Research Article

Received: 06 May 2013, Accepted: 20 May 2013

ISSN: 2321-2969

Int. J. Pharm. Biosci. Technol. To cite this Article: Click here

International Journal of Pharma Bioscience and Technology. 2013; 1(2): 89-101

Journal home page: www.ijpbst.com

PEPPERMINT OIL BASED DRUG DELIVERY SYSTEM OF ACECLOFENAC WITH IMPROVED ANTI-INFLAMMATORY ACTIVITY AND REDUCED ULCEROGENECITY
Anuradha S. Pol1*, Pratikkumar A. Patel2, Darshana Hegde1
1

* Department of Pharmaceutics, Bombay College of Pharmacy, Kalina, Santacruz (E), Mumbai 400 098. Maharashtra, India
2

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, SVKM's Narsee Monjee Institute of Management Studies, Vile Parle, Mumbai, Maharashtra, India. Corresponding Author* E-mail address- anna_1907@yahoo.co.in

ABSTRACT: Aceclofenac (ACF), a nonsteroidal anti-inflammatory (NSAID) BCS class II drug belonging to the class of phenyl acetic acid derivatives exhibiting antipyretic, anti-inflammatory and analgesic activities. Many strategies have been employed for improving solubility and thus bioavailability of this drug moiety. But this is a first report on peppermint oil based oral SMEDDS of ACF for achieving a synergistic anti inflammatory activity by combining NSAIDS with essential oils such as mint oils. Thus, the present investigation was designed with an aim to improve the solubility, dissolution rate, oral bioavailability and eventually anti-inflammatory activity of ACF by incorporating into peppermint oil based SMEDDS. The solubility of ACF was determined in various lipid based excipients viz, essential oils and other lipophiles, surfactants and cosurfactants. Further emulsification studies were carried out in order select specific oilsurfactant-cosurfactant combinations for plotting the pseudo ternary phase diagrams which were then constructed to identify the existence of microemulsion region. The formulations of ACF-SMEDDS were optimized using pseudo-ternary phase diagrams analysis and studied for drug loading and lipid content. The average globule size of ACF-SMEDDS was less than 100 nm and was confirmed by transmission electron microscopy. The optimized formulation exhibited about 99% release of ACF from the SMEDDS filled in capsules. Furthermore, ACF SMEDDS showed 80 7.30 % inhibition after 4 hr of treatment against carrageenan induced paw. In addition to this SMEDDS showed least ulcer score as compared to other treatment group. Thus, the developed SMEDDS were found to exhibit less GI tract toxicity and showed superior anti inflammatory action compared to plain drug. Key words: Aceclofenac, NSAID, self microemulsifying drug systems, peppermint oil, rat paw edema INTRODUCTION The oral route is the most preferred route of drug delivery due to the obvious advantages associated with it. Drug discovery in recent years have led to invention of drug molecules having poor aqueous solubility which in turn result in poor oral bioavailability, high intra- and inter-subject variability and lack of dose proportionality [1]. Delivery of about 35-40% of the drug compounds by oral route is hampered because of its high lipophilicity. Several formulation strategies have Pol et al

been developed to enhance solubilization of lipophilic/hydrophobic drugs for improving their oral delivery. Lipid based oral delivery is one of the most promising approach in this direction. These systems incorporate the lipophilic drug into inert lipid vehicles such as oils, micellar systems, liposomes, lipid emulsions, specialized emulsions (multiple emulsions and microemulsions) and emulsion preconcentrates. The term emulsion

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Int. J. Pharm. Biosci. Technol. preconcentrates include self-emulsifying and selfmicroemulsifying drug delivery systems [2]. in oil or oil/surfactant blend. SMEDDS are preferred over preformed microemulsions due to their improved physical stability, volume consideration and ease of formulating them into hard or soft gelatin capsules for oral delivery [7]. Materials and Methods Materials Aceclofenac and Diclofenac acid was procured from USV Ltd. Mumbai, India. Peppermint oil was a gift sample from Keva Flavors Pvt. Ltd, Mumbai, India. Cremophor EL and Solutol HS 15 was a generous gift sample from BASF, Mumbai, India. Gelucire 44/14 and Labrasol were received as a generous gift from Gattefosse, Mumbai, India. Hard gelatin capsules were procured from Associated Capsules Ltd Mumbai, India. Sodium lauryl sulfate and ethyl cellulose were procured from Colorcon. Mumbai, India. Methanol (HPLC grade), acetonitrile (HPLC Grade), Tween 80, dichloromethane and triethanolamine (AR grade) were purchased from s. d. Fine Chemicals, Mumbai, India. All the excipients and reagents were used as received. Double distilled water was filtered through 0.45 m membranes and was prepared freshly whenever required. Preformulation studies Solubility studies The saturation solubility of ACF in various oils, surfactants, and cosurfactants was determined by shake flask method [8]. Emulsification studies for screening potential excipients Screening of surfactants for emulsifying ability Peppermint oil IP was selected as oily phase based on the results of solubility studies. Briefly, the oil was mixed with surfactants in 1:1 ratio and the vortexed for 5 minutes to ensure proper mixing. From the resultant isotropic mixture 50 mg of mixture was weighed accurately and diluted to 20 ml with double distilled water to yield a fine emulsion. The resulting emulsions were observed visually for physical appearance, optical clarity and separation of dispersed phase over a period of 8 hr. The resulting systems were also evaluated for turbidity by using the turbidimetric method described by Date et al [9]. Screening of cosurfactants Cosurfactants were screened to evaluate their relative efficacy in presence of surfactant and oil. The oil: surfactant: co-surfactant ratio was kept constant as 1:2:1. The mixtures were evaluated for

Fig.1. Chemical structure of Aceclofenac Aceclofenac (ACF) is a phenyl acetic acid- based nonsteroidal anti-inflammatory (NSAID) drug with potent antipyretic, anti-inflammatory and analgesic activity. It is belongs to class II BCS classification (low solubility/high permeability), possesses very slight solubility in water. Currently, it is commercially available as conventional and sustained release tablets. The drug is reported to exhibit slow and/or incomplete dissolution from this dosage form in gastrointestinal fluids, leading to low and variable bioavailability [3]. Improvement of dissolution of ACF from its oral dosage forms is thus, an important issue for enhancing its bioavailability and therapeutic efficacy [4]. Various approaches such as suspension in oily formulation wherein a drug is suspended in oily base using suspending agent such as beeswax, solubilization in aqueous soluble base and solid dispersions have been studied. But these formulations exhibit certain drawbacks such as delayed dissolution in case of oily suspension and less significant increase in dissolution rate when solubilised in aqueous soluble bases [5]. In the light of the aforementioned issues, there is a need of a delivery system which would improve the oral delivery of this hydrophobic drug by circumventing its poor aqueous solubility and enhance its dissolution rate thereby leading to faster onset of action with reduction in GI mucosal toxicity. Microemulsions are defined as thermodynamically stable, transparent, isotropic, low viscosity colloidal dispersions and are mixtures containing at least three components, water, oil and surfactant. Self-microemulsifying drug delivery systems (SMEDDS) are microemulsion preconcentrates which offer lipophilic drugs to the gastrointestinal tract in a dissolved state due to spontaneous emulsification, avoiding the dissolution step and are reported to render more reproducible plasma concentration profiles and enhanced bioavailability [6]. These properties render SMEDDS as a good carrier for delivery of hydrophobic drugs exhibiting adequate solubility Pol et al

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Int. J. Pharm. Biosci. Technol. turbidity by turbidimetric method described by Date et al [9]. Construction of pseudoternary phase diagram Pseudoternary phase diagrams were constructed to define the extent of the microemulsion region. Ternary mixtures with varying compositions of surfactant, co-surfactant and oil were prepared; each of them, representing an apex of the triangle [10]. The surfactant concentration was varied from 20 to 65 % (w/w) and co-surfactant concentration was varied from 10 to 40% (w/w). The percentage of surfactant, co-surfactant and oil used herein was decided on the basis of the requirements stated in the literature for the spontaneously emulsifying systems [11]. Optimization parameter studied Influence of drug loading The selected systems were studied at dose levels of 50mg, 75 mg, 100mg, 115 mg and 130 mg. Formulations equivalent to 5 mg of drug were dispersed in 500 ml of double distilled water and were observed for their dispersability, globule size and its polydispersity. In addition, they were examined for drug precipitation and phase separation over a period of 24 h. Effect of lipid content Suitable formulations were selected for respective surfactant-cosurfactant combination based on the results of earlier studies; effect of drug loading on globule size. The effect of different concentrations of lipid on the formulation with respect to change in globule size was evaluated. Formulations were also observed for visible signs of drug precipitation and phase separation. Formulations of SMEDDS The optimized formulations were prepared by dissolving ACF in oily phase by vortexing for 5 min followed by surfactants and cosurfactants and final blend was sonicated for 5 min to remove any air bubbles. A fixed amount of the SMEDDS was filled in transparent hard gelatin capsules of required sizes using a micropipette. The systems were then evaluated for further studies. Characterization Self-microemulsifying formulations were evaluated visually (before reconstitution/dilution) for appearance. Self-microemulsification efficiency The self-emulsification capacity was assessed for formulations, using a standard USP XXIII Pol et al Pg. 91 dissolution apparatus II. Each capsule was added to 500 ml of 0.1 N HCl at 37C 0.5C. Gentle agitation was provided by a standard stainless steel dissolution paddle rotating at 50 rpm. The lipid-based formulations were assessed visually for the rate of microemulsification and the final appearance of the dispersion. Globule size analysis The capsule was pierced and the contents were diluted ten times with double distilled water. Formulations were evaluated for globule size by photon correlation spectroscopy using a Beckman coulter N5 plus submicron particle size analyzer. pH determination The formulations were diluted 100 times with double distilled water and the pH of resulting microemulsions (with and without ACF) was determined in triplicate at 25 2C using a digital pH meter (Universal Enterprises). Dilution test Optimized formulations were diluted 100, 250, 500, and 1000 times in different buffer media (pH 1.2, pH 3.0, pH 6.8, double distilled water, and ringer solution). The test was carried out in test tubes maintained at 37 1C in a water bath shaker with a view to simulate body temperature and gastric motility in gastrointestinal tract. Furthermore, formulations were assessed for transparency, phase separation, globule size and precipitation of drug at intervals of time up to 8 h. Zeta potential measurement SMEDDS (40 mg) was diluted 250 times with pH 1.2 buffer and pH 6.8 buffer. ZetaPALS instrument was used to measured surface charge (zeta potential) and electrophoretic mobility of the blank and ACF-loaded microemulsions at 25C. Transmission electron microscopy (TEM) The morphology of the oil droplets in the nanoemulsion formulations was visualized with TEM CM 200 Philips operating at 200 Kv. Combination of bright field imaging at increasing magnification and of diffraction modes was used to reveal the form and size of the microemulsion. In order to perform the TEM observations, the microemulsion formulations (blank and ACF loaded) were diluted with 0.45 m filtered distilled water (1/100). A drop of the diluted microemulsion was placed on the film grid coated with copper and samples were observed after drying.

Int. J. Pharm. Biosci. Technol. Thermodynamic stability testing The formulations were subjected to centrifugation at 2000 rpm for 25 min and assessed for their physical stability i.e. transparency, phase separation and globule size analysis. The optimized SMEDDS formulations were subjected to the freeze-thaw cycle and were stored at -20C for 24 hr and then at 40C for another 24 hr. Their physical stability was assessed by evaluating the globule size of the formulations. Drug Content ACF from the SMEDDS was extracted in methanol and diluted suitably upto 25 ml. The total ACF content in the capsules was estimated in triplicate using the validated UV method at 275 nm. In vitro release profile: The conditions followed for the in vitro release study of ACF drug and ACF SMEDDS are listed in table 1.

Table 1. Conditions for in vitro release studies Apparatus Speed In vitro release medium Sampling interval Sampling volume Replenishing fluid Temperature Analytical Method Six station USP XXIII Dissolution Testing Apparatus II 75 rpm 0.1N HCl containing 1% SLS (900 ml) 5,10,15,20,30,45 and 60 min 5 ml 0.1N HCl containing 1% SLS 37C 0.5C UV Spectrophotometry (max =275 nm)

In-vivo pharmacodynamic evaluation Evaluation of Anti-Inflammatory Activity of developed SMEDDS Study design Healthy female Sprague-Dawley (SD) rats weighing in the range of about 150-220 gm were used for the study. The animals were housed in metabolic cages under controlled conditions in the animal house of Bombay College of Pharmacy (Mumbai, India). The general and environmental conditions were strictly monitored. Animal handling routines were performed according to Good Laboratory Practice. All the animals were given free access of food (pellet diet supplied by Hindustan Lever Ltd) and water was made available ad libitum. The research protocol of the animal experimentation was approved by the Institutional Animal Ethics Committee of Bombay college of Pharmacy, Mumbai, India. Modified Plethysmometer was used to measure the paw volume of the rats. The overnight fasted animals were divided into 9 groups (n=6). Treatment groups Group I (vehicle control): 2.5 ml of 0.5% sodium carboxymethylcellulose (CMC) Group II: Placebo Group III: Formulation FA-70 (7 mg/kg) in distilled water Group IV: Formulation FA-100 (10 mg/kg) in distilled water Group V: Pure aceclofenac (10 mg/kg) in 0.5% CMC Pol et al

Group VI: Pure diclofenac acid (9 mg/kg) in 0.5% CMC Group VII: Composition of marketed tablet (Movon, Ipca, Mumbai) (10 mg/kg) in 0.5% CMC. Procedure for evaluation of anti-inflammatory activity [12] After 30 mins of oral administration, all groups were challenged by a subcutaneous injection of 0.1 mL of 1% solution of carrageenan in saline into the plantar site of the left hind paw. The paws were marked with ink at the level of the lateral malleolus. The paw volumes were measured by immersing the paws upto the mark in the mercury well of a plesthysmometer. The paw volumes were measured using a plethysmometer, prior to administration of carrageenan and after 30 mins, 1, 2, 3, 4, 6 and 7.5 h of administration. The percent inhibition of edema was calculated. % inhibition 100 1 Where, A= Paw volume after administration of carageenan at time t X = Paw volume before carrageenan administration of

.Eq.1

B =Mean volume of control rats administration of carageenan at time t. Y = Mean volume of control rats administration of carageenan.

after before

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Int. J. Pharm. Biosci. Technol. Procedure for ulcerogenicity determining gastric tests for comparison between all groups with significance P > 0.05. RESULT AND DISCUSSION Preformulation studies The objective of the preliminary studies was to screen and select suitable components for developing SMEDDS formulations of ACF from a large pool of excipients. Solubility studies Solubility studies were carried out with an aim of identifying suitable oily phase and surfactant/cosurfactants for the development of ACF SMEDDS. [9]. It is even more important for ACF, as the target dose to be incorporated in SMEDDS is substantially high (100 mg). Equilibrium solubility of ACF in various oils, surfactants and co surfactants is presented in Fig. 2 & 3. All the essential oils showed good solubilizing capacity compared to other lipophiles. However, discoloration was observed in isotropic mixtures of ACF with essential oils. The only exception to this behavior was exhibited by peppermint oil IP which displayed good physical stability with no color or odor change even after one week.

The rats were sacrificed eight hours after administration; their stomachs were removed, incised along the greater curvature, and gently washed with saline and then mounted in phosphate buffer saline. The extent of erosion of stomach mucosa was assessed from a scoring system designed by MerazziUberti Turba as follows: 0- No erosions; 1- One to three small erosions (4 mm or smaller); 2- More than three small erosions or one large erosion; 3- Two large erosions; 4- Three to four large erosions; 5- More than four large erosions or lesion proliferation. The results were expressed in terms of an ulcer index, which is the average severity of erosions per rat each group on the scale from 0 to 5 [13]. Statistical Analysis The statistical significance of the difference between mean values was assessed by ANOVA followed by Boneferronis multiple comparison

Akoline MCM Capmul MCM L Capmul MCM Capmul Capmul OILS Labrafil S6 Lauroglycol 90 Lauroglycol FCC Capryol 90 Eucalyptus oil IFF Peppermint oil Peppermint oil IP 0 50 100 150 Labrafil 1944 CS

Concentration of ACF mg/g Fig. 2: Solubility of ACF in various oily phases Solubility expressed as mean S.D. (n = 3) Thus ACF exhibited high solubility in the volatile oils compared to other lipophiles. This observation indicated that terpenes based solubilisers (e.g volatile oils) can also be used for Pol et al drug candidates exhibiting limited solubility in the commonly used lipophiles. These oils could be explored in the development of lipid- based selfemulsifying drug delivery systems. Based on the Pg. 93

Int. J. Pharm. Biosci. Technol. results, peppermint oil IP was selected as the oily phase for further studies. Amongst the various surfactants and cosurfactants screened, (Fig. 3), ACF was found to exhibit solubility in surfactants in the following sequence: Tween 80 > Gelucire 44/14> Solutol HS 15. ACF exhibited very good solubility in various water miscible organic solvents such as, Polyethylene glycol 400, Transcutol P followed by Labrasol. [14].

Fig. 3: Solubility of ACF in various surfactants and cosurfactants Solubility expressed as mean S.D. (n = 3) Emulsification studies Screening of surfactants for emulsifying ability The optical clarity of the aqueous dispersions can be measured using standard quantitative techniques for turbidity assessment. Optical clarity corresponds to high transmittance, as opalescent dispersions will scatter incident radiation to larger extent as compared to transparent dispersions. The intensity of light passing through such dispersion is attributed to the scattering of light which occurs due to absence of optical homogeneities in the medium. Hence, % transmittance could directly be used to predict relative droplet size of the emulsion.. Based on this underlying principle, aqueous dispersions with high transmittance (lower absorbance) were considered optically clear and oil droplets were thought to be in a state of finer dispersion [15, 16]. It was necessary to identify the combinations of surfactants and lipophiles that could produce stable microemulsions. The turbidimetric studies were performed for evaluating the ability of various surfactants and co-surfactants to emulsify the selected oily phases. The percentage transmittance values of various dispersions are listed in Table 2. Emulsification studies clearly distinguished the ability of various surfactants to Pol et al emulsify peppermint oil. These studies indicated that Cremophor EL and Solutol HS 15 were comparatively more efficient in emulsifying peppermint oil followed by Tween 80 and Gelucire 44/14. Table 2: Emulsification efficiency of various non-ionic surfactants Surfactants % Transmittance Cremophor EL 96.45 0.52 Solutol HS 15 97.83 0.78 Tween 80 95.83 0.07 Gelucire 44/14 85.83 0.89 Data expressed as mean (n = 3) Interestingly, all the hydrophilic co-surfactants appeared to improve the emulsification ability of Cremophor EL and Solutol HS 15. Transcutol P was found to exhibit maximum emulsification ability amongst all the co-surfactants tried. Solutol HS 15 exhibited good self-microemulsifying potential as indicated by the transmittance values (almost 100%). These high transmittance values along with the optically clear appearance of SMEDDS dispersion confirmed the finer globule size of the formed_SMEDDS.

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Table 3: Emulsification efficiency of various non-ionic surfactants Surfactants Tween 80 Gelucire 44/14 Solutol HS 15 Cremophor EL % Transmittance values with given Cosurfactants PEG 400 Labrasol Transcutol P 97.59 0.04 98.94 0.08 99.09 0.04 89.26 0.54 92.115 0.88 96.625 0.54 100.1 0.13 100.082 0.54 100.43 0.13 99.72 0.09

Construction of pseudo-ternary phase diagrams Pseudoternary phase diagram is a useful approach to exemplify the various interactions that occur on varying the components used in the formulation of microemulsions. These pseudoternary phase

diagrams can be modified further by plotting the components of SMEDDS except water, whose quantity is kept constant throughout the experiment by diluting the preconcentrates with constant amount of distilled water.

Table 4: Combinations evaluated by pseudo-ternary phase diagrams. Formula A B C D E F G Surfactant Tween 80 Solutol HS 15 Tween 80 Solutol HS 15 Cremophor EL Tween 80 Solutol HS 15 Cosurfactant Labrasol Labrasol Transcutol P Transcutol P Transcutol P PEG 400 PEG 400 Combination Tween 80 Labrasol- Peppermint oil Solutol HS 15 Labrasol- Peppermint oil Tween 80 -Transcutol P-Peppermint oil Solutol HS 15 Transcutol P- Peppermint oil Cremophor EL-Transcutol P- Peppermint oil Tween 80- PEG 400- Peppermint oil Solutol HS 15- PEG 400- Peppermint oil structure of the solute. Enhanced drug solubility in microemulsion and micellar systems usually arises from the solubilization at the interface. The solute associated with interface, in turn, may affect the size and shape of the microemulsion droplets. Phase diagrams studies indicated a remarkable influence of ACF on globule size of systems. Incorporation of ACF in peppermint oil led to a considerable reduction in the area of microemulsion formation (Fig. 5 and 7). Due to its low aqueous solubility, ACF is likely to participate in the microemulsion formation by orienting at the interface. The reduction in the area of microemulsion formation could be due to ACFinfluenced interaction of surfactant and cosurfactant with oil. The phase diagram studies led to the selection of formulations FA containing Solutol HS 15: Labrasol: Peppermint oil combination in 40:30:30 proportions. The selections were made on the basis of lower surfactant concentrations, high content of oily phase, high drug loading and globule size less than 150 nm. These conditions were selected based on a hypothesis that if the concentration of surfactant is high, drug concentration at the interphase would probably be greater and chances of drug precipitation may be more as compared to systems containing high concentration of oily phase.

Initially, optimization studies were performed with the help of phase diagram. The changes in behavior of the systems for phase separation and for drug precipitation were evaluated macroscopically and microscopically. Various phase diagrams depicted in Figs. 4 to 7 reflected the influence of drug content and respective combinations of surfactant and cosurfactants. Although PEG 400 showed best solubilizing capacity for ACF, the region of microemulsion was found to decrease for systems containing PEG 400 as the cosurfactant. The systems with Transcutol P as cosurfactant showed larger microemulsion region but precipitation of ACF was observed eventually in all combinations except combination containing Cremophor EL-Transcutol PPeppermint oil [Fig.7]. The combinations of surfactants with Labrasol gave more stable systems with respect to drug precipitation. But since globule sizes for systems in combination A were higher than 150 nm (which was selected as the criteria for globule size) they were rejected. Microemulsion regions for the combinations B and E were found satisfactory as depicted in the phase diagrams (Fig. 4 and 5). The systems were then evaluated along with their placebo systems for the effect of drug on microemulsion region formation. Location of the solubilized drug in microemulsion systems depends on the hydrophobicity and

Pol et al

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ME

ME

Fig.4: Pseudo ternary phase diagrams of combination B

Fig.5: Pseudo ternary phase diagrams of combination B without drug

ME

ME

Fig.6: Pseudo ternary phase diagrams of combination E

Fig.7: Pseudo ternary phase diagrams of combination E without drug

The outer parallelogram indicates the area, which was explored for locating microemulsification region. The filled blue region indicates the region in which microemulsions of desired size were obtained. Influence of drug loading Effect of drug loading on globule size and polydispersity index of microemulsions generated from optimized formulations is shown in Fig.8. The formulations containing dose higher than 110 mg were found to show an increase of 25 nm in the globule size Fig.8) and drug precipitation over a period of 12 h.

Influence of oil content The effect of increase in the oil concentration on globule size of selected systems is shown in Figs.8. As depicted in Fig.8, increase in the content of oily phase upto 250 mg in system was not found to show a considerable change in globule size of the resulting microemulsions. This could be explained as, when the concentration of oil was increased, the concentrations of surfactant and cosurfactant were probably insufficient to reduce the surface tension at the interphase, resulting in separation of non- emulsified oil droplets and thus coalescence of oil droplets and phase separation.

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Globule size of FA P.I of FA 180 160 140 120 100 80 60 40 20 0 50 75 100 115 130 ACF dose in mg 1.25 1.2 Polydispersity index 1.15 1.1 1.05 1 0.95 0.9 Mean globule size in nm

90 Mean globule size in nm 60 30 0 200 225 250 350 Content of peppermint oil in mg

1.2 1.15 1.1 1.05 1 0.95 Polydispersity index

(a) (b) Fig. 8: (a) Effect of drug loading on globule size# and P.I. of optimized formulations FA (b) Effect of oil content on globule size and P.I of system (n=3). Characterization Appearance The filled capsules showed no signs of leakage, discoloration, pinholes and shell distortion. ACF loaded SMEDDS appeared as clear, transparent, homogenous liquids at room temperature. No traces of particulate matter nor drug precipitation were observed.

Fig. 9: ACF capsules and process of formulation release and self-emulsification from capsule.

Uniformity of weight None of the capsules were found to deviate from the average capsule by more than 7.5 % and were found to comply with I.P96 standards for uniformity of weight for capsules. Drug Content The drug content of various self-microemulsifying formulations was found to be within the range of 99-101% which was in agreement with pharmacopoeial specifications. Self-microemulsification efficiency Formulations were found to release the contents immediately upon rupturing and self-emulsify within a minute. Fig.9 shows process of drug release and self-emulsification from capsule. Globule size analysis The results of globule size of the formulations (with and without ACF) were found to be 25.6 2.34 and 91.9 10.58 nm respectively. The effect of drug Pol et al

incorporation in SMEDDS was found to be influenced by the drug-system physicochemical properties. After dilution of preconcentrates with various aqueous phases, the resulting microemulsions were found to be clear, transparent and appeared like homogenous single-phase liquids. pH determination ACF incorporation into SMEDDS lowered the pH of the systems towards acidic side; this change may be attributed to the acidic nature of the drug. After the dilutions of the preconcentrates with distilled water, the pH values were found to increase. This may be probably due to encapsulation of major quantity of drug into microemulsion and so decrease in acidic nature of the systems. Dilution test The ability of a microemulsion to be diluted without any drug precipitation is essential for its use as a drug delivery vehicle since, after administration, it will almost certainly be diluted Pg. 97

Int. J. Pharm. Biosci. Technol. by body fluids. These studies were performed to study the robustness of the formulations towards different media (varying in pH & electrolyte content) simulating gastric tract conditions. After dilution, the resulting microemulsions were found to remain clear, transparent and appeared like homogenous single-phase liquids. All the preconcentrates were found to be dispersed within one minute. In addition, the composition and pH of the aqueous phase was found to have no Water 180 160 140 120 100 80 60 40 20 0 0 1 Time (Hr) Fig. 10: Effect of pH and electrolytes of aqueous phase on globule size (nm) # Globule size (nm) expressed as mean, (n = 3) where relative standard deviation was < 10 %. centrifugation and freeze-thaw cycle process and no phase separation or drug precipitation was observed. Particle size and polydispersity remained unaffected after freeze-thaw process, thus confirming the stability of developed microemulsions. In vitro release profile The dissolution profile of ACF from various optimized SMEDDS, marketed (Movon Capsule 100mg) and plain ACF was determined. The selection of the particular medium for studying in vitro release profile of the drug was based on the solubility studies results reported in literature. The in vitro drug release profile of the ACF in 0.1 N HCl + 1 % SLS from various formulations are shown in Fig. 11. The dissolution rate of ACF from the developed SMEDDS was found to be significantly higher than that from the marketed formulation. The results indicated a faster in-vitro release of the drug from the developed formulations (T 80% = 5 mins) as compared to that from marketed formulations (T 80% = 45 min). The plain drug released 72.76% of drug in one hour whereas the marketed formulation and the developed formulations exhibited a release of 81.774 % and 99.23 to 100.17 % respectively. Pg. 98 2 4 8 Globule Size (nm) pH 1.2 effect on the properties of microemulsions and did not show any separation or drug precipitation till 8 hr. The ACF SMEDDS showed fairly similar mean globule size (within range of 50100 nm) when diluted with various media differing in pH and electrolyte concentration. In general globule size of microemulsions was found to remain stable for 4 hr. (Fig. 10). Thus from these studies it can be predicted that ACF SMEDDS could retain its stability after dilutions in GI tract. pH 3 pH 6.8 Ringer Solution

Zeta potential measurement The electrical surface charge of the droplets is produced by the ionization of interfacial filmforming components. The surface potential and the resulting Zeta potential of emulsion droplets will depend on the extent of ionization of the emulsifying agents. Zeta potential of the system was found to be -47.77 7.64 mV and 8.04 4.3 in pH 1.2 and 6.8 respectively, thus indicating stability of developed smedds after dilution in gastric environment. Elecrtophoretic mobility was found to be -3.7 2.6 and 0.63 0.34 in pH 1.2 and 6.8 respectively. Transmission electron microscopy (TEM) The microemulsion appeared dark and the surroundings were bright (data not shown), a positive image was seen using TEM. In addition, the morphology of the droplet was spherical and there was no evidence of ACF precipitation in either the oil phase or the aqueous phase. Thermodynamic stability Centrifugation and freeze-thaw cycling are accelerated tests used to determine the stability of microemulsions under stress conditions. All the formulations were found to remain stable after Pol et al

Int. J. Pharm. Biosci. Technol. inflammatory drugs including ACF are effective at this phase of edema formation [18]. The antiinflammatory activity of plain ACF, plain diclofenac and the developed SMEDDS formulations over a period of 7.5 hours is depicted in Fig. 12. A significant difference (P < 0.05) in the percent inhibition values was obtained between the developed ACF SMEDDS and plain ACF at 4 hr. The percentage inhibition of edema by developed SMEDDS was comparable to that of plain diclofenac while placebo formulation also showed inhibition in inflammation during first half of the study. The ACF SMEDDS and plain diclofenac showed inhibition of 80 7.30 % and 88.01 2.97 respectively after 4 hr of treatment against carrageenan induced paw edema at the dose of 10mg/kg. These observations may be attributed to the increase in absorption of ACF from the formulated SMEDDS, indicating a possibility of increased bioavailability and synergistic activity with peppermint oil. As volatile oils such as mint oils have shown to exhibit in vivo antiinflammatory activity. A statistically insignificant difference was observed in the anti-inflammatory activity of the formulation containing lower dose of drug (FA-70) and that containing 100 mg of ACF (FA-100). Thus, it could be concluded that reduction of dose could be effectively tried in ACF SMEDDS without significant reduction in the anti-inflammatory activity.

100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 0 5

F A ( 100 mg) Marketed formulation Plain drug

% Drug release

10 15 20 Time in min

30

45

60

Fig. 11: In vitro release profiles of different formulations of ACF in 0.1N HCl containing 1% SLS Evaluation of Anti-Inflammatory Activity in rat paws edema Carrageenan induced edema in rat is a well established model used experimentally to evaluate anti-inflammatory activity of any drug/extract from natural/synthetic origin. The edema develops in three distinct phases. The first phase involved release of histamine, whereas in the second phase kinin and bradykinin are released, and the last phase in manifested by involvement of prostaglandins. Most anti

100 90 Percentage Inhibition 80 70 60 50 40 30 20 10 0 Placebo

3 Hr
*

4 Hr

75
*

vehicle control F A-70

Blnk A F A-100

65 55 Percentage
ACF ACF Plain ACF Plain SMEDDS SMEDDS diclofenac 70 100

45 35 25 15 5 -5 0 1 2 3 4 5 Time (hr) (b) 6 7 8

(a)

Fig. 12: Effect of ACF SMEDDS on rat paw edema wherein (a) Comparative graph representing effect of ACF formulations and plain ACF and Diclofenac on paw edema induced by carageenan in Sprague dawley rats [FA-70 is SMEDDS with ACF 70mg and FA-100 is SMEDDS with ACF 100mg] and (b) Comparative graph of percent inhibition ( * P < 0.01 Pol et al Pg. 99

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Evaluation of gastric ulcerogenicity The gastric ulcer scores of developed selfmicroemulsified systems were found to be significantly lower than the marketed formulation (Fig. 13) of ACF (P < 0.05). The formulation exhibited the lowest score (0.670.577), as compared to other groups whereas the highest score was observed by the marketed formulation (5.0 0.07) Fig.16. The mean score for the degree of injury produced by plain ACF was 3.67 1.15 (n = 6). The formulation exhibited a significant difference in the score index as compared to that of plain ACF. Thus, it could be concluded that the delivery of the drug as a self-microemulsified system resulted in reduction in the ulcerogenic potential of the drug. It is reported that crystals of

NSAIDs being poorly soluble in gastric acid remain in contact with the stomach wall for a longer period of time, resulting in a dangerously high local concentration. This leads to local irritation of the stomach wall and to ulceration. In general, it is expected that the drug delivered in a microemulsion vehicle is in a solubilized form, thus resulting in accelerated absorption. Moreover, when delivered as SMEDDS, the drug may probably not come in direct contact with the stomach wall, leading to decreased ulceration. The incorporation of ACF in SMEDDS, thus provided better protection against GI tract ulceration as compared to the marketed formulation [18,19,20].

Degree of Injury

0 Vehicle Control Placebo ACF ACF SMEDDS 70 SMEDDS 100 Plain ACF Plain Diclofenac Marketed Formulation

Fig. 13: Effect of various treatments on degree of injury to stomach

CONCLUSION In the present investigation, improvement in aqueous solubility of Aceclofenac was achieved by incorporation into SMEDDS. Use of Peppermint oil, a terpene based solubilizers was explored for preparation of ACF SMEDDS. ACF SMEDDS exhibited excellent stability in different pH media and electrolyte content. In vitro dissolution study demonstrated a significant improvement in the dissolution profile of ACF. The developed SMEDDS were found to exhibit less GI tract Pol et al

toxicity and showed superior anti inflammatory action compared to plain drug as well as diclofenac. REFERENCES 1. Shaji J, Joshi V. Self-Micro Emulsifying Drug Delivery System (SMEDDS) for improving bioavailability of hydrophobic drugs and its potential to give sustain release dosage form, Indian Journal of Pharmaceutical Education. 2005; 39:130-135. Pg. 100

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How to cite this article APA style Pol, A. S., Patel, P. A., & Hegde, D. (2013). Peppermint oil based drug delivery system of aceclofenac with improved anti-inflammatory activity and reduced ulcerogenecity. International Journal of Pharma Bioscience and Technology, 1(2), 89101. Elsevier Harvard style Pol, A.S., Patel, P.A., Hegde, D., 2013. Peppermint oil based drug delivery system of aceclofenac with improved anti-inflammatory activity and reduced ulcerogenecity. Int. J. Pharm. Biosci. Technol. 1, 89101. Vancouver Style Pol AS, Patel PA, Hegde D. Peppermint oil based drug delivery system of aceclofenac with improved anti-inflammatory activity and reduced ulcerogenecity. Int. J. Pharm. Biosci. Technol. 2013;1(2):89101. To receive bibliographic information in RIS format (For Reference Manager, ProCite, EndNote): Send request to: ijpbst@yahoo.com