Enhancement of the Solubility of Herbal Polyphenolic Compounds in the Presence of Lactic Acid Butyle

2.1. Materials and methods

2.1.1. Materials Curcumin (97%) , Morin (2′,3,4′,5,7-Pentahydroxyflavone, 97 % ) was purchased from Merck (Darmstadt, Germany). Polyethylene glycole monooleate and tween 20 were obtained SigmaAldrich (Taufkirchen Germany). Lactic acid (Merck), Poly (ethylene glycol) butylether (PEG 200) (Aldrich) and P-toluene sulfonic acid (Aldrich), Acetic acid, sodium acetate, methanol and sodium hydroxide were supplied from Fluka (Fluka AG, Buchs, Switzerland). All the chemicals used in the study were of analytical reagent grade. The solutions have been prepared by using ultrapure water from Millipore Milli Q System (resistivity equal to 18 MΩ). Phosphate buffer solutions with different pH values were prepared by using phosphoric acid, and then adjusted with sodium hydroxide. 2.1.2. Esterfication of Poly(ethylene glycol) butyl ether with lactic acid 10 g of Poly(ethylene glycol) butyl ether and 4.37 g (48.54 mmol) of Lactic acid and catalytic amount of p-toluene sulfonic acid were mixed and transferred into Dean-Stark apparatus. The reaction mixture was heated removing of water at 180 0C. Then, the viscose and yellow reaction content was cooled and put into 50 mL of hexane. The phase separation was observed and the PEG- lactic acid ester (PLE) was removed by using separating funnel. The product was dried under vacuum at 40 0C for removing of hexane. The yield was found about 12.50 g. The PLE is soluble in water and common organic solvents such as ethanol, acetone, ethyl acetate and DMF. 2.1.3. Fourier Transform Infrared Spectroscopy (FTIR) FTIR Spectrophotometer (Nicholet) was used to analyze the structure of the synthesized PEG butyl ether-lactic acid ester and the interactions between curcumin, morin and PEG ester. 2.1.4. UV/VIS measurements UV/VIS measurements were performed with a UV-Vis Spectrophotometer (Perkin-Elmer Lambda 25) spectrometer. Measurements were performed at 425 nm for curcumin and 325 nm for morin solutions.

2.1.4. Preparation of the Calibration Curve For the calibration curve of curcumin, 5 mg of curcumin and morin were dissolved in 5 mL of ethanol to prepare the stock solutions. Dilute standard solutions were prepared from the stock solution for UV/VIS measurements. 2.1.5. Preparation of the emulsion systems The PEG-ester emulsions were prepared in different compositions with a total of 10 mL each, and placed in beakers. 5 mg of the compound (curcumin, morin) was added to each and stirred with a mechanical stirrer at room temperature for 5 minutes at 600 rpm. Resulting homogeneous mixture filtered through 0.45 mm membrane filter for UV/VIS measurements for the determination of curcumin and morin contents. 2.1.6. Polarized Optical Microscopy (POM) POM studies were performed with Leica DM2500P equipped with a LTSE350 Liquid Crystal Prosystem TMS 94 Hot Stage.

3.1. Synthesis of PEG-ester

PEG based polymeric lactate ester was synthesized starting from poly (ethylene glycol) butyl ether (Mn ~ 206) and lactic acid esterfication reaction in the presence of catalytic amount of PTSA at 180 0C. The reaction was proceeded to remove water from reaction medium.

Scheme 2. Synthesis of PEG-ester The characterization of PEG-ester was performed by using FT-IR spectroscopy technique. According to the Fig.1 butyl ether PEG lactate represent ester -C=O stretching vibration at 1739.77 cm−1. Also, -OH peak at 3435.36 cm−1, -C-O- ether peak at 1097.92 cm−1 and strong aliphatic C-H stretching vibration peak at 2867 cm-1 were observed. 1226

Wavenumbers (cm-1) Figure 1. FTIR spectra A) PEG-ester (a), the mixture of PEG-ester and curcumin (b), curcumin (c) B) PEG-ester (a), the mixture of PEG-ester and morin (b), morin (c)

3.2. Interactions PEG ester with morin and curcumin by FTIR measurements

Interactions between curcumin and PEG lactic acid ester and its emulsion system with both curcumin and morin were investigated by FTIR (Fig.1-A and Fig. 1-B). The OH band at 3435 cm1 was observed both PEG-lactac ester and its emulsions. The peaks at 1627.99 cm -1 and broad peak at 1652 cm -1 are attributed to the groups -C=C- and/or -C=O for curcumin and morin respectively. Also, the peak which was observed at 1597 cm-1 in Fig.1-A-b belongs to the symmetrical aromatic ring stretching vibrations (-C = C-) for curcumin emulsion. According to IR spectrum of curcumin, the bands at 1425.32, 1231.90 and 1275.27 cm-1 were found to shifted to 1455, 1201.52 and 1249.75 cm-1, respectively. On the other hand, the original morin FTIR peak 1227

of 1598, 1505, 1303, 1166, 826, 793 and 562-686 cm-1 disappear in the PEG-morin emulsion. On the other hand, the stretching vibration of -C–O–C- band for morin shifts from 1310 to 1353 cm1 . Except for hydroxyl peak, curcumin and morin IR bands are also present in the mixture formulation, but high wavelength shifts, disappears some peaks or reduction in intensities indicate that the interaction may be due to non-covalent hydrogen bonds, possibly with OH and C = O and enol groups.

3.3. Calibration graphs for curcumin and morin

Standard solutions of 0.5 mg / mL of curcumin and morin dissolved in ethanol were prepared. The standard solutions were diluted and the UV absorbances were measured at 425 nm for curcumin and 325 nm for morin. Obtained calibration graphs are shown in Fig. 2-a and Fig. 2-b. The curcumin and morin content of the prepared emulsions were determined by using the calibration graph equations. The linear calibration plot was obtained in the concentration of curcumin from 0.001 mg/mL to 0.016 mg/mL with the linear regression equation y = 46.638x + 0.0109 (R² = 0.9971) and in the concentration of morin from 0.025 mg/mL to 0.30 mg/mL with the linear regression equation y = 0.301x + 0.216 (R² = 0.9980) . 1.2

Figure 2. Calibration graphs of curcumin (a), and morin (b). 1228

3.4. Water solubility of curcumin and morin in the presence of PEG -ester

Synthesized PEG-ester was used for solubility enhancement of both curcumin and morin. To investigate the solubility of curcumin and morin in water in the presence of PEG, solutions in various curcumin/PEG-ester ratios (and also morin/PEG-ester ratios) were prepared. In a total of 10 mL solution, 0.005 g of polyphenolic substance (curcumin, morin) was kept constant and a series of solutions were prepared in which PEGester amounts were increased. Eight samples containing a fixed amount of olyphenolic sustance and different amounts of PEGester were prepared. PEGester amounts were selected as 5, 10, 50, 80, 100, 120, 150, 200 mg / mL. The amount of curcumin and morin dissolved in the prepared solutions were determined by using UV/VIS spectrophotometric measurements. Absorbance measurements were performed at 425 nm for curcumin and 325 nm for morin. The amounts of curcumin and morin were determined by using the obtained calibration curve equations. Solubilities obtained with various amounts of PEG-ester are given in Table 1. The solubilities of curcumin and morin against different concentrations of PEG-ester in 10 mL of water were plotted in Fig. 3. Fig. 3 shows a significant increase in the solubility of curcumin and morin at a concentration of about 100 mg /mL PEGester. The increase in solubility is slowed down after 100 mg/mL PEG-ester concentration. Therefore, 100 mg/mL was chosen as the optimum PEG-ester concentration for the 0.005 g polyphenolic substances (curcumin and mornin).

Figure 3. Solubility of curcumin (a) and morin (b) at different PEG-ester concentrations in 10 mL of water. 1229

Table 1. Solubility obtained at various PEG-ester concentrations for curcumin and morin. PEG-ester (mg/mL) 5 10 50 80 100 120 150 200

Curcumin (mg/mL) 0.0002 0.0009 0.0079 0.0149 0.0151 0.0204 0.0221 0.0245

Morin (mg/mL) 0.4243 0.7347 1.3895 1.4801 1.5743 1.9328 2.0442 2.1718

3.5. Effect of pH

The influence of pH on the water solubility curcumin and morin was investigated. Phosphate buffer solutions were used to investigate the effect of pH on the water solubility of curcumin and morin. Curcumin and morin degrade above pH 7. Thus buffer solutions between pH 2 and 7 were used to study the pH effect. The solubility of curcumin and morin was determined by UV/VIS measurements in emulsions prepared using buffer solutions with these pH values instead of water. Each solution contains 1000 mg PEG-ester and 2.5 mg curcumin (or morin). Table 2 shows the solubility of curcumin and morin in different pHs of these solutions. The table also shows that the solubilty is independent of pH. The PEG-ester has a nonionic property. The increase in the solubilities of the polyphenolic substances obtained in this study may be due to the hydrogen bonding interaction between the polyphenols and the PEG-ester. Table 2. Solubility of curcumin and morin at different pHs pH 2 3 4 5 6 7 Water

Curcumin (mg/mL) 0.0116 0.0137 0.0125 0.0127 0.0125 0.0103 0.0151

Morin (mg/mL) 1.2344 1.0420 1.0604 1.1146 1.0927 1.2191 1.5743

3.6. Characterization of the emulsion systems by Polarized Optical Microscopy (POM)

POM observation was performed to characterize the emulsion systems. Examples of POM photographs are shown in Fig. 4. POM micrographs reveal that the particles are dispersed in a very smooth and fairly even manner in the emulsions. Particle sizes vary between 10 and 40 µm.

Figure 4. POM micrographs of the curcumin (a) and morin (b) emulsions with PEG-ester prepared by the described procedure at room temperature.

3.7. Stability of the Emulsions

The curcumin and morin emulsions were prepared by using 1000 mg PEG-ester, 5 mg curcumin (or morin) in a total 10 mL of solution. Then, they were allowed to stand at room temperature (20°C ± 2). The contents of the curcumin and morin emulsions were analyzed every day for one week and then once a month for two months. There was no decrease in the content of the curcumin and the morin at the end of the period showing that the amount of curcumin and morin remained constant in these emulsions.