Solubility and Dissolution Enhancement of Silymarin with Fulvic Acid Carrier

Objective: Solubility is a key parameter as it is one of the main causes for poor bioavailability. The objective was to improve the solubility and dissolution profile of poorly-soluble silymarin with a water-soluble carrier fulvic acid. Materials and methods: Phase solubility studies were carried out for the determination of stoichiometric ratio between silymarin and fulvic acid by Higuchi and Connors method. The binary systems made by physical mixing and kneading methods were characterized by drug content, solubility studies, solid-state characterization by DSC, FT-IR, dissolution studies and ex vivo permeation studies. Results and discussion: The phase-solubility studies between silymarin and fulvic acid revealed AL type of curve, indicating linear increase in drug solubility with increase in carrier concentration and from these apparent stability constant and Gibbs free energy transfer values were calculated. It was found that the reaction conditions became more favorable as the concentration of fulvic acid increased from 0.2% to 2% w/v, indicating the spontaneity of solubilization process at higher carrier concentrations. Physical mixture and kneading showed increased solubility and dissolution rates compared with pure drug. In DSC, no melting peak of silymarin was seen, indicating that it was in amorphous form inside the carrier and FT-IR studies demonstrated interactions between drug and carrier. Conclusion: Improvement in solubility, dissolution profiles and permeation was observed in physical mixture and kneading as compared to pure drug establishing the role of fulvic acid as a promising carrier which can be used to formulate silymarin with better in vitro and in vivo performances. Javed S, Kohli K, Ahsan W (2016) Solubility and Dissolution Enhancement of Silymarin with Fulvic Acid Carrier. Int J Drug Dev & Res 8: 009-014 Volume 8(1): 009-014 (2016)-010 Int J Drug Dev & Res ISSN: 0975-9344 concentrations of FA (0.2-2% w/v) in stopper conical flask (10 mL) and the resulting mixture was equilibrated in a thermostatic shaking water bath for 48 h on a rotary flask shaker. The suspension so obtained were passed through a membrane filter (0.45 μm) to remove undissolved solid particles and the filtrate was suitably diluted and analyzed spectrophotometrically (Shimadzu, UV 1601) at 288 nm against blanks prepared using the same concentration of fulvic acid in distilled water. From the solubility data, the apparent stability constant (Ka) and Gibbs free energy transfer (∆G°tr) were calculated with formulas as described by Ahuja et al. in the literature [20]. Apparent stability constant (Ka) of the complexes was calculated from the phase-solubility diagrams according to the following equation: ( ) Slope S Slope Ka − = 1 0 where; So is the solubility of drug at RT in the absence of carrier/ligand and slope means the corresponding slope of the phase-solubility diagrams, that is, the slope of the drug molar concentration versus carrier/ligand molar concentration graph. Values of Gibbs free energy of transfer, ∆G°tr, of SILY from plain water to aqueous solutions of the carrier fulvic acid were calculated according to the following relationship: 0 0 2.303 .log tr s S G RT S D = where; So and Ss are the molar solubilities of SILY in 1% w/v aqueous solution of the fulvic acid carrier and in the plain water respectively. Preparation of Silymarin-Fulvic acid binary systems Physical mixture: Physical mixture of SILY and FA were prepared by grinding the appropriate amount of mixture for a period of 60 minutes in a clean dry glass pestle and mortar and the resulting mass was passed through a 100 mesh sieve to obtain a uniformly sized powder. The homogenous mixture so obtained was stored in an air tight vial in a dessicator [21,22]. Kneading: Weighed appropriate amounts of SILY and FA were triturated for 15 min a dry clean glass mortar and pestle. During the process, the water content of the paste was empirically adjusted by ethanol and triturated to maintain the consistency of the paste. Trituration was continued until the product started drying on the walls of the mortar. The product was further dried in the hot air oven at 60°C for 15 min, powdered, passed through 100 mesh sieve, transferred in an air tight vial and stored in a dessicator [21,22]. Characterization parameters Drug content estimation: The quantities of binary mixtures (equivalent to 5 mg of SILY) were dissolved in water. Appropriate dilutions were made and drug content was measured spectrophotometrically at λmax 288 nm. Aqueous solubility determination: Excess amount of complex was kept in an amber colored bottle containing 10 mL of distilled water and stirred using a thermostatic mechanical shaker (25°C) for 72 hr [18]. Appropriate dilutions were made and drug solubility was measured spectrophotometrically at λmax 288 nm. Differential scanning calorimetry: The DSC thermograms of SILY and FA alone and their binary systems prepared by both the methods were obtained using a Pyris6 DSC equipment using aluminium crimp cells with about 2 mg of samples, under dynamic N2 atmosphere (flow rate: 20 mL/min) and at a heating rate of 10°C/min in temperature range from 30-300°C and analyzed [18,21,22]. Fourier transform infrared spectroscopy: The FT-IR spectrograms of SILY and FA alone and their binary systems prepared by both the methods were obtained using FT-IR Perkin Elmer equipment. Pellets of the samples were prepared after grinding and dispersing the powder in micronized IR grade KBr powder in a mortar and pestle, and scanned over a wave number range of 4000-400 cm-1 as reported in literature [18,21,22]. Dissolution study: Dissolution of both the binary systems (equivalent to 25 mg of SILY) was carried out using the USP rotating paddle dissolution apparatus (DS 8000, Labindia Pvt. Ltd, India). The dissolution media was 900 mL double distilled water, pH 6.5 at 37°C, with stirring speed of 100 rpm and paddle depth of 25 mm. Dissolution studies were performed on pure drug (25 mg) and the binary systems containing an equivalent amount of the drug. Aliquots of 5 mL were withdrawn periodically and were replaced with 5 mL of fresh dissolution medium up to 120 minutes. The aliquots were analyzed spectrophotometrically at 288 nm. The percentage drug release versus time graph was obtained and enhancement in dissolution with respect to pure drug was calculated [10]. Ex vivo rat gut sac permeation studies: To determine the effect of complexation on permeability of SILY, non-everted rat gut sac method was employed and compared with that of plain SILY after modification of the method as described in literature [23]. Here, a 5 cm long noneverted intestinal tissue sac basically ileum was taken and thoroughly flushed with 0.9% normal saline to remove any debris from inside. The tissue was mounted on a double jacketed glass assembly containing 50 mL of tyrode buffer solution under controlled temperature 37 ± 2°C with continuous aeration and stirring. The tissue was filled with 1 mL of plain drug dispersion or complexed drug (equivalent to 10 mg of SILY) by both the methods. The samples were withdrawn from outside the sac in intervals, filtered with syringe filters (0.5 μm) and analyzed spectrophotometrically at λmax 288 nm. Results and Discussion Solubility and phase solubility-interpretations Solubility is an important parameter that affects the absorption and bioavailability of drugs. Solubility of SILY in plain water at 25°C was found to be 50 μg/mL indicating it as practically insoluble in water [24]. The phase-solubility studies were carried out at room temperature to determine the stoichiometric proportion of SILY with FA. According to Higuchi and Connors there can be five types of phase-solubility relationship [19]. A-type phase-solubility profiles are obtained when the solubility of the substrate (i.e., drug) increases with increasing ligand (carrier) concentration. These are further of 3 types: AL-type, APtype and AN-type. B-type phase-solubility profiles indicate formation of complexes with limited solubility in the aqueous complexation medium. These are further of two types: BSand BI-types as shown in Figure 1. From the color of the solutions in the vials (Figure 2), it was quite clear ostensibly that silymarin solubility increased linearly in different fulvic acid concentrations as compared with plain water and these solutions were analyzed spectrophotometrically at 288 nm as shown in Table 1. The SILY solubility increased from 50 μg/mL (in plain water) upto 765 μg/mL (in 2% w/v fulvic acid solution) which was approximately 15 times higher when compared to solubility of Javed S, Kohli K, Ahsan W (2016) Solubility and Dissolution Enhancement of Silymarin with Fulvic Acid Carrier. Int J Drug Dev & Res 8: 009-014 Volume 8(1): 009-014 (2016)-011 Int J Drug Dev & Res ISSN: 0975-9344 SILY in plain water, attributable to the great complexing ability of FA, increased wettability and micellar solubilization of SILY with hydrophilic groups on the water side and hydrophobic nucleus inside the core. In aqueous solutions carriers like FA are able to form inclusion complexes with many drugs by taking up drug molecule or more frequently some lipophilic moiety of the molecule, into the central cavity. The hydrophilic carrier interacts with drug molecules mainly by electrostatic forces and occasionally by other types of forces like hydrogen bonds. No covalent bonds are formed or broken during the complex formation, and drug molecules in the complex are in rapid equilibrium with free molecules in the solution. For the stoichiometric ratio determination of drug and carrier, a graph was plotted between solubility of SILY (μg/mL) versus FA concentrations (μg/mL) (Figure 3). According to Higuchi and Connors, the straight line curvecharacteristics of AL type was obtained with r2 value (0.9845) and from this the ratio between the drug SILY and carrier FA was adjusted as 1:1 molar ratio. Apparent stability constant: The value of apparent stability constant, Ka, was computed for 1:1 drugcarrier interaction, since the curve obtained in the present study was AL type with resultant slope less than unity. From the graph, the slope of the straight line was found to be 0.034879 (3.48 × 10-2) and the apparent stability constant (Ka) for silymarin was found to be: ( ) 0.034879 50 1 0.0034879 a K = Ka=0.00072279(7.22 × 10 -4 K-1) High values of Ka obtained for SILY-FA binary solutions revealed strong binding affinity between drug and solubilizer. Gibbs free energy transfer: The solubility values were also used to Figure 1: Phase solubility relationships according to Higuchi and Connors. Figure 2: Effect of fulvic acid concentrations on solubility of silymarin. % w/v FA FA (μg/mL) Solubility of Silymarin (μg/ mL) *Increase in solubility (%) Increase in solubility (times)