Materials and Methods

Transcription

1 3. MATERIALS AND METHODS 3.1. MATERIALS Curcumin was purchased from Sigma Aldrich, Mumbai, India. Flurbiprofen was obtained as a gift sample from Alpha Remedies, Ambala, India. Chitosan (low mol. wt., viscosity cp) and Eudragit S100 (Evonik Rohm Pharma, Germany, Viscosity mpa s, mol. wt. 135,000) were received as a gift sample from Matrix laboratory, Hyderabad. The deacetylation degree of chitosan according to the specifications from the provider was higher than 80%. Span 80 (Mol. wt , viscosity mpa s) and liquid paraffin, anhydrous zinc chloride, carboxy methyl cellulose were purchased from Loba chemie Mumbai, India. All chemicals and reagents used were of analytical grade. Double distilled water was used throughout the study PREFORMULATION STUDIES OF CURCUMIN Preformulation study is defined as the process of optimizing the delivery of drug through determination of physicochemical properties of the active compound that could affect drug performance and development of an efficacious, stable and safe dosage form. A thorough understanding of these physicochemical properties may provide a rationale formulation design. The nature of the drug highly affects the processing parameter like method of preparation, entrapment efficiency, compatibility and pharmacokinetic response of the formulation. Thus, in order to ensure optimum condition for clinically beneficial delivery system, various preformulation studies were performed. The procured sample of Curcumin was characterized in terms of its Melting point, Thin Layer Chromatography technique (TLC), Fourier Transform Infra-Red (FTIR) spectral study, Solubility studies and calibration curves for Curcumin Authentication of the procured curcumin Several parameters were taken into consideration for drug authentication. Drug sample was authenticated by melting point, thin layer chromatography (TLC) and FTIR Melting point determination Capillary fusion method was used for determining the melting point of curcumin. In this one sided sealed capillary was taken and sufficient amount of drug was filled into the capillary. The capillary was kept inside the melting point apparatus and temperature was increased gradually. School of Pharmaceutical Sciences 54

2 When the drug started melting and when melted properly, the temperature was noted down with the help of a thermometer. Similarly readings were taken six times and compared with reference value Thin Layer Chromatography Thin Layer Chromatography is a technique used to identify compounds alone or present in a mixture. As stationary phase precoated silica gel (F 254 ) aluminum plates (2x4cm) were used. A spot of the standard solution was put 0.5cm from the bottom and placed in TLC chamber, which was previously saturated with the solvent system chloroform: methanol (9:1). The solvent was allowed to rise at least 3/4th of the plate and the distance travelled by solvent front was noted. The Rf value was calculated by using the formula: Rf value= Distance travelled by the solute from the origin/distance travelled by the solvent front from the origin The R f value was calculated and compared with literature R f value (Vedamurthy et al., 2010) FTIR [Fourier Transform Infra-Red Spectroscopy] FTIR is most powerful technique for qualitative identification of compound. It gives the information about the functional group present in the particular compound. The main application of FTIR-spectrophotometer is the determination of the identity of a compound by means of spectral comparison with that of reference spectra and verification of the presence of functional group in an unknown molecule. Spectra were recorded from 4000 to 650 cm -1 scanning range and the observed spectrum was compared with reference FTIR spectra. FTIR of CUR was determined by FTIR Instrument of Agilent Technologies 630 Cary using Micro Lab software Estimation procedures of Curcumin Calibration curve of Curcumin by UV-Visible Spectrophotometer (Systronic 2202) a) Medium: 0.1N HCl (ph 1.2) The absorption maxima (λ max ) for Curcumin in 0.1 N HCl was determined by scanning the drug solution within the range of nm using UV-Visible Spectrophotometer (Systronic 2202). It was found that the drug exhibited λ max at 432 nm. School of Pharmaceutical Sciences 55

3 Preparation of stock solution: 100 mg of curcumin was accurately weighed and solubilized in 5 ml of methanol and the volume was made up to 100 ml with 0.1N HCl, to result in a concentration of 1mg/mL. Working standards were prepared using this stock solution. Preparation of working standard: Stock solution of 10 ml was taken in volumetric flask (100 ml) and volume was made up with 0.1N HCl to obtain concentration of 100µg/mL. Further dilutions were made to give 5, 10, 15, 20, 25 and 30 µg/ml concentration of curcumin respectively. These dilutions were scanned and the absorbance was measured at 432 nm using UV-Visible Spectrophotometer (Systronic 2202). To obtain the standard calibration curve, the resulted absorbance values were plotted against the respective concentrations. Average values of absorbance were calculated by repeating the procedure six times. b) Medium: Phosphate buffer (ph 6.8) The absorption maxima (λ max ) for Curcumin in phosphate buffer ph 6.8 were determined by scanning the drug solution within the range of nm using UV-Visible Spectrophotometer (Systronic 2202). It was found that the drug exhibited λ max at 432 nm. Preparation of stock solution: Accurately weighed 100 mg of curcumin was solubilized in 5 ml of methanol and the volume was made up to 100 ml of phosphate buffer ph 6.8 to obtain a concentration of 1mg/mL. By using this stock solution, working standards were prepared. Preparation of working standard: 10 ml of stock solution was taken in volumetric flask and volume was made up with phosphate buffer ph 6.8 to result in a concentration of 100µg/mL. Further dilutions were made to give 5, 10, 15, 20, 25 and 30 µg/ml concentration of curcumin respectively. These solutions were scanned and the absorbance was measured at 432 nm using UV-Visible Spectrophotometer (Systronic 2202). The resulted absorbance values were plotted against the respective concentrations to obtain the standard curve. Average values of absorbance were calculated by repeating the procedure six times. c) Medium: Phosphate buffer (ph 7.4) The λ max (absorption maxima) for Curcumin in phosphate buffer ph 7.4 were analyzed by scanning the solution of drug within the range of nm using UV-Visible Spectrophotometer (Systronic 2202). It was observed that the drug exhibited λ max at 432 nm. Preparation of stock solution: 100 mg of curcumin was weighed and solubilized in 5 ml of methanol which was further dissolved up to 100 ml with phosphate buffer ph 7.4 to achieve a concentration of 1mg/mL. By using this stock solution, working standards were prepared. School of Pharmaceutical Sciences 56

4 Preparation of working standard: 10 ml of stock solution was taken and volume was made up with phosphate buffer ph 7.4 to obtain a concentration of 100µg/mL. Further dilutions were made to give 5, 10, 15, 20, 25 and 30 µg/ml concentration of curcumin respectively. These solutions were scanned and the absorbance was measured at 432 nm using UV-Visible Spectrophotometer (Systronic 2202). To obtain the standard curve, the resulted absorbance values were plotted against the respective concentrations. Average values of absorbance were calculated by repeating the procedure six times Solubility studies Solubility study of curcumin was performed using orbital shaker in 0.1N HCl (ph 1.2), phosphate buffer (ph 6.8) and phosphate buffer (ph 7.4) by Equilibrium solubility method Equilibrium solubility method Excess amount of curcumin was put into 25 ml of 0.1N HCl (ph 1.2), phosphate buffer (ph 6.8) and phosphate buffer (ph 7.4) separately in conical flasks. Then these flasks were kept in solubility shaker at 37 C. After 1 h all the solutions were filtered and measured using UV spectrophotometer at 432 nm against 0.1N HCl (ph 1.2), phosphate buffer (ph 6.8) and phosphate buffer (ph 7.4). Then the amount of drug solubilized was calculated Compatibility studies DSC Analysis A differential scanning calorimetry (JADE DSC, Perkin Elmer, USA) was used to study the thermal analysis of drug-excipient compatibility. Firstly, the curcumin was mixed with chitosan and Eudragit S100. The drug-excipient mixture was scanned in the temperature range of ºC under an atmosphere of nitrogen. The heating rate was 20ºC/min and the obtained thermograms were observed for any type of interaction (Carstensen et al., 2000) Preparation of Microspheres of Curcumin Emulsion cross linking method: Chitosan polymer was dissolved in 10 ml of aqueous solution of acetic acid (1%) and Curcumin was dispersed in the polymeric solution (Table 3.1). This solution was incorporated into 100 ml of liquid paraffin containing Span 80 (1% v/v). An School of Pharmaceutical Sciences 57

5 w/o emulsion was formed by stirring at 1500 rpm for 2 h with the help of mechanical stirrer (Remi Motors, Mumbai, India). 0.5 ml of glutaraldehyde was added to the emulsion and kept for 1 h. The solvents were removed by stirring under vacuum. Microspheres were obtained by filtration and washed with petroleum ether and dried in hot air oven at 40º C (Jose et al., 2013). All the studies were performed in amber colored glass apparatus and under dark conditions Polymeric coating of chitosan microspheres of CUR: The CUR chitosan microspheres were coated with Eudragit S100 by emulsification-solvent evaporation method. The developed chitosan microspheres loaded with CUR were added in 20 ml of ethanolic solution of Eudragit S100 (10% w/v). Emulsification was achieved with 70 ml of light paraffin including Span 80 (1% v/v) followed by stirring for 2 h at 1500 rpm with mechanical stirrer. The Eudragit S100 coated microspheres were collected by filtration and washing steps were done with petroleum ether. The developed coated microspheres were dried in hot air oven at 40º C for 3 h (Perge et al., 2012). Table 3.1: Composition of colon targeted CUR microspheres Formulation code CUR (g) Chitosan (g) Emulsifier (%) Eudragit (% w/v) F F F F F F F F School of Pharmaceutical Sciences 58

6 3.4. In-vitro evaluation of CUR microspheres Determination of particle size, shape and surface morphology Malvern Mastersizer (Malvern Instruments, Mastersizer 2000, UK) was used to determine the particle size of CUR microsphere formulations (Tsai et al., 2013). The values (Z avg ) were denoted for all formulations as average size range. The prepared microspheres were characterized for shape and surface morphology using scanning electron microscope (SEM-QUANTA 250, FEI Makers, Singapore). The microspheres were mounted on an aluminum stub with carbon-glue and coated with gold using a gold sputter module in a high vacuum evaporator. The photographs were then taken using an excitation voltage of 10 kv. The magnifications selected were sufficient to appreciate in detail the morphology of the samples under study (Yadav et al., 2013) X-ray diffraction X-ray diffractometer (Bruker Axs D8 Advance, Germany) was used for qualitative powder X-ray diffraction of CUR and its microspheres. The instrument was operated at a voltage of 40 kv and a current of 30 ma, with copper as the tube anode material (Philip et al., 2009). The samples were run over a range of 2θ angles from 2º to 80º Determination of percent yield, drug loading and entrapment efficiency The percent yield (PY) was calculated based on mass of CUR and polymer added, using following equation: Mass of the obtained microspheres PY = x 100 Initial mass of drug + Initial mass of polymer 100 mg of prepared microspheres were first crushed with the help of glass mortar pestle followed by dispersion in 100 ml of methanol and kept for overnight for the extraction of drug. The supernatant was diluted with methanol after centrifugation at 2500 rpm for 10 min and using UV spectrophotometer (Systronics 2202), absorbance was measured at 432 nm. After that the mass of CUR in microspheres was calculated by calibration curve. The drug loading was calculated for each batch six times (Singh and Pathak, 2011, Ahmad et al., 2012, Zhao et al., 2013). School of Pharmaceutical Sciences 59

7 Using following equation, drug loading (DL) and entrapment efficiency (EE) were determined. Mass of the CUR in microspheres DL = X 100 Mass of the microspheres Mass of the CUR in microspheres EE = X 100 Initial mass of drug In-vitro swelling In a cellophane membrane dialysis bag (D9402, Sigma Aldrich), 100 mg of chitosan microspheres and Eudragit coated chitosan microspheres were placed in phosphate buffer (ph 7.4). Then microspheres were allowed to swell for a period of 8 h. After that changes in weight were measured by removal of the samples and blotted with a filter paper for 10 s to absorb excess solvent on surface. The degree of Swelling was determined using the following equation: [S α = (W t W 0 ) / W 0 ] Where S α represents the degree of swelling, W t and W 0 represent weights of the sample at equilibrium swelling and the original dry weight, respectively (Ruiz-Caro et al., 2009) In-vitro drug release In-vitro release study of developed formulations was performed in USP dissolution- I (basket type, Electrolab, EDT 08 LX, Mumbai, India) in various simulated GI fluids under anaerobic condition. The simulation of variation in GI ph was achieved by altering the ph of the dissolution medium at different time intervals. The dissolution medium was kept at ph 1.2 with 0.1 N HCl for 2 h, ph 6.8 for next 2 h with phosphate buffer and then at ph 7.4 up to 12 h. Then at regular time intervals 5 ml of samples were withdrawn and replaced with equal amount of the fresh medium to maintain sink condition. The samples were analyzed by UV spectrophotometer at 432 nm (Systronics 2202, India) (Jose et al., 2011). Further the in-vitro release of selected microspheres formulation was carried out in the presence of 0.32% w/v pepsin for first 4 h to School of Pharmaceutical Sciences 60

8 predict the stability of Eudragit S-100 and then in the presence of 10% w/v of rat caecal content from 5 th h, to analyze the biodegradability of chitosan in presence of colonic microflora (Paharia et al., 2007) Determination of kinetic model for release of CUR-loaded chitosan microspheres The in-vitro drug release data of the Eudragit coated chitosan microspheres was fitted to different kinetic models i.e. zero order, first order, Higuchi and Peppas model and on the basis of goodness of fit, the best model was selected (Christoper et al., 2013). The zero-order rate describes systems where drug release is independent of its concentration and is generally seen for poorly water-soluble drug in matrix, transdermal, etc. (Najib et al., 1985). Q t = k 0 t (1) The first-order describes systems in which the release is dependent on its concentration (generally seen for water-soluble drugs in porous matrix) (Desai et al., 1966). ln Q t = ln Q 0 k 1 t (2) The Higuchi model describes the release of the drug from an insoluble matrix to be linearly related to the square root of time and is based on Fickian diffusion. Q t = k Η t 1 / 2 (3) Korsmeyer Peppas derived a simple relationship which describes drug release from a polymeric system. Q t /Q α = Kt n (4) Where Q t /Q α is the fraction of drug released at time t; K is release rate constants and n is release exponent. The n value is used to characterize different release pattern. In this model, the value of n characterizes the release mechanism of drug n corresponds to a Fickian diffusion mechanism, 0.45 < n < 0.89 to non-fickian transport, n = 0.89 to Case II (relaxational) transport, and n > 0.89 to super case II transport. To study the release kinetics, data obtained from in vitro School of Pharmaceutical Sciences 61

9 drug release studies were plotted as log cumulative percentage drug release versus log time (Dash et al., 2010) Preparation of Curcumin- Zn (II) Complex The complex was prepared by mixing curcumin with anhydrous zinc chloride at a molar ratio of 1:1 in ethanol solution as showed in scheme (Fig. 3.1). Curcumin (0.25 g) and anhydrous zinc chloride (0.1 g) was dissolved in 20 ml absolute ethanol solution. The mixture was stirred for 3 h at room temperature. The brick red solid product was filtered and washed by cold water to remove the residue reactants and then the product was dried in vacuum overnight (Zhao et al., 2010). O O C C ZnCl 2 HO OH OCH 3 OCH 3 EtOH RT Cl Cl Zn O O HO OH OCH 3 OCH 3 Fig. 3.1: Scheme for preparation of Curcumin-Zn(II) complex School of Pharmaceutical Sciences 62

10 Thin Layer Chromatography Thin Layer Chromatography of Curcumin-Zn(II) complex was performed using precoated silica gel (F 254 ) aluminum plates (2x4cm) as stationary phase and mobile phase was Chloroform: Methanol [9:1]. The R f value was calculated and compared with R f value of curcumin (Ansari et al., 2005, Modasiya and Patel, 2012) IR Spectroscopy IR spectroscopy of Curcumin and Curcumin-Zn(II) complex was performed on Fourier transformed infrared spectrophotometer. The spectral range was from cm UV-Visible Spectroscopy Ultraviolet-Visible (UV-Vis) absorbance spectroscopy of Curcumin and Curcumin-Zn(II) complex was performed on a UV spectrophotometer (Systronics 2202). Samples were scanned in the range of 200 to 800 nm H NMR spectroscopy 1 H NMR spectra of Curcumin and Curcumin-Zn(II) complex in DMSO-d6 solution were recorded on Bruker DMX-500 spectrometer with 1 H resonance frequency of 400 MHz. The tetramethylsilane (TMS) was used as an external reference of the spectrum with its 1 H chemical shift set at 0 ppm. A 5 mm diameter NMR tube was used (Zhao et al., 2010) Estimation procedures of Curcumin-Zn(II) Complex Calibration curve of Curcumin -Zn(II) Complex by UV-Visible Spectrophotometer The absorption maxima (λ max ) for Curcumin-Zn(II) complex in 0.1 N HCl (ph 1.2), Phosphate buffer (ph 6.8) and Phosphate buffer (ph 7.4) was determined by scanning the drug solution within the range of nm using UV-Visible Spectrophotometer (Systronic 2202). It was found that the drug exhibited λ max at 466 nm. Calibration curves of Curcumin-Zn(II) Complex were prepared in various media such as in 0.1 N HCl (ph 1.2), Phosphate buffer (ph 6.8) and Phosphate buffer (ph 7.4) respectively. School of Pharmaceutical Sciences 63

11 Preparation of stock solution: 100 mg of curcumin-zn(ii) complex was accurately weighed and solubilized in 1mL of methanol and the volume was made up to 100 ml with respective medium, to result in a concentration of 1mg/mL. Working standards were prepared using this stock solution. Preparation of working standard: Stock solution of 10 ml was taken in volumetric flask (100 ml) and volume was made up with respective medium to obtain concentration of 100µg/mL. Further dilutions were made to give 5, 10, 15, 20, 25 and 30 µg/ml concentration of curcumin- Zn(II) complex respectively. These dilutions were scanned and the absorbance was measured at 466 nm using UV-Visible Spectrophotometer (Systronic 2202). The resulted absorbance values were plotted against the respective concentrations to obtain the standard calibration curve. Average values of absorbance were calculated by repeating the procedure six times HPLC method development A RP-High Performance Liquid Chromatography method was developed for the determination of Curcumin-Zn(II) in developed formulation and in plasma. The method was statistically validated. Instrumentation Analysis was performed using HPLC binary system (Agilent technology) composed of binary pumps combined with photodiode array detector set at wavelength range 254 nm. Agilent eclipse XBD column (C 18 (4.6 x 150 mm) bonded with 5 µm particle size) coupled with EZ-Chrome software was used for recording and processing of chromatographic data. Method Calibration Curve Stock solutions of Curcumin-Zn(II) 1 mg/ml were prepared in solvent system and stored at 2-8 C until used. Aliquots from each stock solution were diluted stepwise with solvent system to obtain further dilutions (2 ppm, 4 ppm, 6 ppm, 8 ppm, 10 ppm). 5µL injections of each concentration of drug were injected into the RP-HPLC system separately under the conditions as described above. Evaluation of drug was analyzed at 254nm. Peak areas were recorded for all the peaks and peak areas were plotted against the concentrations to obtain the standard calibration curves. School of Pharmaceutical Sciences 64

12 Optimization of chromatographic conditions The effects of different chromatographic conditions on the instrument response create a situation where one has to compromise between different experimental variables in order to achieve the best chromatographic separation. Chromatographic separations are significantly affected by the mobile phase conditions, such as type and composition of the formulation and therefore before selecting the conditions for the optimization, a number of preliminary trials were conducted with combinations of different organic solvents, compositions and flow rate to check the retention time, shape, resolution and other chromatographic parameters. Among all tried experiments, the mobile phase combination of water and Acetonitrile in the ratio of (50: 50 v/v) with RP-HPLC at flow rate of 0.8 ml/min was found to be most suitable. Best resolution and sensitivity of the method was obtained for Curcumin-Zn(II) at 254nm. Typical chromatogram with optimized condition gives sharp and symmetric peak with specific retention time. Validation of Optimized method After chromatographic method development and optimization, method was validated. Optimized method was validated according to ICH guidelines for linearity, sensitivity, precision and % recovery (ICH Guidelines, 1994; ICH Guidelines, 1996). All the variables of the method were considered for validation including sampling procedure, sample preparation, chromatographic separation, detection and data evaluation. The developed method was validated for various parameters such as linearity, system suitability, specificity, precision, robustness, accuracy, limit of detection and limit of quantification In-Vitro Stability In-vitro kinetic degradation of Curcumin and Curcumin-Zn(II) complex was determined spectrophotometrically at 432 and 466 nm respectively. 20mg of curcumin and curcumin complex was incubated at 37ºC in 100mL of phosphate buffer at ph 7.4. Kinetic degradation of curcumin and curcumin-zn(ii) complex was analyzed for 24 h (Zebib et al., 2010) Solubility Studies Solubility study of Curcumin-Zn(II) was performed using orbital shaker in 0.1N HCl (ph 1.2), phosphate buffer (ph 6.8) and phosphate buffer (ph 7.4) by Equilibrium solubility method. School of Pharmaceutical Sciences 65

13 Equilibrium solubility method Excess amount of Curcumin-Zn(II) was placed into 25 ml of 0.1N HCl (ph 1.2), phosphate buffer (ph 6.8) and phosphate buffer (ph 7.4) separately in conical flasks. Then these flasks were kept in solubility shaker at 37 C. After 1 h all the solutions were filtered and measured using UV spectrophotometer at 466 nm against 0.1N HCl (ph 1.2), phosphate buffer (ph 6.8) and phosphate buffer (ph 7.4). Then the amount of drug solubilized was calculated Preparation of Microspheres of Curcumin-Zn(II) Complex Emulsion cross linking method: Chitosan polymer was dissolved in 10 ml of aqueous solution of acetic acid (1%) and Curcumin-Zn(II) was dispersed in the polymeric solution (Table 3.2). This solution was added to 100 ml of liquid paraffin containing Span 80 (1% v/v). An w/o emulsion was formed by stirring at 1500 rpm for 2 h with the help of mechanical stirrer (Remi Motors, Mumbai, India). 0.5 ml of glutaraldehyde was added to the emulsion and kept for 1 h. The solvents were removed by stirring under vacuum. Microspheres were obtained by filtration and washed with petroleum ether and dried in hot air oven at 40º C. All the studies were performed in amber colored glass apparatus and under dark conditions Polymeric coating of chitosan microspheres of Curcumin-Zn(II) complex Emulsification-solvent evaporation method was used to coat the Curcumin-Zn(II) chitosan microspheres with Eudragit S100. The developed chitosan microspheres loaded with Curcumin- Zn(II) were added in 20 ml of ethanolic solution of Eudragit S100 (10% w/v). Emulsification was achieved with 70 ml of light paraffin including Span 80 (1% v/v) followed by stirring for 2 h at 1500 rpm with mechanical stirrer. The Eudragit S100 coated microspheres were collected by filtration and washing steps were done with petroleum ether. The developed coated microspheres were dried in hot air oven at 40º C for 3 h (Oosegi et al., 2008). School of Pharmaceutical Sciences 66

14 Table 3.2: Composition of colon targeted Curcumin-Zn(II) microspheres Formulation code CUR-Zn(II) (g) Chitosan (g) Emulsifier (%) Eudragit (% w/v) F F F F F F F F In-vitro and in-vivo evaluation of Curcumin-Zn(II) microspheres Determination of particle size, shape and surface morphology Curcumin-Zn(II) microsphere formulations were analyzed for determination of average diameter by Zeta-Sizer (Malvern Instruments, Mastersizer 2000, UK). The values (Z avg ) were denoted for all formulations as average size range. The prepared microspheres were characterized for shape and surface morphology using scanning electron microscope (SEM-QUANTA 250, FEI Makers, Singapore). The microspheres were mounted on an aluminum stub with carbon-glue and coated with gold using a gold sputter module in a high vacuum evaporator. The photographs were taken using an excitation voltage of 10 kv. The magnifications selected were sufficient to appreciate in detail the morphology of the samples under study (Zhang et al., 2013) X-ray diffraction X-ray diffractometer (Bruker Axs D8 Advance, Germany) was used for qualitative powder X-ray diffraction of Curcumin-Zn(II) and its microspheres. The instrument was operated at a voltage of School of Pharmaceutical Sciences 67

15 40 kv and a current of 30 ma, with copper as the tube anode material. The samples were run over a range of 2θ angles from 2º to 80º (Paradkar et al., 2004) Determination of percent yield, drug loading and entrapment efficiency The percent yield (PY) was calculated based on mass of Curcumin-Zn(II) and polymer added, using following equation: Mass of the obtained microspheres PY = x 100 Initial mass of drug + Initial mass of polymer Curcumin-Zn(II) microspheres [100 mg] were crushed and extracted using 100 ml methanol by vortexing and centrifugation of sample at 2000 rpm for 10 min. Then insoluble residue was separated and the supernatant was analyzed spectrophotometrically using UV spectrophotometer (Systronics 2202) at 466 nm after appropriate dilution and by using calibration curve the mass of Curcumin-Zn(II) in microspheres was calculated. The entrapment efficiency and drug loading were calculated six times for each batch (Mennini et al., 2012) by following equation Mass of the CUR- Zn(II) in microspheres DL = X 100 Mass of the microspheres Mass of the CUR-Zn(II) in microspheres EE = X 100 Initial mass of drug In-vitro swelling 100 mg of chitosan microspheres and Eudragit coated chitosan microspheres were placed in phosphate buffer (ph 7.4) in a cellophane membrane dialysis bag (D9402, Sigma Aldrich). Then for a period of 8 h, microspheres were allowed to swell. After that changes in weight were measured by removal of the samples and blotted with a filter paper for 10 s to absorb excess solvent on surface. Following equation was used to determine the degree of swelling. [S α = (W t W 0 ) / W 0 ] School of Pharmaceutical Sciences 68

16 Where S α represents the degree of swelling, W t and W 0 represent weights of the sample at equilibrium swelling and the original dry weight, respectively (Ruiz-Caro et al., 2009) In-vitro drug release Dissolution study of developed formulations was carried out in USP dissolution- I (basket type, Electrolab, EDT 08 LX, Mumbai, India) in various simulated GI fluids under anaerobic condition. The simulation of variation in GI ph was achieved by altering the ph of the dissolution medium at different time intervals. The dissolution medium was kept at ph 1.2 with 0.1 N HCl for 2 h, ph 6.8 for next 2 h with phosphate buffer and then at ph 7.4 up to 12 h. Then at regular time intervals 5 ml of samples were withdrawn and replaced with equal amount of the fresh medium to maintain sink condition. The samples were analyzed by UV spectrophotometer at 466 nm (Systronics 2202) (Jose et al., 2011). Further the in-vitro release of selected microspheres formulation was carried out in the presence of 0.32% w/v pepsin for first 4 h to predict the stability of Eudragit S-100 and then in the presence of 10% w/v of rat caecal content from 5 th h, to analyze the biodegradability of chitosan in presence of colonic microflora (Mohapatra et al., 2011) Determination of kinetic model for release of Curcumin-Zn(II) loaded chitosan microspheres The in-vitro drug release data of the Eudragit coated chitosan microspheres was fitted to different kinetic models i.e. zero order, first order, Higuchi and Peppas model and on the basis of goodness of fit, the best model was selected (Christoper et al., 2013). The zero-order rate describes systems where drug release is independent of its concentration and is generally seen for poorly water-soluble drug in matrix, transdermal etc. (Najib et al., 1985). Q t = k 0 t (1) The first-order describes systems in which the release is dependent on its concentration (generally seen for water-soluble drugs in porous matrix) (Desai et al., 1966). ln Q t = ln Q 0 k 1 t (2) School of Pharmaceutical Sciences 69

17 The Higuchi model describes the release of the drug from an insoluble matrix to be linearly related to the square root of time and is based on Fickian diffusion. Q t = k Η t 1 / 2 (3) Korsmeyer Peppas derived a simple relationship which describes drug release from a polymeric system. Q t /Q α = Kt n (4) Where Q t /Q α is the fraction of drug released at time t; K is release rate constants and n is release exponent. The n value is used to characterize different release pattern. In this model, the value of n characterizes the release mechanism of drug n corresponds to a Fickian diffusion mechanism, 0.45 < n < 0.89 to non-fickian transport, n = 0.89 to Case II (relaxational) transport, and n > 0.89 to super case II transport. To study the release kinetics, data obtained from in vitro drug release studies were plotted as log cumulative percentage drug release versus log time (Dash et al., 2010) In-vivo study In-vivo study using acetic acid induced colitis All the in-vivo studies were conducted with prior approval of Institutional Animal Ethical Committee (01/CRI/08/2013). In-vivo study was performed by using acetic acid induced experimental ulcerative colitis model. Swiss Mice (body weight 18-22g), n=6 were selected and caged individually provided food and water ad-libitum. Animals were randomly distributed in three groups, i.e. Control group, Curcumin-Zn(II) treated group and Curcumin-Zn(II) microspheres treated group, each consisting six animals. The mice were firstly anesthetized with ether and 0.2 ml (4%) (v/v) of Acetic acid was given by intra-rectal route to induce ulcerative colitis in mice which resembled with the IBD. For three days, mice were kept without any treatment for development of full IBD model. The animals of group 2 and group 3 were received Curcumin-Zn(II) (20 mg/kg of body weight) and Curcumin-Zn(II) microspheres [equivalent to 20 mg/kg body weight of Curcumin-Zn(II)] orally in 1%w/v CMC (2 ml) daily for three days. The control group was received only 1%w/v CMC orally. Animals were sacrificed after 24 h of last drug administration. The colon portion of mice was isolated and washed with saline, School of Pharmaceutical Sciences 70

18 photographed and scored by blinded observer using scoring system of Wallace and Keenan: i) 0- no damage, ii) 1- localized hyperemia without ulcers, iii) 2- linear ulcer with significant inflammation, iv) 3- linear ulcer with inflammation at one site, v) 4- two or more sites of ulceration and/or inflammation, vi) 5- two or more major sites of inflammation and ulceration extending more than 1 cm along the colon (Tahan et al., 2011, Kazi et al., 2009). The colonic portions were also assessed for weight/length ratio (Kandhare et al., 2012). Further the histopathological study was performed on sections of colon tissues. The colonic tissues about 2 cm long after isolation were preserved in formalin (10% v/v) solution to prevent autolysis. The preserved colonic sections were fixed on paraffin slide, stained and analyzed by microscope (Azad et al., 2011) In-vivo organ bio-distribution study All the in-vivo studies were conducted with prior approval of Institutional Animal Ethical Committee (01/CRI/08/2013). Curcumin-Zn(II) loaded microspheres were administered to mice orally in 1%w/v CMC for this study. After 12 h the mice were sacrificed and the stomach, small intestine and colon were isolated. Then the organs were homogenized by using Tissue Homogenizer with 2mL of phosphate buffer (ph 7.4). To this homogenate 2 ml of methanol was added and kept for 1 h. The drug content was determined by using HPLC. (Ahmed et al., 2012) In-vivo pharmacokinetic study The in-vivo pharmacokinetic studies were performed in albino rats weighing g (n=6). All the animals were kept on overnight fasting. Curcumin-Zn(II) microspheres F14 [equivalent to 20 mg/kg body weight of Curcumin-Zn(II)] in 1%w/v CMC (2 ml) were administered by oral route. Blood samples were collected from retro-orbital plexus at predetermined time (0, 1, 2, 4, 6, 8, 10, 12, 24 h) in eppendorf tubes containing EDTA. Plasma was separated by centrifuging the tubes at 5000 rpm for 15 min. The drug was analyzed in plasma by HPLC. The pharmacokinetic parameters were calculated by using Kinetica 2000 software. C max (maximum plasma drug concentration) and T max (time taken to reach maximum plasma drug concentration) were obtained from plasma drug concentration-time profile data. The area under the curve (AUC) of plasma concentration-time profile was calculated by trapezoidal rule. The total AUC was calculated by extrapolating the AUC (0-t) to infinity by dividing the last concentration by elimination rate School of Pharmaceutical Sciences 71

19 constant (k e ) and adding it to AUC (0-t). K e was measured by linear regression of log concentration in the elimination phase of plasma drug concentration-time profile. The half-life (t 1/2 ) was calculated by formula t 1/2 = 0.693/K e. In vitro-in vivo correlation (IVIVC) was established by plotting the graph between percentage drug dissolved vs. percentage drug absorbed. The percentage drug dissolved for optimized formulation F14 was analyzed by HPLC method and the Wagner-Nelson method was used to calculate the percentage drug absorbed (Takka et al., 2003) Stability studies Stability studies of F14 formulation was carried out as per ICH Q 1 A guidelines by placing at 40ºC ± 2ºC and 75% ± 5% RH for 3 months. The samples were analyzed periodically for physical appearance and in-vitro dissolution for 3 months. The sampling intervals were 0, 1, 2 and 3 month (Borhadea et al., 2012) PREFORMULATION STUDIES OF FLURBIPROFEN Authentication of the procured Flurbiprofen Several parameters were taken into consideration for drug authentication such as melting point determination, thin layer chromatography (TLC) and FTIR Melting point determination Melting point determination of flurbiprofen (FLB) was carried out by capillary fusion method. In this method first of all one end of the capillary was fused. Then the drug was filled with other end of the capillary. After that the capillary was placed in the melting point apparatus and melting point of the drug was noted down with the help of a thermometer. Similarly readings were taken six times and compared with reference value Thin Layer Chromatography Thin Layer Chromatography of FLB was performed using precoated silica gel (F 254 ) aluminum plates (2x4cm) as stationary phase. A spot of the standard solution was put 0.5cm from the bottom and placed in TLC chamber, which was previously saturated with the solvent system chloroform: acetone (4:1). The solvent was allowed to rise at least 3/4th of the plate and the School of Pharmaceutical Sciences 72

20 distance travelled by solvent front was noted. The spots were detected by spraying the acidified potassium permanganate (KMnO4) on to the plate. The Rf value was calculated for the standard solution using the formula: Rf value= Distance travelled by the solute from the origin/distance travelled by the solvent front from the origin The R f value was calculated and compared with reported R f value of FLB (Rajesh et al., 2013) FTIR [Fourier Transform Infra-Red Spectroscopy] FTIR is most powerful technique for qualitative identification of compound. It gives the information about the functional group present in the particular compound. The main application of IR-spectrophotometer is the determination of the identity of a compound by means of spectral comparison with that of reference spectra and verification of the presence of functional group in an unknown molecule. FTIR of FLB was determined by FTIR Instrument of Agilent Technologies 630 Cary using Micro Lab software. Small amount of drug was placed on the sampling platform followed by lowering the upper nobe, so that drug comes into the close contact with the sampling diamond. Graph was collected by running the software and interpretation was made. Spectra were recorded from 4000 to 650 cm -1 scanning range Estimation procedures of Flurbiprofen a) Calibration curve of Flurbiprofen by UV-Visible Spectrophotometer (Systronic 2202). b) Development and standardization of HPLC (Agilent Technology 1200, Germany) procedure for estimation of Flurbiprofen Calibration curve of Flurbiprofen by UV-Visible Spectrophotometer a) Medium: 0.1N HCl (ph 1.2) The absorption maxima (λ max ) for flurbiprofen in 0.1N HCl were determined by scanning the drug solution within the range of nm using UV-Visible Spectrophotometer (Systronic 2202). It was found that the drug exhibited λ max at 254 nm. Preparation of stock solution: 100 mg of flurbiprofen was accurately weighed and solubilized in 5 ml of methanol which was further dissolved up to 100 ml of 0.1N HCl to result in a concentration of 1mg/ml. Working standards were prepared using this stock solution. School of Pharmaceutical Sciences 73

21 Preparation of working standard: Stock solution of 10 ml was taken in volumetric flask and volume was made up with 0.1N HCl to obtain concentration of 100µg/mL. Further dilutions were made to give 5, 10, 15, 20, 25 and 30 µg/ml concentration of flurbiprofen respectively. These dilutions were scanned and the absorbance was measured at 254 nm using UV-Visible Spectrophotometer (Systronic 2202). To obtain the standard calibration curve, the resulted absorbance values were plotted against the respective concentrations. Average values of absorbance were calculated by repeating the procedure six times. The data was statistically evaluated to obtain the regression coefficient. b) Medium: Phosphate buffer (ph 6.8) The absorption maxima (λ max ) for flurbiprofen in phosphate buffer ph 6.8 were determined by scanning the drug solution within the range of nm using UV-Visible Spectrophotometer (Systronic 2202). It was found that the drug exhibited λ max at 254 nm. Preparation of stock solution: Accurately weighed 100 mg of flurbiprofen was solubilized in 5 ml of methanol which was further dissolved up to 100 ml of phosphate buffer ph 6.8 to obtain a concentration of 1mg/ml. By using this stock solution, working standards were prepared. Preparation of working standard: 10 ml of stock solution was taken in volumetric flask and volume was made up with phosphate buffer ph 6.8 to result in a concentration of 100µg/mL. Further dilutions were made to give 5, 10, 15, 20, 25 and 30 µg/ml concentration of flurbiprofen respectively. These solutions were scanned and the absorbance was measured at 254 nm using UV-Visible Spectrophotometer (Systronic 2202). The resulted absorbance values were plotted against the respective concentrations to obtain the standard curve. Average values of absorbance were calculated by repeating the procedure six times. The data was statistically evaluated to obtain the regression coefficient. c) Medium: Phosphate buffer (ph 7.4) The λ max (absorption maxima) for flurbiprofen in phosphate buffer ph 7.4 were analyzed by scanning the solution of drug within the range of nm using UV-Visible Spectrophotometer (Systronic 2202). It was observed that the drug exhibited λ max at 254 nm. School of Pharmaceutical Sciences 74

22 Preparation of stock solution: 100 mg of flurbiprofen was weighed and solubilized in 5 ml of methanol which was further dissolved up to 100 ml of phosphate buffer ph 7.4 to achieve a concentration of 1mg/ml. By using this stock solution, working standards were prepared. Preparation of working standard: 10 ml of stock solution was taken and volume was made up with phosphate buffer ph 7.4 to obtain a concentration of 100µg/mL. Further dilutions were made to give 5, 10, 15, 20, 25 and 30 µg/ml concentration of flurbiprofen respectively. These solutions were scanned and the absorbance was measured at 254 nm using UV-Visible Spectrophotometer (Systronic 2202). To obtain the standard curve, the resulted absorbance values were plotted against the respective concentrations. Average values of absorbance were calculated by repeating the procedure six times. To obtain the standard deviation of the said values and regression coefficient, the data was statistically evaluated HPLC method development A RP-High Performance Liquid Chromatography method was developed for the determination of FLB in developed formulation and in plasma. The method was statistically validated. Instrumentation Analysis was performed using HPLC binary system (Agilent technology) composed of binary pumps combined with photodiode array detector set at wavelength range 254 nm. Agilent eclipse XBD column (C 18 (4.6 x 150 mm) bonded with 5 µm particle size) coupled with EZ-Chrome software was used for recording and processing of chromatographic data. Method Calibration Curve Stock solutions of FLB 1 mg/ml were prepared in solvent system and stored at 2-8 C until used. Aliquots from each stock solution were diluted stepwise with solvent system to obtain further dilutions (2 ppm, 4 ppm, 6 ppm, 8 ppm, 10 ppm). 5µL injections of each concentration of drug were injected into the RP-HPLC system separately under the conditions as described above. Evaluation of drug was analyzed at 254nm. Peak areas were recorded for all the peaks and peak areas were plotted against the concentrations to obtain the standard calibration curves. Optimization of chromatographic conditions The effects of different chromatographic conditions on the instrument response create a situation where one has to compromise between different experimental variables in order to achieve the School of Pharmaceutical Sciences 75

23 best chromatographic separation. Chromatographic separations are significantly affected by the mobile phase conditions, such as type and composition of the formulation and therefore before selecting the conditions for the optimization, a number of preliminary trials were conducted with combinations of different organic solvents, compositions and flow rate to check the retention time, shape, resolution and other chromatographic parameters. Among all tried experiments, the mobile phase combination of water and Acetonitrile in the ratio of (50: 50 v/v) with RP-HPLC at flow rate of 0.8 ml/min was found to be most suitable. Best resolution and sensitivity of the method was obtained for FLB at 254nm. Typical chromatogram with optimized condition gives sharp and symmetric peak with specific retention time. Validation of Optimized method After chromatographic method development and optimization, method was validated. Optimized method was validated according to ICH guidelines for linearity, sensitivity, precision and % recovery (ICH Guidelines, 1994; ICH Guidelines, 1996). All the variables of the method were considered for validation including sampling procedure, sample preparation, chromatographic separation, detection and data evaluation. The developed method was validated for various parameters such as linearity, system suitability, specificity, precision, robustness, accuracy, limit of detection and limit of quantification Solubility studies Solubility study of FLB was performed using orbital shaker in 0.1N HCl (ph 1.2), phosphate buffer (ph 6.8) and phosphate buffer (ph 7.4) for 72 h by Equilibrium solubility method Equilibrium solubility method Excess amount of flurbiprofen was put into 25 ml of 0.1N HCl (ph 1.2), phosphate buffer (ph 6.8) and phosphate buffer (ph 7.4) separately in conical flasks. Then these flasks were kept in solubility shaker for 72 h at 37 C. After 72 h all the solutions were filtered and measured using UV spectrophotometer at 254nm against 0.1N HCl (ph 1.2), phosphate buffer (ph 6.8) and phosphate buffer (ph 7.4). Then the amount of drug solubilized was calculated. School of Pharmaceutical Sciences 76

24 Compatibility studies DSC Analysis A differential scanning calorimetry (JADE DSC, Perkin Elmer, USA) was used to study the thermal analysis of drug-excipient compatibility. Firstly, the FLB was mixed with chitosan and Eudragit S100. The drug-excipient mixture was scanned in the temperature range of ºC under an atmosphere of nitrogen. The heating rate was 20ºC/min and the obtained thermograms were observed for any type of interaction. (Carstensen et al 2000) Preparation of Microspheres of Flurbiprofen Emulsion cross linking method: Chitosan polymer was dissolved in 10 ml of aqueous solution of acetic acid (1%) and Flurbiprofen was dispersed in the polymeric solution (Table 3.3). This solution was added to 100 ml of liquid paraffin containing Span 80 (1% v/v). An w/o emulsion was formed by stirring at 1500 rpm for 2 h with the help of mechanical stirrer (Remi Motors, Mumbai, India). 0.5 ml of glutaraldehyde was added to the emulsion and kept for 1 h. The solvents were removed by stirring under vacuum. Microspheres were obtained by filtration and washed with petroleum ether and dried in hot air oven at 40º C (Jose et al., 2011, Lamprecht et al., 2004) Polymeric coating of chitosan microspheres of FLB: The chitosan microspheres of FLB were coated with Eudragit S100 by emulsification-solvent evaporation method. The prepared FLB loaded chitosan microspheres were added in 20 ml of ethanolic solution of Eudragit S100 (10% w/v). Emulsification was achieved with 70 ml of light paraffin containing Span 80 (1%v/v). The emulsion was stirred for 2 h at 1500 rpm with mechanical stirrer. The Eudragit S100 coated microspheres were obtained by filtration and washed with petroleum ether. Then the coated microspheres were dried in oven at 50º C (Jose et al., 2011). School of Pharmaceutical Sciences 77

25 Table 3.3: Composition of colon targeted FLB microspheres Formulation code FLB (g) Chitosan (g) Emulsifier (%) Eudragit (% w/v) F F F F F F F F In-vitro and in-vivo evaluation of FLB microspheres Determination of particle size, shape and surface morphology Particle size of FLB microsphere formulations was determined by Malvern Mastersizer (Malvern Instruments, Mastersizer 2000, UK) (Orlu et al., 2006). The values (Z avg ) were expressed for all formulations as mean size range. The shape and surface morphology of the microspheres was characterized using scanning electron microscope. The microspheres were mounted on an aluminum stub with carbon-glue, and coated with gold using a gold sputter module in a high vacuum evaporator. Samples were then observed with the SEM (LEO, 435 VP, U.K.) at 20 kv (Kietzmann et al., 2010) X-ray diffraction Qualitative powder X-ray diffraction of FLB and its FLB microspheres was performed using X- ray diffractometer (Bruker Axs D8 Advance, Germany). The instrument was operated at a voltage of 40 kv and a current of 30 ma, with copper as the tube anode material. The samples were run over a range of 2θ angles from 2º to 80º. School of Pharmaceutical Sciences 78

26 Determination of percent yield, drug loading and entrapment efficiency The percent yield (PY) was determined based on the weight of the FLB and polymer added, by applying the following equation: Mass of the obtained microspheres PY = x 100 Initial mass of drug + Initial mass of polymer 100 mg of microspheres were first crushed using a glass mortar pestle and powdered microspheres were dispersed in 100 ml of methanol and kept for overnight for drug extraction. Then after centrifugation at 2000 rpm for 15 min, the supernatant was appropriately diluted with methanol and absorbance was measured at 254 nm using UV spectrophotometer (Systronics 2202). The drug loading was determined for each batch six times (Patel et al., 2011). The drug loading (DL) and entrapment efficiency (EE) were calculated using following equation: Mass of the FLB in microspheres DL = X 100 Mass of the microspheres Mass of the FLB in microspheres EE = X 100 Initial mass of drug In-vitro swelling In a cellophane membrane dialysis bag (D9402, Sigma Aldrich), 100 mg of chitosan microspheres and Eudragit coated chitosan microspheres were placed in phosphate buffer (ph 7.4). Then microspheres were allowed to swell for a period of 8 h. After that changes in weight were measured by removal of the samples and blotted with a filter paper for 10 s to absorb excess solvent on surface. The degree of Swelling was determined using the following equation: [S α = (W t W 0 ) / W 0 ] Where S α represents the degree of swelling, W t and W 0 represent weights of the sample at equilibrium swelling and the original dry weight, respectively (Muraa et al., 2012). School of Pharmaceutical Sciences 79