1, asian edition 2007, page no CVS Subrahmanyam, Micromeritics: Text book of physical pharmaceutics, second edition 2009, page no D. A. Bhagwat, S Kawtikwar, Dinesh M Sakarkar. sustained release matrices of verapamil hcl using glyceryl monosterate and stearic acid research J. Pharm. and Tech. Dec2008;1(4):p Aulton M.E., Pharmaceutics, The science of dosage form design, 2nd edition, Churchill Livingstone, London, 2002; 15 42, , Kalsi P S. Spectroscopy of organic compounds. New Age International publication. NewDelhi 2004; 6th Edition, Divyakumar Bora, Priyanka Borude, Kiran Bhise*. Formulation And Evaluation Of Self Microemulsifying Drug Delivery Systems Of Low Solubility Drug For Enhanced Solubility And Dissolution. International Journal of Pharmaceutical Innovation. 17. Gupta A.K.*, Mishra D.K. and Mahajan S.C.Preparation And In-Vitro Evaluation Of Self Emulsifying Drug Delivery System Of Antihypertensive Drug Valsartan. Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 2, Issue 3: March: 2011, Bonacucina, G., Cespi, M., Palmieri, G.F., Characterization and Stability of Emulsion Gels Based On Acrylamide/Sodium Acryloyldimethyl Taurate Copolymer. AAPS PharmSciTech. 2, Crunkhorn, P., Mencock, S.C., Mediators of inflammation induced in rat paw carregeenan. Br. J. Pharmacol. 42, ICH Harmonized Tripartite Guidelines, Stability Testing of New Drug Substances and Products. ICH Committee 2003; 8. Abbreviation DMSO - Di Methyl Sulphoxide HLB - Hydrophilic Lipophilic Balance %DR - Percent Drug Release %EE - Percent Entrapment Efficiency Smix - Surfactant and Co-surfactant Mixture Address for correspondence: Tejinder Kaur Marwaha, M.Pharma Scholar, Department of Pharmaceutics, MCE Society s Allana College of Pharmacy, Azam Campus, Pune Maharashtra (India) tej.kaur20@gmail.com JREIM

14 88 Tajender Kaur and Kiran Bhise Mns No: JREIM Tejinder Kaur Marwaha TITLE: Formulation Design and Evaluation of Anti-inflammatory Activity of Commiphora mukul Loaded Emulgel Article Type : Original Research Authors: Tejinder Kaur Marwaha, Kiran S Bhise Affiliation: Allana College of Pharmacy; Allana College of Pharmacy Corresponding author tej.kaur20@gmail.com Sender Author: Tejinder Kaur Marwaha (tej.kaur20@gmail.com) [ SEND ] Mns No: JREIM Rec.Date: Dec 17, :19 Accept Date: Jun 11, :07 Initial Version: Original WORD document (docx) Initial Version (PDF): Original PDF Full Text (.pdf) (932 KB) Revised Version : (.docx)(.pdf) Obligatory Files: TitlePageofArticle.docx JREIMAuthorsDeclarationForm.doc JREIMcoveringletter.doc CVofMainAuthor.docx Tejinder Kaur Marwaha, M.Pharma Scholar, Department of Pharmaceutics, MCE Society s Allana College of Pharmacy, Azam Campus, Pune Maharashtra (India) tej.kaur20@gmail.com