Formulation optimization and in situ absorption in rat intestinal tract of quercetin-loaded microemulsion

Colloids and Surfaces B: Biointerfaces 71 (2009) 306–314

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Formulation optimization and in situ absorption in rat intestinal tract of quercetin-loaded microemulsion Yan Gao a , Yuqiang Wang a , Yukun Ma b , Aihua Yu a , Fengqun Cai a , Wei Shao a , Guangxi Zhai a,∗ a b

Department of Pharmaceutics, College of Pharmacy, Shandong University, 44 Wenhua Xilu, Jinan 250012, China Department of Pharmacy, The Second People’s Hospital of Jinan, Jinan 250001, China

a r t i c l e

i n f o

Article history: Received 1 December 2008 Received in revised form 12 March 2009 Accepted 12 March 2009 Available online 24 March 2009 Keywords: Microemulsion Quercetin Simplex lattice mixture design

a b s t r a c t A new microemulsion system has been developed to increase the solubility and oral absorption of quercetin, a poorly water-soluble drug. The formulation of quercetin-loaded microemulsion was optimized by a simplex lattice experiment design. The optimized microemulsion formulation consisted of oil (7%, w/w), surfactant (48%, w/w), and cosurfactant (45%, w/w). Under this condition, the mean droplet diameter of microemulsion was 38.9 nm and solubility of quercetin in the microemulsion was 4.138 mg/ml. The in situ absorption property of quercetin-loaded microemulsion in rat intestine was studied and the results showed there was significant difference in absorption parameters such as Ka , t1/2 and uptake percentages between microemulsion and micelle solution containing quercetin. The study on absorption percentage in different regions of rat intestine attested that the colon had the best permeability, followed by ileum, duodenum in order. It can be concluded that microemulsion can improve the solubility and oral absorption of quercetin, a poorly water-soluble drug. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Appropriately 40% of new molecular entities fail to be new drugs because of poor biopharmaceutical properties, such as solubility and permeability [1]. Solubility is an important factor that governs absorption of orally administered drugs. With recent advances in pharmaceutical field, solubility is easily quantifiable in vitro and can be manipulated by formulation strategies, such as microemulsion (ME) [2] and self-microemulsifying drug delivery systems (SMEDDS) [3]. ME has attracted much attention in terms of its intensified application from drug delivery potential to food industry [4]. In general, ME exhibits some superior physical characteristics over other colloidal systems such as emulsion and suspension. ME possesses a series of favorable characteristics such as nanometric size, transparency, low viscosity, thermodynamic stability over a wide range of pH and ionic environments, ease of preparation, high solubilization capacity, protecting for the entrappment of drugs from degradation, hydrolysis, and oxidation, finally increase of bioavailability [4–6]. Quercetin (QT, 3,3 ,4 ,5,7-pentahydroxyflavone), a plant flavonoid (Fig. 1) extracted and isolated from Sophora japonica L., is a common component of most edible fruits and vegetables and demonstrates a broad range of physiological activities, such

∗ Corresponding author. Tel.: +86 531 88382015. E-mail address: [email protected] (G. Zhai). 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.03.005

as anti-inflammatory, anti-proliferative effects on a wide range of human cancer cell lines, osteoporosis, and inhibition of glycolysis, macromolecule synthesis, pulmonary and cardiovascular diseases, and also against aging [7–11]. In recent years, QT has gained much attention and arouses the enthusiasm of research work on cancer prevention and reduction of cardiovascular diseases. However, QT has a very low oral bioavailability (16.2%) due to its low solubility in water (0.17–7.7 ␮g/ml) and artificial gastric juice (5.5 ␮g/ml) and artificial intestinal juice (28.9 ␮g/ml) [12–16]. So its application is restricted in clinic. Therefore, formulation strategies have been designed to increase the solubility and improve the oral absorption of QT. For example, QT was incorporated into solid lipid nanoparticles and its oral absorption was 5.71 times that of the crude drug suspended in 4% CMC-Na solution in rats [17]. ME could improve solubility and oral bioavailability of poorly soluble drugs such as berberine, paclitaxel and acyclovir [18,19]. Ghosh’s study showed an increase of bioavailability (12.78 times) after oral administration of the acyclovir-loaded ME as compared with the commercially available tablets [20]. However, the ME formulation for improving the solubility and oral absorption of QT has not been evaluated. In present work, the QT-loaded ME was prepared and characterized. ME formulation was optimized by a simplex lattice design. In situ absorption property of QT-loaded ME in rat intestine was studied by intestine perfusion method, and the main absorption site in rat small intestine was determined by a closed loop experiment.

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and surfactant solution [22]. Under same conditions of clearance and transparency of obtained solution, the less amounts of added cosurfactant showed better compatibility with the oil and surfactant solution [23]. Cosurfactants such as dehydrated ethanol, 1,2-propylene glycol, glycerine and PEG400 were chosen to drop to the mixed systems composed of emulsifier Tween 80 (10%, w/v) and oil such as ethyl oleate, paraffin oil, castor oil or peanut oil, and the results were recorded as shown in Table 1. 2.3. Construction of phase diagrams and formulation of QT-loaded ME

Fig. 1. The structure of quercetin.

2. Materials and methods 2.1. Reagents and animals QT, Cremorphor EL and poloxamer 188 were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). Emulsifier OP was procured from Guangcheng chemical reagent Co. Ltd. (Tianjin, China). Ethyl oleate, castor oil, paraffin oil and PEG400 were purchased from Shanghai Chemical Co. (Shanghai, China). Peanut oil was purchased from Beijing Changcheng Chemical Co. (Beijing, China). All other reagents and buffer solution components were analytical grade preparation. Distilled and deionized water were used in all experiments. 2.2. Screening of formulation compositions for ME 2.2.1. Solubility of QT in oils and surfactants To find out the suitable oil and surfactant as compositions of ME, the solubility of QT in various oils such as paraffin oil, castor oil, peanut oil, ethyl oleate, and surfactants including Cremorphor EL-40, Tween 80, poloxamer 188 and emulsifier OP was measured. An excess amount of QT was added to 3 g oil or 20 ml 12.5% (w/v) surfactant solutions and then the resulting mixture was shaken in a water bath at 37 ◦ C for 24 h followed by centrifugation for 10 min at 12,000 rpm [21]. The supernatant was diluted with ethanol appropriately and the drug concentration was determined by high performance liquid chromatography (HPLC) analysis. The oil and surfactant that showed higher solubility for QT were selected as the compositions of ME. 2.2.2. Compatibility tests Cosurfactants were screened by the compatibility tests with the tested cosurfactants mixing with the systems of different oils and chosen surfactant solution. 6 ml Tween 80 micelle solution (10%, w/v) was mixed with 0.05 g oil, and then the resulting mixture was titrated by cosurfactant under proper magnetic stirring till the appearance of resulting solutions was subjected to a change from turbid to clear and transparent. Samples were left to equilibrate for at least 10 min before being examined for transparency. The added amounts of cosurfactant were recorded. The transparency of resulting solution was determined by ocular inspection and was used as an index to evaluate the compatibility of oil, cosurfactant

2.3.1. Construction of pseudo-ternary phase diagrams Pseudo-ternary phase diagrams method was used to obtain concentration range of the components for ME. The weight ratios of surfactant to cosurfactant (Km) were chosen at 1:3, 1:2, 1:1, 2:1, 3:1 and 4:1, and at each specific Km, the oily mixtures were prepared with different weight ratio of oil to the mixture of surfactant and cosurfactant at 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, respectively. Water was added by drop to each oily mixture under proper magnetic stirring at 37 ◦ C until the mixture became clear. The concentrations of components were recorded to complete the pseudo-ternary phase diagrams, and then the contents of oil, surfactant, cosurfactant and water were selected based on these results. 2.3.2. Preparation of QT-loaded ME systems To prepare QT-loaded ME, appropriate oil, surfactant and cosurfactant in accordance with the ME domain in the phase diagrams mingled together, and equilibrated with gently magnetic stirring for 30 min to get the initial concentrate. Then appropriate QT was dissolved in the initial concentrate under ultrasonication. The resulting mixture was added by water precisely drop by drop with magnetic stirring at room temperature. To obtain blank ME, no QT was added in the above process before adding water. 2.4. Formulation optimization of ME The influence of the surfactant, cosurfactant and oil on the ME properties was studied using a simplex lattice design [24–28]. The total concentration of water, surfactant and oil phase in the formulation was kept constant while the ratio of the three was varied according to Fig. 2. 3-Component system is represented by an equilateral triangle in two-dimensional space. Seven batches were prepared as followed: three vertexes (A, B, C), three halfway points between vertices (AB, BC, AC), and the center point (ABC). Each vertex represents a formulation containing the maximum amount of one component, with the other two components at a minimum level. The halfway point between the two vertices represents a formulation containing the average of the minimum and maximum amounts of the two ingredients represented by two vertices. The center point represents a formulation containing one-third of each ingredient. Each of the seven formulations was processed 3 times in order to estimate the precision of the production method. The concentrations of surfactant, cosurfactant and oil were selected as

Table 1 The compatibility of surfactant, cosurfactant and oil. Cosurfactant

Dehydrated ethanol 1,2-Propylene glycol Glycerine PEG400

Amounts of added cosurfactant (g)/clarity (yes/no) 6 ml Tween 80 (10%) 6 ml Tween 80 (10%) 10 ml + peanut oil 0.05 g 10 ml + castor oil 0.05 g

6 ml Tween 80 (10%) 10 ml + ethyl oleate 0.05 g

6 ml Tween 80 (10%) 10 ml + paraffin oil 0.05 g

4.6978/no 4.3594/no 3.0355/no 3.0237/no

1.4078/yes 1.8439/yes 1.3998/yes 1.4195/yes

4.5260/no 2.8303/no 3.3748/no 2.4567/no

2.8286/no 1.9431/no 2.2265/no 1.8215/no

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Fig. 2. Equilateral triangle representing simplex lattice design for three components (A–C).

independent variables. The solubility of QT in ME and the mean particle size of ME were taken as responses. In the simplex lattice model, an equation was used to calculate the responses of seven formulations, solubility and particle size of all possible formulations can be predicted [29,30]. Graphs of these properties in the form of contour plots were constructed with Matlab Software [31,32]. With the aid of Matlab Software, the model equation was developed to be the best explanation for the relationship between the solubility or particle size and the measured characteristics. 2.5. Characterization of ME The morphology of QT-loaded ME was examined using transmission electron microscopy (TEM, JEM-100CX, Jeol, Japan). Samples were prepared by depositing a drop of diluted samples onto a filmcoated copper grid and later staining with a drop of 2% aqueous solution of phosphotungstic acid for contrast enhancement and allowed to dry before examined under the TEM. The viscosities of various ME vehicles were determined by the NDJ-8S digital viscometer (Shanghai Precision and Scientific Instrument, Shanghai, China) at 25 ◦ C with a No. 1 rotor set at 60 rpm. Electrical conductivity was determined by DDS-11C digital display conductivity meter (Shanghai Instrument, China). The phase systems of the ME were determined on the basis of electrical conductivity. Refractive indices were measured with a thermostated Abbe refractometer (Shijiazhuang Optical Instrument Factory, China). Particle size was measured using a Beckman Coulter N5 particle size analyzer with 25 mW He–Ne laser (Beckman Coulter, Inc., English). To determine the solubility of QT in ME, excess QT was added into 1 ml initial concentrate, then 4 ml water was added and the resulting suspension was shaken at 37 ◦ C for 72 h using an oscillator (THZ-82, Changsi Commercial Ltd., China), and then the suspension was centrifuged at 12,000 rpm. The supernatant was diluted with dehydrated ethanol appropriately and the QT was determined by HPLC with detection wavelength at 370 nm. The stability of the optimized QT-loaded ME was assessed by measuring the changes in the clarity, phase separation and concentration of QT at 4 ◦ C during 6 months storage. The centrifuge tests at 12,000 rpm for 30 min were carried out to assess the physical stability of ME [33]. 2.6. Absorption property of QT-loaded ME in rat intestine 2.6.1. Absorption experiment of QT-loaded ME in rat intestine The absorption property of QT-loaded ME was conducted with the established in situ intestinal perfusion methods in rats [34,35]. Male Wistar rats weighing 200 ± 20 g were purchased from Exper-

imental Animal Center of Shandong University (Shandong, China). Briefly, rats were fasted for 12 h before experiment with free access to water. The rats after anaesthetized were placed under an infrared lamp to keep normal body temperature. The whole small intestine was surgically exposed and ligated for perfusion and cannulated with plastic tubing (diameter 0.4 cm). The cannulated segment was rinsed with 37 ◦ C normal saline and attached to the perfusion assembly which consisted of a peristaltic pump (BT00-100M, Baoding Longer Precision Pump Co., Ltd., China) and a 100 ml volumetric cylinder containing 100 ml sample solution. Care was taken to handle the small intestine gently in order to maintain an intact blood supply. The entire surgical area was covered with a piece of sterilized absorbent gauze wetted with normal saline. At the beginning of the test, sample solution was perfused through the intestine at a flow rate of 5 ml min−1 . Ten minutes later, the volume of perfusion solution in the circulation system as the 0 min volume was recorded and the flow rate was adjusted to 2.5 ml min−1 . During 6 h perfusion period, at predetermined time interval, 1 ml of sample solution was taken out, the volume of solution in the circulation system was recorded, and then 2 ml Krebs–Rings solution was added in. Samples were frozen immediately and stored at −20 ◦ C until analysis. Before analysis, samples were thawed at 25 ◦ C and treated with 9 ml methanol, and the resulting solution was centrifuged at 12,000 rpm for 10 min, 20 ␮l of supernatant was introduced into HPLC. The absorption constant (Ka ) is calculated using Fick’s law: Ka = [−ln(X/X0 )]/t, where X0 is the amount of drug before perfusion, X is the residue amount of drug after perfusion at different intervals. Ka can be obtained as the slope from the regression curve of −ln(X/X0 ) versus time, and t1/2 can be calculated when X = X0 /2 [36]. The perfusion solution, Krebs–Rings buffer, contained 7.8 g NaCl, 0.35 g KCl, 1.37 g NaHCO3 , 0.02 g MgCl2 , 0.22 g NaH2 PO4 and 1.48 g glucose in 1000 ml purified water. 1 ml of QT-loaded initial concentrate with 10 mg QT were diluted to 100 ml with Krebs–Rings buffer, obtaining an approximate 100 mg/l QT-loaded ME. As a comparison, QT-loaded micelle composed of Tween 80 and ethanol at the same ratio as ME without oil, was dissolved in the same perfusion solution and processed with the above method. 2.6.2. Stability of QT in blank intestinal circulating solution According to the rat’s intestinal perfusion experiment in situ above, the blank intestinal circulating solution can be obtained after 6 h circulation. 1 ml of QT-loaded initial concentrate was diluted to 100 ml by blank perfusion solution, and then the resulting solution was treated with a water bath at 37 ◦ C for 6 h. The concentrations at 0 h and 6 h were determined and compared with each other [37,38]. 2.6.3. Effect of physical adsorption in intestine Three everted segments of empty small intestine about 10 cm long after cleaned were isolated. Then the harvested intestine segments were put into 100 ml perfusion solution with known drug concentration and incubated for 6 h in a water bath at 37 ◦ C. The concentration of QT in the resulting solution was determined and compared with the original concentration [39]. 2.6.4. Closed loop experiment for the study of main site of QT-loaded ME absorption Drug permeability to different regions of rat intestine, duodenum, jejunum, ileum and colon, was measured by the closed loop experiment [40]. After the abdominal cavity was opened, an intestinal loop (length, 10 cm) was made at four regions (duodenum, proximal jejunum, distal ileum, and colon) by cannulation with a silicone tube (i.d., 3 mm). Duodenum segment was selected at 1 cm away from pylorus, the jejunum of 15 cm from pylorus, the ileum

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of 20 cm from the cecum and colon from appendix to the rectum direction. The intestinal contents were removed by a slow infusion of a few of saline and air. Following the above procedure in the perfusion experiment, 25 ml of QT-loaded ME solution (5 mg/100 g rats body weight) was introduced into intestinal loops with the help of a peristaltic pump at a flow rate of 2.5 ml min−1 and both ends of the loop were ligated. After 2 h, the effluent solution in the loop was collected to the volumetric flask and diluted by Krebs–Rings buffer solution which had washed the tested intestine segments. The resulting solution was filtered through 0.45 ␮m millipore filter and measured by HPLC. The drug absorption was evaluated by the percentage of dose absorbed through subtracting the remaining amount of the drug from the administered amount.

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2.7. HPLC analysis The samples were analyzed using the HPLC system including a separations module (Waters 2695), a diode array detector (Waters 2996) and a reversed phase C18 column (5 ␮m, 4.6 mm × 250 mm, Dikma). The mobile phase was a mixture of 30 mM sodium dihydrogen phosphate solution/acetonitrile/methanol at a ratio 65:29:6 (v/v) with the flow rate at 1 ml min−1 and the detection wavelength was set at 370 nm. Aliquots of 20 ␮l of each sample were injected into the column, and all operations were carried out at room temperature. The peak area correlated linearly with QT concentration in the range of 2–10 ␮g/ml with the lowest detection limit at 0.5 ␮g/ml, and the average correlation coefficient was 0.9997.

Fig. 3. Pseudo-ternary phase diagrams of ME composed of oil (ethyl oleate), surfactant (Tween 80), cosurfactant (dehydrated ethanol) and water at various Km values.

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Table 2 Actual and transformed values, mean particle size and solubility of seven different formulations as per simplex lattice design. Formulation number

1 2 3 4 5 6 7

Formulation components (%)/transformed fraction values S

CoS

O

80%/1 45%/0 45%/0 62.5%/0.5 62.5%/0.5 45%/0 56.7%/0.33

15%/0 50%/1 15%/0 32.5%/0.5 15%/0.5 32.5%/0.5 26.7%/0.33

5%/0 5%/0 40%/1 5%/0 22.5%/0.5 22.5%/0.5 16.7%/0.33

Mean particle size (d ± SD, nm)

16.1 11.9 333.3 6.7 117.2 179.5 246.2

± ± ± ± ± ± ±

6.5 5.5 159.9 3.2 39.2 81.4 106.7

Solubility (s ± SD, g/l)

4.85 5.10 3.33 3.17 3.92 3.07 4.44

± ± ± ± ± ± ±

0.17 0.15 0.16 0.22 0.10 0.12 0.13

Data of solubility and mean particle size were shown as mean ± SD (n = 5).

2.8. Statistical analysis Statistical data were analyzed using the Student’s t-test with P < 0.05 as the minimal level of significance. 3. Results and discussion 3.1. Screening components for ME The solubility of QT in various vehicles was analyzed in order to screen suitable components for ME. In four tested surfactant solutions (12.5%, w/v), QT had the highest solubility in Tween 80 solution (887.7 ␮g/ml), followed by Cremorphor EL (522.4 ␮g/ml) and emulsifier OP (320.8 ␮g/ml). It was almost insoluble in poloxamer 188 (73.3 ␮g/ml). Therefore, Tween 80 was chosen as surfactant for ME.

In the four tested oils, the solubility of QT was highest in castor oil (835.1 ␮g/g), followed by peanut oil (772.4 ␮g/g), and the solubility of QT in paraffin oil or ethyl oleate was 702.3 ␮g/g. So castor oil was initially considered as a good oil phase. However, in compatibility tests, compatible ME was exclusively obtained when Tween 80 was matched with ethyl oleate (shown in Table 1). This phenomenon was in accordance with the rule of “the likes dissolves each other” since both of them had a long oleic acid structure which suggested that Tween 80 and ethyl oleate had better compatibility, predicting a stable ME can be obtained from them. This observation could further explain the structure of the droplets of microemulsion. Tween 80 could insert into the oil drop surface uniformly and form a homogeneous interface that prevented them from aggregation [41]. Similar results were obtained for 9-nitrocamptothecin in self-microemulsifying drug delivery system with Tween 80 and ethyl oleate [42].

Fig. 4. The contour plots of response. (A) The contour plot of mean particle size; (B) the contour plot of solubility; (C) the mixing contour plot of mean particle size and solubility, the shadow section represents the optimized domain for ME.

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When the mixture of Tween 80 and ethyl oleate was titrated with different cosurfactants, the stability of ME varied greatly. In tested cosurfactants, dehydrated ethanol or glycerine can form stable ME. Since dehydrated ethanol has a good ability in forming ME with ethyl oleate and Tween 80, and its aqueous solution has a good solubility of QT which can form a concentration gradient, it was chosen as cosurfactant. In conclusion, ethyl oleate, Tween 80 and dehydrated ethanol were subsequently chosen as the oil phase, surfactant and cosurfactant for the formulation of QT-loaded ME in this study. 3.2. Construction of pseudo-ternary diagrams Appropriate concentration ranges of components to form ME were obtained via the construction of pseudo-ternary phase diagrams. The pseudo-ternary phase diagrams with various weight ratios of Tween 80 to ethanol (Km) values were shown in Fig. 3. As Km decreased, the water amount needed to form ME was less and the area of ME decreased. It could be explained by the lower interfacial tension, increased mobility of membrane interface and reduced viscosity of the system as the proportion of ethanol increased. A similar result was obtained from an oleic acid-based microemulsion system with Cremorphor EL as surfactant and ethanol as cosurfactant [21]. In the preparation process of ME, if water content was low and the system was transparent or had a slightly opalescence condition in the process of titration, W/O ME was formed and no birefringence phenomenon was observed in polarized microscopy. If surfactant had a higher ratio, the system would be in a viscid state. ME in the form of W/O went on being titrated by water to a certain extent until it became turbid and then transparent. Samples were left to equilibrate for at least 10 min before being visually examined for transparency. At this point it formed O/W ME.

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Fig. 2 and Table 2. According to simplex lattice model and the chosen concentration scope of surfactant, cosurfactant and oil, the seven formulations for different initial concentrates shown in Table 2 were prepared. Aliquots of each initial concentrate were diluted with 10 ml distilled water. Droplet size was measured using a Beckman Coulter N5 particle size analyzer with 25 mW He–Ne laser (Beckman Coulter, Inc., English) and the results were processed by Matlab data-processing system. The concentrations of surfactant (S), cosurfactant (CoS) and oil (O) were chosen as the independent variables while the solubility of QT and the mean droplet size of formed ME by diluting ME with distilled water were taken as responses (Y), respectively. The equation for simplex lattice model is described as follows: Y = b1 [S] + b2 [CoS] + b3 X3 + b12 [S][CoS] + b13 [S][O] +b23 [CoS][O] + b123 [S][CoS][O]

(1)

where Y is the dependent variable and b1 is the estimated coefficient for the factor[S], [CoS] or [O]. The main effects ([S], [CoS] and [O]) represent the average results of changing one factor at a time from its low to high value, and the interactions [S][CoS], [CoS][O], [S][O], and [S][CoS][O] show how the responses change when two or three factors change simultaneously. With the help of Matlab Software, the results of analysis are shown in Eqs. (2) and (3): Ysize = 438.71[S] + 1.83 × 103 [CoS] + 7.75 × 103 [O] − 5.00 × 103 [S][CoS] − 1.61 × 104 [S][O] − 4.27 × 104 [CoS][O] + 9.53 × 104 [S][CoS][O] (r = 0.9999)

(2)

Ysolubility = 13.33[S] + 46.53[CoS]

3.3. Formulation optimization

+60.82[O] − 102.98[S][CoS] − 137.67[S][O]

A simplex lattice experiment design was adopted to optimize the formulation variables, the composition of ME. Based on above experiments and previous reports [42], the ratio of Tween 80 was set in the range of 45–80%, 5–40% for oil and 15–50% for cosurfactant. In the study, the actual concentrations of surfactant, cosurfactant and oil were transformed based on the simplex lattice method so that the minimum concentration corresponds to zero and the maximum concentration corresponds to one as shown in

−433.73[CoS][O]+880.77[S][CoS][O] (r = 0.9999) (3) Eqs. (2) and (3) can be used to calculate the predicted size and solubility for other formulations in the design space. From Eqs. (2) and (3), it was evident that particle size and solubility of QT were related to the ratios of surfactant, cosurfactant and oil. As shown

Fig. 5. Transmission electron micrographs of blank ME (A) and QT-loaded ME (B).

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Table 3 The parameters for physicochemical characteristics of the optimized QT-loaded ME. Characteristics

Mean droplets size (nm)

Viscosity value (mm2 s−1 )

Zeta value (mV)

Electrical conductivity (␮s/cm)

pH

Refractive indices

Blank ME QT-loaded ME

35.7 38.9

2.06 2.13

−2.72 −6.75

98.8 98.6

6.56 6.24

1.365 1.366

in Table 2, with the increase of the ratio of oil, particle size had a significant enlarging trend, while the solubility of QT decreased on the whole. It indicated that QT mainly dispersed into the emulsifying membrane layer composed of surfactant and cosurfactant. In other words, the decrease of the particle size contributed to the significant increase of total surface area of the emulsifying membrane layer. And the much larger surface area performed as a “drug store” dissolving much more drug, as a result, the solubility of QT increased. Based on Eqs. (2) and (3), contour plots of mean particle size and solubility were constructed when responses (Y) were set as certain values as shown in Fig. 4. In Fig. 4(A), two mean particle size curses stand for 10 nm and 100 nm, respectively. Trace contours were constructed when Ysolubility was equal to 4 g/l and 5 g/l as shown in Fig. 4(B). According to the requirements for solubility and size scope of ME, the overlapping part between Ysize located at the scope of 10–100 nm and Ysolubility located at the field of 4–5 g/l was the best choosing area for the concentrations for surfactant and cosurfactant as shown in Fig. 4(C). Based on the consideration of solubility for QT and stability of formed ME, the appropriate ratio of the components in initial concentrate was chosen for the optimized formulation, consisting of oil (7%), surfactant (48%) and cosurfactant (45%). Under the condition of above compositions, the predicted values of mean particle size and solubility of dilution ME calculated by Eqs. (2) and (3) were 36.2 nm and 4.411 g/l, respectively, which were close to that of the experiment (38.9 nm and 4.138 g/l). Besides, the above optimal formulation was located in the shadow area forming ME in the pseudo-ternary phase diagram which was constructed by mixing ethyl oleate, Tween 80 and dehydrated ethanol at different ratio and recording the ratio of components when clear and transparent solutions were formed. It can be concluded that the simplex lattice method can accurately predict the experiment results.

3.4. Characteristics of optimized ME The droplet of optimized ME appeared in perfect round shape without aggregation under TEM (Fig. 5). The parameters for physicochemical characteristics of the optimized formulation were shown in Table 3. The incorporation of QT in the ME system increased the droplets size slightly in the system, which was in accordance with the literature report [43]. Based on the solubility test, QT molecule should be mainly dissolved or dispersed into the emulsifying membrane layer (composed of surfactant and cosurfactant) with small amount of QT partitioning into oil phase as shown in Fig. 6, which leads to the change of ME droplet size. The zeta value of QT-loaded ME was 2.5 times higher than that of the blank ME, the possible reason is that there are five hydroxy groups in QT molecule structure as shown in Fig. 1. These groups in QT molecule were conducive to the lower pH of the QT-loaded ME than the blank ME due to H+ dissociation of QT, leaving the oxygen atoms in hydroxy groups to some extend the property of negative charge and the negative zeta value. QT-loaded ME was stable at 4 ◦ C for 6 months storage. No changes of clarity and phase separation were observed. The assay of QT was subjected to no significant change after the storage. Furthermore, the centrifuge tests showed that all ME systems had good physical stability. 3.5. Absorption property of quercetin-loaded ME in rat intestine Among all absorption screening methods, in situ intestinal perfusion study in rats was considered as a simple and relevant method of absorption assessment and the absorption properties were most similar to human beings [44]. After incubation with the harvest blank perfusion solution for 6 h at 37 ◦ C, the QT concentration was (99.07 ± 0.61)% of the original solution (n = 6). Similar result was obtained when QT-loaded

Fig. 6. Schematic illustration (not to scale) of possible packing of QT in O/W microemulsion.

Y. Gao et al. / Colloids and Surfaces B: Biointerfaces 71 (2009) 306–314 Table 4 The absorption parameters of ME and micelle at different concentrations in rat’s intestine in situ after perfusing for 6 h (n = 5). Dosage/dose (50 mg/kg)

Ka (h−1 )

t1/2 (h)

Uptake percentage (%)

ME Micelle

0.0681 ± 0.0068 0.0408 ± 0.0045

10.27 ± 1.089 17.13 ± 1.765

34.53 ± 1.64 21.20 ± 2.32

ME was treated with harvest turned intestine mucous membrane ((98.8 ± 0.46)%, n = 3). Those results indicated that QT was stable in blank intestinal perfusion solution and rat’s intestine could be regarded as having no physical adsorption with drugs. The intestinal absorption kinetics of QT-loaded ME and micelle solution at the dose of 50 mg/kg were investigated. The results were shown in Table 4. From Table 4, it could be concluded that there were significant differences in absorption parameters such as Ka , t1/2 (P < 0.01) and uptake percentage (P < 0.05) between ME and micelle solution. QT in the ME solution could be absorbed more and faster in rat intestine than that in micelle solution. This was in accordance with the results reported by Yuan et al. and Griffin et al. [45,46]. The special lipid composition of ME formation, ethyl oleate, was considered to account for the better absorption. Because there are a lot of Peyer’s patches and M cells in rat intestine, absorption via the lymphatic tissues is an important route for the drugs with low orall bioavailability [47–49]. Lipids, as compositions of intestine mucous membrane, have an important effect on absorption enhancement via the lymphatic route [50]. It was proved that compared with micelle without oil, ME could produce higher and more prolonged concentration of poorly soluble drug in intestinal lymph [44]. In the experiment of absorption property in rat intestine, the micelle solution containing the same surfactants and cosurfactants had a similar solubilizing capacity of QT to ME, thus lack of oil phase ethyl oleate was the main reason why its Ka was less than that of ME. In rat intestinal perfusing experiment, the concentration changes could also be affected by the water content of the intestine. A volumetric marker that ever has been considered zero absorption like Phenol red used to be added into the sample solution. However, recent research reports showed that phenol red was partly absorbed in small intestine and could interfere with the transport or analytical measurement of some definite drugs especially the poorly water-soluble drugs [37,39]. Therefore, other simpler methods such as gravimetric method and recording directly volume were developed [38,51]. In this paper, the non-absorbed drug was calculated using the method of recording directly volume. 3.6. Main site of QT–ME absorption after oral administration Drug permeability to different regions of rat intestine, duodenum, jejunum, ileum and colon, was measured by the in situ single pass perfusion method. The regional differences in the absorption of drugs were shown in Table 5, and there was significant difference in uptake percentage among different intestine segments (P < 0.05). The absorption percentage of QT-loaded ME in the colon was the highest, followed by ileum, duodenum in order. Jejunum had the lowest permeability for QT-loaded ME. Since the duodenum has the largest epithelial surface area due to the villous structure (fourTable 5 Absorption of drugs at various regions in rat intestine. Region

Mean uptake percentage (%)

Duodenum Jejunum Ileum Colon

37.52 34.40 40.82 47.30

± ± ± ±

0.99 2.62 1.41 2.83

313

folds to the colon and two-folds to the ileum) but the slightly lower absorption percentage in the study, it suggested that the epithelial surface area was not a determinant of intestinal permeability of QTloaded ME. From an opposing perspective, it can be explained by the different mucus layers at the surface of the epithelium and the fluidity of the epithelial cell membrane of the different regions of rat intestine. It has been demonstrated that mucus layer is thicker in the upper intestine than in the lower or colon in fasted rat [52]. The layer that functions as a rate controlling barrier limits the absorption of QT. Moreover, previous study [40] showed that the lower portion of the small intestine is more fluid than the upper. Therefore, it was considered that the difference in the membrane fluidity contributed to the higher permeability of QT in the colon. QT-loaded ME had good ileum absorption to some extent because ileum is abound in collecting lymphatic vessel. ME had a tendency to adhere to collecting lymphatic vessel surface and transferred via M cell. The concrete mechanism is still ambiguous and further profound researches are required. 4. Conclusion In this paper, ME system for improving the solubility and oral absorption of QT was developed and the ME formulation was optimized by a simplex lattice experiment design. The solubility of QT in ME (4.138 mg/ml) increased significantly compared with that of QT in water (0.17–7.7 ␮g/ml). The results of in situ absorption of QT in rat intestine suggested that the ME played an important role in absorption enhancing effect. Further investigation of the concrete mechanism is needed for in vivo studies. In conclusion, microemulsion may represent potential delivery system for sparingly soluble anticancer drugs. Acknowledgement This work is partly supported by a research grant (No. 2004BS03019) from Department of Shandong Science and Technology, PR China. References [1] R.A. Prentis, Y. Lis, S.R. Walker, Br. J. Clin. Pharmacol. 25 (1988) 387. [2] M.B. Cheng, J.C. Wang, Y.H. Li, X. Zhang, D.W. Chen, S.F. Zhou, Q. Zhang, J. Control. Release 29 (2008) 41. [3] H. Araya, M. Tomita, M. Hayashi, M. Tomita, M. Hayashi, Drug Metab. Pharmacokinet. 21 (2006) 45. [4] A. Kogan, N. Garti, Adv. Colloids Interf. Sci. 123–126 (2006) 369. [5] A. Kogan, A. Aserin, N. Garti, J. Colloids Interf. Sci. 315 (2007) 637. [6] T.P. Formariz, L.A. Chiavacci, V.H.V. Sarmento, C.V. Santilli, E.S. Tabosa do Egito, A.G. Oliveira, Colloids Surf. B: Biointerf. 60 (2007) 28. [7] J.V. Formica, W. Regelson, Food Chem. Toxicol. 33 (1995) 1061. [8] A.W. Boots, G.R. Haenen, A. Bast, Eur. J. Pharmacol. 585 (2008) 325. [9] D.H. Lee, M. Szczepanski, Y.J. Lee, Biochem. Pharmacol. 75 (2008) 2345. [10] W.M. Loke, J.M. Proudfoot, S. Stewart, A.J. McKinley, P.W. Needs, P.A. Kroon, J.M. Hodgson, K.D. Croft, Biochem. Pharmacol. 75 (2008) 1045. [11] M. Mamani-Matsuda, T. Kauss, A. Al-Kharrat, J. Rambert, F. Fawaz, D. Thiolat, D. Moynet, S. Coves, D. Malvy, M.D. Mossalayi, Biochem. Pharmacol. 72 (2006) 1304. [12] K.A. Khaled, Y.M. El-Sayed, B.M. Al-Hadiya, Drug Dev. Ind. Pharm. 29 (2003) 397. [13] B.L. Wei, C.M. Lu, L.T. Tsao, C.N. Lin, Planta. Med. 67 (2001) 745. [14] M.R. Lauro, M.L. Torre, L. Maggi, F. De Simone, U. Conte, R.P. Aquino, Drug Dev. Ind. Pharm. 28 (2002) 371. [15] K. Azuma, K. Ippoushi, H. Ito, H. Higashio, J. Terao, J. Agric. Food Chem. 50 (2002) 1706. [16] D. Fasolo, L. Schwingel, M. Holzschuh, V. Bassani, H. Teixeira, J. Pharm. Biomed. Anal. 44 (2007) 1174. [17] H.L. Li, X.B. Zhao, Y.K. Ma, G.X. Zhai, L.B. Li, H.X. Lou, J. Control. Release 133 (2009) 238. [18] S.Y. Gui, L. Wu, D.Y. Peng, Q.Y. Liu, B.P. Yin, J.Z. Shen, Pharmazie 63 (2008) 516. [19] A.O. Nornoo, H. Zheng, L.B. Lopes, B. Johnson-Restrepo, K. Kannan, R. Reed, Eur. J. Pharm. Biopharm. 71 (2009) 310. [20] P.K. Ghosh, R.J. Majithiya, M.L. Umrethia, R.S. Murthy, AAPS PharmSci. Technol. 7 (2006) 77. [21] W.W. Zhu, A.H. Yu, W.H. Wang, R.Q. Dong, J. Wu, G.X. Zhai, Int. J. Pharm. 360 (2008) 184.

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