Enhanced solubility and dissolution of curcumin by a hydrophilic polymer solid dispersion and its insilico molecular modeling studies

Journal of Drug Delivery Science and Technology 29 (2015) 226e237

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Research paper

Enhanced solubility and dissolution of curcumin by a hydrophilic polymer solid dispersion and its insilico molecular modeling studies Avinash B. Gangurde*, Harish S. Kundaikar, Sharadchandra D. Javeer, Divakar R. Jaiswar, Mariam S. Degani, Purnima D. Amin Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, University under Section 3 of UGC Act- 1956, Elite Status & Centre of Excellence e Government of India, TEQIP Phase II Funded, Matunga (E), Mumbai 400 019, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 December 2014 Received in revised form 8 August 2015 Accepted 10 August 2015 Available online 13 August 2015

Curcumin (CUR) is highly lipophilic drug that shows degradation at alkaline pH which restricts its oral bioavailability. The aim of the present study was to enhance the oral bioavailability of CUR by increasing its solubility and dissolution rate. Solid dispersions (SDs) of CUR in aqueous and organic solvent using Eudragit EPO (EuD) were prepared by spray drying and rota evaporation technique. The solubility of plain CUR in acidic pH 1.2 is only 0.02% whereas SDs containing EuD have solubilities of 40.29% and 18.78% by spray drying and rota evaporation technique respectively. Physical characterization by SEM, IR, DSC, and XRD studies, revealed the changes in solid state during the formation of dispersion and justified the decreased crystallinity of CUR SDs. Dissolution studies showed that pH values influenced the release profile lower the pH values higher the release speed (pH 1.2). CUR in pH 1.2 showed negligible release even after 120 min (2e5%) whereas, SDs showed 20e45% drug release after 60 min. Further, insilico docking study was carried out followed by molecular dynamic simulations to understand the molecular level binding interactions between drug and polymer. The insilico studies demonstrates the role of van der Waals interactions in binding of CUR to EuD. © 2015 Elsevier B.V. All rights reserved.

Keywords: Curcumin Eudragit EPO Spray drying Solid dispersion Docking study

1. Introduction Curcumin (CUR) [1, 7-bis (4-hydroxy-3-methoxyphenyl)-1, 6heptadiene-3, 5-dione] is a naturally occurring hydrophobic polyphenol extracted from the plants of the Curcuma longa, its structure was showed in (Fig. 1) [27]. It has variety of biological activities and pharmacological actions, such as anticancer, antiviral, antiarthritic, antiamyloid, antioxidant and anti-inflammatory properties [8]. In spite of these wide span of activities of CUR, its therapeutic efficiency has been highly limited due to poor solubility in water (the maximum solubility was reported to be 11 ng/ml in plain aqueous buffer pH 5.0) [11]. Low bioavailability of CUR after oral delivery in addition to poor aqueous solubility, is attributed to high pre-systemic metabolism in gastrointestinal tract (GIT), degradation in GIT at neutral and alkaline pH, rapid systemic metabolism to sulfate and glucuronide conjugates leading to short half-life [4,34].

* Corresponding author. Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, N. P. Marg, Matunga (E), Mumbai 400 019, India. E-mail address: [email protected] (A.B. Gangurde). http://dx.doi.org/10.1016/j.jddst.2015.08.005 1773-2247/© 2015 Elsevier B.V. All rights reserved.

Different strategies were used to overcome these problems include conversion of CUR to amorphous microparticles by improving its solubility in acidic pH and improving CUR stability in aqueous media [28]. Numerous formulation approaches have been made to design soluble formulation of CUR, which include loading CUR into liposome or nanoparticle, self-emulsifying drug delivery system (SMEDDS) and CUR cyclodextrins complexation [26]. However, slow process of complexation, high molecular weight of cyclodextrins and pH of the processing medium may limit their practical utility. Different hydrophilic polymers such as PVP K-30, PEG 4000 and PEG 6000 have been tried for solubility and dissolution rate enhancement of CUR by different solid dispersion (SDs) techniques [2,28]. The hydrophilic polymers used in this study was Eudragit EPO (EuD). EuD which is a cationic copolymer with dimethyl-amino ethyl methacrylate as a functional group. The polymer has a maximum solubility in gastric fluids up to pH 5. Spray drying is a technique used for development of microparticulate SDs for improving the stability and solubility of compounds [6,33]. Spray drying technique gives uniform and spherical shape particles that are small in size and that have narrow

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227

2.3. Preparation of spray dried microspheres 2.3.1. Solid dispersion preparations The composition of prepared formulations were depicted in (Table 1). SDs were prepared by different methods to enhance solubility of CUR in acidic medium using the pH dependent polymer EuD. The CUR-EuD SDs were prepared by spray drying and rota evaporation technique. Fig. 1. The structure of curcumin.

distributions. Spray drying technique is used in the food industry due to its high productivity, its relatively low cost of production, the increased microbiological stability of phytopharmaceutical products and decreases the risk of chemical and/or biological degradations [25]. Spray drying has been used to produce biopharmaceuticals and biomaterial products [32]. Cationic polymer (EuD) based SDs by using scalable spray drying and rota evaporation techniques are the appropriate option for solubility enhancement of CUR. The present work examines the influence of hydrophilic polymer EuD on solubility and dissolution rate of CUR followed by insilico prediction of the interactions between the drug and polymer to understand the possible mechanism of binding and enhancement of solubility and dissolution rate of CUR. The work also involved studying the effect of method of preparation of SDs on the dissolution rate of CUR.

2. Materials and methods

2.3.2. Using organic solvent A CUR SDs using organic solvent were obtained by dissolving a weighed quantity active, EuD and HPMC E5 in ethanol/acetone and dried using spray dryer (CUR01 & CUR02) (Jay Instruments and Systems Pvt. Ltd., India) and Rota Evaporator (CUR04 & CUR05) (Heidolph, Lab rota Efficient 400 WB eco, India). The operating parameters for spray drying were: inlet temperature 54e56
C; outlet temperature 42e45
C; feed rate 2e5 ml/ min; atomization air pressure 2e3 kg/cm2; and aspiration rate40e45%. For, rota evaporator drying temperature was set to 60
C. 2.3.3. Using aqueous solvent A CUR SDs using aqueous solvent was obtained by preparing o/ w emulsion of CUR (CUR03) using Cremophore RH 40 as oil, HPMC E5 as recrystallization inhibitor and Tween 80 as solubilizer and further spray dried. The operating parameters were: inlet temperature 105e110
C; outlet temperature 55e60
C; feed rate 1e5 ml/ min; atomization air pressure 2e3 kg/cm2 and aspiration rate4045%.

2.1. Materials 2.4. Standard curve of CUR in 0.1N HCl and in different pH medium CUR was obtained as a gift sample from K. Patel Phytoextratct Ltd. (Mumbai, India). EuD (Eudragit EPO) from Evonik industries Ltd. (Mumbai India) was used as the hydrophilic carrier soluble at gastric fluid pH 5. Methocel® E5-LV and Cremophore RH 40 were obtained from Dow Chemical's (Mumbai, India), and BASF India respectively. Tween 80 from SD Fine Chemicals Ltd. as surfactant. Cross carmellose sodium was obtained as gift sample from Anshul Life Sciences Ltd., Avicel PH 101 and lactose was procured from Signet Chemicals Ltd. Mumbai. Solvent selected for solubilization of CUR was acetone (RANKEM AR grade), absolute ethanol (AR grade) and distilled water. All other reagents used were of analytical reagent grade.

The standard curve of CUR was prepared in different pH medium (pH 1.2/pH 4.5, pH 6.8 and pH 7.8). A 1000 mg/ml stock solution of CUR was prepared in with addition of 10 ml of glacial acetic acid. From this stock solution, 1 ml aliquot was withdrawn to prepare a 10 mg/ml solution of CUR in different pH medium. The 10 mg/ml solution of CUR was diluted suitably to 10 ml each medium to obtain respective concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 mg/ml of CUR. The absorbance of these solutions was recorded at 420 nm on a Shimadzu UV Spectrophotometer (UV e 1650 PC). The absorbance versus concentration data (n ¼ 3) was treated by least squares linear regression analysis. 2.5. Drug content analysis

2.2. Physical mixture (PM) The physical mixtures (PM) of drug with polymer EuD were prepared by simple blending of CUR50% with the hydrophilic polymer 49% and HPMC E5 1%. The blends obtained were passed through a sieve value of 250 micron size.

10 mg of the sample was weighed accurately and dissolved in 10 ml glacial acetic acid (AR). The solution was sonicated for 10 min and the sample was centrifuged at 10,000 rpm for 5 min. The supernatant was diluted with suitable quantity of methanol. The absorbance of supernatant solution was recorded at 420 nm using

Table 1 Formulation composition of CURSDs. Formulation %

CUR01 (Rota Evap.)

CUR02 (Spray drying)

CUR03 (Spray drying)

CUR04 (Rota evap.)

CUR05 (Spray drying)

CUR Eudragit EPO HPMC E5 Cremophore RH 40 Tween 80 Acetone Ethanol Purified water

50 49 1 e e e q.s. e

50 49 1 e e e q.s e

50 40 1 12 2 e e q.s.

50 49 1 e e q.s. e e

45 49 1 e e q.s. e e

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UV visible spectrophotometer (Shimadzu UV-1650, Tokyo, Japan). The drug content was determined by using a standard plot of absorbance versus concentration. 2.6. Saturation solubility An excess amount of the plain CUR and SDs formulation was added to 10 ml of the 0.1 N HCl and sonicated at ambient room temperature for 25 min. All test tubes were covered with aluminum foil and subjected to sonication for 20 min at room temperature, these were then kept in orbital shaking thermo stable incubator (Boekel Scientific, Germany) for 48 h at 37
C ± 0.5
C with rotation speed of 75 agitations/min. The supernatant solution were then passed through a whatman filter paper (Grade 1). After equilibration, the samples were filtered through 0.45 mm pore size nylon filters suitably diluted, and analyzed by UV at 420 nm. All solubility measurements were performed in triplicate. 2.7. Solid state characterization The SDs and plain CUR were passed through a number 60# (250 micron). The formulations were characterized by differential scanning calorimetry (DSC), X-ray diffraction, and Fourier transform infrared (FTIR) spectroscopy analyses. Also, morphological characterization was done using scanning electron microscopy (SEM). 2.7.1. Differential scanning calorimetry (DSC) Differential scanning calorimeter (DSC-PYRIS-1, Perkin Elmer, USA) was used to study the drug polymer interactions and thermal behavior of drug. The experiments were performed in a dry nitrogen atmosphere. Samples (5.0e10.0 mg) of CUR and CURSDs were accurately weighed into crimped aluminum pans and heated at 10
C/min under a nitrogen purge (20 mL/min) from 0 to 250
C. The samples were cooled rapidly from 250
C/min to 40
C and reheated at 10
C/min (second heating cycle) to 250
C. An empty crimped aluminum pan was used as the reference cell. The instrument was calibrated prior to testing the samples, using indium. 2.7.2. Powder X-ray powder diffraction (PXRD) The crystallinity between two samples was measured using a Miniflex apparatus (Rigaku, Japan) with Cu Ka radiation. Samples were held on quartz frame. Diffraction pattern were obtained at a voltage of 40 kV and at a current of 20 mA. The slide was then placed vertically at 0
angle in the X-ray diffractometer so that the X-ray beam fell on it properly. The results were recorded over a range of 0e40
(2q) using the Cu-target X-ray tube and Xe-filled detector. The operating conditions were: voltage 40 kV; current 20 mA; scanning speed 1/min; temperature of acquisition: room temperature; detector: scintillation counter detector and sample holder: non-rotating holder. 2.7.3. FT-IR spectroscopy (FTIR) Fourier transform infrared analysis was performed on samples of neat CUR and CUR-SDs using a Shimadzu MIRACLE IR -Affinity-1 FTIR spectrophotometer. Samples were mixed with dry potassium bromide using a mortar and pestle, compressed to prepare a disk and analyzed over a range 4000e400 cm1. 2.7.4. Scanning electron microscopy (SEM) To understand any changes in the surfaces, the morphology of pellets like size and shape was analyzed by using XL 30 Model JEOL 5400 SEM (Japan) during analysis. Double sided carbon tape was affixed on Al stubs over which sample of neat CUR and prepared CUR-SDs was placed. The radiation of platinum plasma beam using

JFC-1600 auto fine coater was targeted on Al stubs for its coating to make layer of 2 nm thickness above the sample for 25 min. These prepared coated stubs were then placed in the vacuum chamber of a SEM and adjusted to maximum magnification to obtain excellent quality scanning images. Then, those samples were observed for morphological characterization using a gaseous secondary electron detector (working pressure: 0.8 Torr, acceleration voltage: 20.00 kV). SEM images were obtained at maximum and visible magnification to understand the surface interaction between drug and polymer. 2.8. Dosage form development Capsule and tablet formulations of solid dispersed product were also prepared. For this all the excipients including drug containing SDs were sieved through 40-mesh and mixed in geometrical order. Mixing was continued for 10 min. Blends were evaluated for their physical properties such as flow, density and compressibility. The bulk density and tapped bulk density were determined by using density apparatus (VEEGO, India). Compressibility was defined by Carr's indices (CI) and Hausner's ratio values. Carr's indices (CI) and the Hausner's ratio were calculated from the obtained density values. Flow property (angle of repose) was determined by fixed funnel method [14]. Tablet was prepared using 12 mm standard concave round punch using single punch tablet machine (Cadmach, India). Tablets were evaluated for hardness, thickness, and disintegration time and dissolution rate. Capsule formulation contains the same composition as tablet shown in (Table 2). 2.9. In-vitro dissolution studies First of all powder dissolution study was carried out by using eight station USP apparatus II (Electro lab, TDT- 08 L, India) in 900 ml of different pH buffers (pH 1.2, pH 4.5, pH 6.8 and pH 7.4) at a temperature of 37 ± 0.5
C at 50 rpm. A powdered sample (equivalent to 200 mg dose) was introduced directly into the dissolution medium. At regular time intervals of 15, 30, 45, 60, 90 and 120 min, 10 ml of aliquots sample were withdrawn and same amount replaced by fresh medium to maintain sink condition. The withdrawn samples were suitably diluted and analyzed through UVeVisible spectrophotometer at 427 nm. All studies were carried out in triplicates. For tablet and capsule dissolution study, conditions were kept same. 2.10. Dissolution kinetic studies Dissolution kinetic studies of prepared formulation were carried out using zero order, first order, and Higuchi, Hixson Crowell and Korsemeyer Pappas equation model. Regression coefficient factor

Table 2 Formulation composition for Tablet and Capsule CUR Equivalent to 200 mg. Ingredients

Neat CUR

CUR01

CUR02

CUR03

CUR04

CUR05

430 [196] 15 19 10 3 3 470

515 [160] 15 19 10 3 3 565

430 [196] 15 19 10 3 3 470

430 [196] 15 19 10 3 3 470

SDs Quantities in mg CUR EuD in SDs Cross carmellose Na Avicel PH102 Lactose Talc Magnesium stearate Total

200 e 15 19 10 3 3 250

430 [196] 15 19 10 3 3 470

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(r2) and other factors were calculated to understand the release kinetic performance of prepared SDs formulations [3].

For the contact angle measurement, G10 Contact angle meter (KRUSS, Germany) was used. Static contact angle method was adopted to measure it. A solid disc with flat surface was prepared of plain CUR and CUR-SDs prepared by spray drying and rota evaporation technique. A single drop of distilled water was dropped on the flat disc surface and the contact angle was measured immediately and after 60 s of equilibrium. Measurements were carried out in triplicate for each sample. 2.12. Moisture uptake study Moisture uptake by drug and prepared SDs was studied using Moisture balance MB 50C (Citizen, India). Moisture content of CUR and SDs was calculated as percentage. After moisture content analysis neat CUR and SDs were placed in crucible at accelerated conditions of temperature 40 ± 2
C and humidity 75 ± 5% RH in environmental test chamber for 24 h (Thermo lab, India). These samples were then analyzed for drug content by UV spectroscopy. The method is useful to determine the effect of moisture on degradation of drug and prepared SDs systems [13]. 2.13. Stability study Stability studies were conducted by placing samples in closed glass vials which were stored in a controlled temperature environment inside stability chamber with relative humidity (RH) of 75% and 40
C temperature [5]. Samples were removed after 6 months and tested for crystalline content using DSC and XRD. Drug release experiments were also conducted on samples stored for 6 months and compared with those tested immediately following manufacture. Tablet and capsule formulation was also kept for 6 months stability study under above mentioned conditions. 2.14. Antioxidant assay 2.14.1. Free radical scavenging activity of curcumin solid dispersion by stable 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical Antioxidant potential of curcumin SD were determined by the hydrogen-donating ability of stable free radical DPPH [7,29]. DPPH produces a violet color in methanol. When the free radical reacts with an antioxidant, its free radical property is lost and its color changes to light yellow. Extent of color change in presence of curcumin and standard ascorbic acid was monitored by UV absorption at its absorption maxima in methanol of 517 nm. A blank was prepared using the reaction mixture (without samples and DPPH solution), and absorbance positive control was prepared with various concentrations of CUR-SDs (3.0 mL) were mixed with 1.0 mL of 0.2 mM DPPH [1]. The obtained solution was shaken vigorously and allowed to stand in the dark at room temperature for 30 min. All measurements were performed in triplicate. The percent antioxidant inhibition (ability to scavenge DPPH radical) was calculated using Eq. (1).

Acontrol  Atest  100 Acontrol

to 50% inhibition of DPPH reduction was reported as the IC50 value. 2.15. Molecular modeling interaction studies

2.11. Contact angle measurement

% Inhibition ¼

229

(1)

whereas, A ¼ absorbance; Acontrol ¼ absorbance of blank solution Atest ¼ absorbance of the reference standard. The percentages of inhibition were plotted against the concentrations for each sample, and the concentration that corresponded

The monomer and dimer unit structures of EuD and the diketo and keto-enol forms of CUR were drawn by using 2D sketcher utility of Maestro software [24] and subjected to ligand preparation [19] to refine the structures. The docking studies [15] were carried out using flexible XP (extra precision) docking at the centroid of the generated polymer grids and subjected to post-docking minimization, to understand the binding interactions between the drug and polymer and optimize the poses of the drug and polymer in the complex. The energy minimization of these drugepolymer complexes was done [22,23] to get the lowest energy conformations of the complexes. The molecular dynamics (MD) [12] simulation studies were further carried out on these lowest energy conformations, using NVT ensemble under vacuum for 10 ns. The trajectory data was collected and analyzed to understand the structural interaction modes of the drug with polymer. 3. Results and discussion CUR is most widely used bioactive compound which has countless disease preventing and health promoting activities. The safety of CUR confirmed by Phase I clinical trials doses upto 12 g daily with no significant toxicity [17,31]. However, the poor water solubility, low stability and low systemic bioavailability of CUR has retards its uses in human health (J, [21]. Therefore, to increase solubility and chemical stability of CUR are important target to enable its use for enhancing human health. Preparation of amorphous solid dispersion is a promising way to improve oral drug solubility and bioavailability. Crystalline drugs exhibit poor water solubility, since the lattice energy must be overcome in order for dissolution to occur [9,20]. We investigated the impact of polymer structure upon stabilization of CUR SDs in the solid phase, dissolution, and stabilization of CUR against crystallization from the supersaturated solution phase generated upon SDs dissolution. We evaluated the effect of CUR: EuD SDs and compared with Neat CUR using different techniques. SDs is dispensation of active ingredients in molecular, amorphous or microcrystalline forms bordered by inert carriers [10]. In the present investigation amorphous SDs of CUR were prepared by two different technique. Amorphous drug substances are physically unstable due to their high energy state and tend to recrystallize upon storage. The solubility of amorphous drug substances is higher solubility than the thermodynamically stable crystalline forms, because their internal bonding forces are weak. Solutions obtained from amorphous forms are supersaturated, and crystallization occurs once a crystal of the stable form develops. This recrystallization process is starts when the amorphous drug gets into contact with the dissolution medium. In order to stabilize these systems, various polymer carriers have been used because they readily generate amorphous forms and may be able to retain the amorphous nature of the drug upon storage. During dissolution recrystallization of the amorphous drug is blocked by the addition of polymers, which form a hydrodynamic boundary layer around the drug molecules being discharged from the solid solution. HPMC is a suitable polymer for recrystallization inhibitor in SDs [18]. Molecular complexes of CUR with a hydrophilic polymer (EuD) were prepared by spray drying or rota evaporation method. The formation of the complex was optimized using acetone or water as solvent, recrystallization inhibitor HPMC E5 and surfactant Tween 80 to achieve enhanced loading and increased solubility.

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3.1. Preparation of SDs Plain CUR is observed to show low solubility. Physically mixed CUR and polymer mixture showed similar powder characteristics as the plain drug whereas spray dried drug was found to be porous and less dense in nature. The SDs prepared by rota evaporation technique were dense and irregular in shape.

3.4. In-vitro dissolution study

3.2. Drug content and saturation solubility studies The drug content of different batches was found to be uniform and within acceptable range from 35% to 45% as indicated in (Table 3). The dissolution rate of drug dependent upon aqueous solubility of a drug and compounds with aqueous solubility lower than 0.1 mg/ml often present dissolutions restricted to absorption [16]. Saturation solubility study was done in 0.1 N HCl without surfactant. A comparative study of SDs was done with that of pure CUR and PMs of CUR: EuD in 1:1 ratio. In 1:1 ratio of CUR: EuD, increase in solubility was seen using SD method. This may be attributed to the improved wetting of CUR in the presence of EuD probably due to the formation of intermolecular hydrogen bonding between the carbonyl group of EuD and the hydrogen atom in the hydroxyl group of CUR. An approximate linear increase in solubility of the drug with the weight fraction of EuD was observed. In PMs a significant increase in solubility was not observed. SDs prepared by spray drying and rota evaporation method showed increase in solubility as compared to PM and plain drug in both the solubility study media. Increase in solubility might be observed due to hydrophilic nature and surface active property of EuD. 3.3. Physical characterization of SDs powder and tablet formulations Flow and compaction behavior play an important role in manufacturing, processing and packaging techniques. It was found Table 3 Drug content and Saturation solubility of SDs (n ¼ 3, mean ± SD). Samples

Solubility in water (mg/ml)

Plain CUR PM of CUR: EuD(1:1) CUR01 CUR02 CUR03 CUR04 CUR05

0.527 0.261 0.512 0.398 198.94 0.538 0.416

± ± ± ± ± ± ±

0.092 0.089 0.094 0.078 0.782 0.098 0.084

Solubility in pH 1.2 buffer (mg/ml) 0.163 37.13 224.12 412.32 288.69 459.56 730.78

± ± ± ± ± ± ±

0.080 0.512 0.791 0.753 0.892 0.812 0.912

that plain CUR has poor flow properties and needs manual tapping to make it freely flowable but has good compaction behavior. The compaction and flow behavior further improved with SDs prepared by rota evaporation and spray drying technique. All other parameters after formulation were found to be within acceptable range as mentioned in (Table 4).

Drug content (%) 95 46.32 45.75 45.71 35.57 46.12 46.29

Neat CUR and CUR: EuD release profile was investigated under different pH values were showed in (Fig. 2). Release pattern showed that pH values influenced the release profile lower the pH values higher the release speed (pH 1.2). The nature of interaction between CUR: EuD is responsible for the release characteristic as H bond is highly influenced by the pH of the aqueous medium [21]. The release profiles were monitored for 120 min and it was notable that CUR: EuD SDs showed the initial burst release and then steady pattern of drug release depending upon pH of dissolution medium. The dissolution rate of pure CUR was slow because of its hydrophobicity due to which the powder floats on the dissolution medium and prevents its deep contact with dissolving medium. All prepared SDs showed increase in dissolution rate but higher dissolution was observed with spray drying technique using acetone as solvent. Further, the SDs prepared by spray drying method showed better dissolution behavior as compared to rota evaporation method. This improvement in solubility and dissolution rate of SDs can be contributed to several factors such as excellent wettability, rapid dispersion in dissolving medium, the solubilizing effect of the hydrophilic carrier and dissolution medium. Higher solubility was observed for drug prepared by spray drying technique using acetone as solvent because of better solubility of CUR in acetone and it forms spherical porous micro particle of CUR. HPMC E5 was used as recrystallization growth inhibitor which avoids crystal lattice formation by enhancing the viscosity of the dissolution medium on all sided of the drug molecules which decreases their diffusion. Additionally, HPMC E5 has better ability to get adsorbed on newly formed surface and reduction in interfacial tension between solid and dissolving liquid surface which increases solubility of drug. Capsule and tablet formulation prepared from SDs prepared by spray drying method were also evaluated for dissolution rate. Tablet provides a better dissolution rate as compared to capsule formulation and dissolution pattern was found to be similar as solid dispersed powder shown in (Fig. 3A and Fig. 3B). A limited dissolution rate was observed due to crystalline nature and attainment of saturation level of CUR in dissolving medium, this

Table 4 Flow properties of tablet and capsule formulations. Sample Blend properties

Neat cur CUR01 CUR02 CUR03 CUR04 CUR05

Sample Formulation (Tablet)

Plain cur CUR01 CUR02 CUR03 CUR04 CUR05

Bulk density (g/cc)

Tap density (g/cc)

Angle of repose (q)

0.374 0.352 0.403 0.349 0.428 0.350

0.424 0.423 0.512 0.452 0.507 0.430

29.24 23.34 23.45 25.87 24.34 26.45

Hardness (kg/cm2)

Diameter (mm)

3e4 3e4 3e4 3e4 3e4 3e4

12.06 12.12 12.14 12.18 12.19 12.14

± ± ± ± ± ±

0.27 0.26 0.25 0.21 0.24 0.23

± ± ± ± ± ±

1.45 1.23 1.05 1.31 1.07 1.43

Thickness (mm) 3.42 4.10 4.12 4.17 4.11 4.06

± ± ± ± ± ±

0.10 0.12 0.14 0.15 0.13 0.11

Compressibility (%)

Hausner's ratio

11.79 16.78 21.28 22.78 15.58 18.60

1.13 1.20 1.28 1.29 1.18 1.22

Disintegration time (min.)

Friability (%)

20 13 12 7 10 9

0.8 0.6 0.8 0.6 0.7 0.8

A.B. Gangurde et al. / Journal of Drug Delivery Science and Technology 29 (2015) 226e237

100

Neat CUR

pH 1.2

CUR01

80

100

Neat CUR

80

CUR02

% Releasee

% Release

CUR03 CUR04 CUR05

40

CUR03

60

CUR04 CUR05

40

20

20

0

0 0

15

30

45 60 75 Time (min.)

90

0

105 120

15

30

45 60 75 Time (min.)

90

105 120

100

100 Neat CUR

Neat CUR

pH 6.8

CUR01

80

CUR02 % Release

CUR03

60

CUR04 CUR05

40

pH 7.4

CUR01

80

CUR02 % Release

pH 4.5

CUR01

CUR02

60

231

CUR03

60

CUR04 CUR05

40 20

20

0

0 0

20

40 60 80 TIime (min.)

100

0

120

20

40 60 80 TIime (min.)

100

120

Fig. 2. In vitro drug release profile of Neat CUR and SDs 0.1N HCl, pH 4.8, pH 6.8, pH 7.4 (n ¼ 3, mean ± SD).

100

100 Neat cur

Neat cur

A

CUR01

80

80

CUR02

CUR03

% Release

% Release

CUR02 60

CUR04 CUR05

40

B

CUR01

CUR03

60

CUR04 CUR05

40

20

20

0

0 0

15

30

45 60 75 Time (min.)

90 105 120

0

15

30

45 60 75 Time (min.)

90

105 120

Fig. 3. In vitro drug release profile of A) capsule and (B) tablet formulation prepared using SDs in 0.1N HCl (n ¼ 3, mean ± SD).

result was supported by data obtained from DSC and PXRD analysis.

3.5. Dissolution kinetics studies The dissolution kinetic studies were conducted and drug release profiles from all these SDs and CUR-SDs formulations of tablets and capsules best expressed by Higuchi equation, as shown in (Tables 5e7). Higher correlation as indicated by r2, was observed for Higuchi matrix release kinetics in all formulations, suggesting that diffusion is a probable mechanism of drug release.

3.6. Wetting property Contact angle of CUR and its SDs were analyzed in order to see wettability changes through surface modification. The contact angle of PMs with EuD was calculated to find out whether simple mixing of an excipient is enough to increase the wettability of CUR or whether the CUR has to be processed with the excipients. Pure CUR has a poor wettability as shown by high contact angle as shown in (Table 8). Converting the solid state by transferring the CUR into its amorphous form leads to a lower contact angle. The contact angle in PMs is lower than that of SDs of CUR with EuD. The poor wettability in PMs leads to lowering in the dissolution rate.

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Table 5 Order of drug release of prepared SDs determined by the regression coefficients. Formulation code

CUR01 CUR02 CUR03 CUR04 CUR05

Zero order

First order

Higuchi

Hixon-Crowell

Krosemeyer-Peppas

r2

r2

r2

r2

r2

0.9532 0.9577 0.8591 0.9952 0.7963

± ± ± ± ±

0.24 0.21 0.31 0.19 0.35

0.9564 0.9681 0.8634 0.9962 0.8234

± ± ± ± ±

0.25 0.27 0.26 0.22 0.36

0.9862 0.9912 0.9695 0.9618 0.9346

± ± ± ± ±

0.23 0.21 0.25 0.23 0.28

0.9549 0.9647 0.8617 0.9957 0.8134

± ± ± ± ±

0.27 0.28 0.21 0.18 0.21

0.9635 0.9317 0.8803 0.9971 0.9381

n-value ± ± ± ± ±

0.23 0.29 0.31 0.20 0.28

9.798 20.26 11.46 1.907 9.567

± ± ± ± ±

1.23 2.12 0.98 0.56 1.12

Table 6 Order of drug release of tablet formulation determined by the regression coefficients. Formulation code

Zero order

First order

2

0.9633 0.8652 0.9444 0.9542 0.9001

± ± ± ± ±

0.23 0.38 0.25 0.23 0.31

0.9652 0.8730 0.9461 0.9582 0.9163

Hixon-Crowell

2

r

r CUR01 CUR02 CUR03 CUR04 CUR05

Higuchi

2

2

r ± ± ± ± ±

0.23 0.38 0.23 0.34 0.31

0.9744 0.9490 0.9944 0.9816 0.9862

r2

r ± ± ± ± ±

0.22 0.25 0.19 0.20 0.22

0.9640 0.8704 0.9453 0.9570 0.9102

Krosemeyer-Peppas

± ± ± ± ±

0.24 0.32 0.23 0.23 0.29

0.9704 0.9186 0.9515 0.9678 0.9538

n-value ± ± ± ± ±

0.22 0.21 0.25 0.26 0.24

6.867 10.76 11.63 8.045 10.05

± ± ± ± ±

1.21 1.54 1.52 0.93 1.21

Table 7 Order of drug release of capsule formulation determined by the regression coefficients. Formulation code

CUR01 CUR02 CUR03 CUR04 CUR05

Zero order

First order

Higuchi

Hixon-Crowell

Krosemeyer-Peppas

r2

r2

r2

r2

r2

0.9522 0.9403 0.8493 0.9592 0.8641

± ± ± ± ±

0.21 0.25 0.36 0.21 0.39

0.9562 0.9481 0.8503 0.9635 0.8807

± ± ± ± ±

0.27 0.21 0.39 0.23 0.31

0.9860 0.9943 0.9370 0.9876 0.9699

Table 8 Contact angle measurement of SDs prepared by spray drying and rota evaporation technique (n ¼ 3, mean ± SD). Samples

SDs t¼0s

t ¼ 60 s

Neat CUR PM of CUR: EuD(1:1) CUR01 CUR02 CUR03 CUR04 CUR05

70
45
30
25
38
35
20


65
42
28
20
32
30
15


± ± ± ± ± ± ±

2.42 1.23 1.23 1.19 1.67 1.17 1.23

± ± ± ± ± ± ±

2.12 1.11 1.14 1.15 1.43 1.23 1.18

3.7. Moisture uptake study Pure CUR and CUR inside the SD formulation are hygroscopic in nature which is useful to understand the degradation effect of moisture. The SD were heated in the chamber using UV-light source for 3 min at 120
C to determine the moisture content in percentage which was showed on screen. It was hypothesized that the content of moisture absorption is directly proportional to the bulk of hygroscopic surface area on the SD particles. Thus, moisture absorption would be indicative of the intimacy of mixing of CUR with polymer matrix in SD. It also indicates that the extent of complexation and coverage particle surface of drug in SD form. The moisture content in SD was found to be in the range of 0.92 ± 0.23 to 2.24 ± 0.24% which is minute and has no harmful effect on drug efficacy. The drug content analysis carried out after treatment was observed to be in the range of 35.86 ± 0.6 to 45.12 ± 1.2% analyzed by UV spectroscopy at 427 nm.

± ± ± ± ±

0.21 0.17 0.26 0.21 0.24

0.9545 0.9450 0.8496 0.9616 0.8745

± ± ± ± ±

0.25 0.23 0.21 0.23 0.40

0.9623 0.9624 0.8587 0.9708 0.9299

n-value ± ± ± ± ±

0.21 0.22 0.38 0.23 0.24

0.030 0.304 0.283 0.037 2.112

± ± ± ± ±

0.01 0.11 0.28 0.07 0.92

3.8. Solid state characterization 3.8.1. Differential scanning calorimetry (DSC) DSC represent change of thermal behavior of interaction during preparations. DSC was carried out in order to determine whether the crystalline amounts are still detectable or whether drug is completely molten or dissolved in the carrier. DSC thermogram of pure CUR showed an endothermic peak around 179.92
C (DHsf) with enthalpy of fusion being 97.89 J/g which can be attributed to crystal melting point of CUR. While the EuD showed no special endothermic transition peak In the SDs of CUR03 & CUR04 this endotherm was broadened and shifted slightly to lower temperature 168
C which indicate the presence of crystalline CUR as showed in (Fig. 4). The DSC of CUR05 did not exhibit any significant melting peak. The disappearance of the peak suggests that CUR is completely entrapped in the polymer matrix and points to the possibility of significant reduction in drug crystallinity in the polymer matrix. The absence of endothermic peaks in the SDs CUR05 indicated conversion of crystalline CUR to amorphous form. 3.8.2. Powder X-ray powder diffraction (PXRD) The X-ray diffraction pattern of CUR, EuD, SDs and PMs are shown in (Fig. 5). CUR showed distinct peak at 2q equal to 8.96, 14.56, 17.36, 25.64 and a series of small peak at 18.22, 21.22, 23.4, 26.8, 26.98, 27.42, 28.22 and 29.28. The EuD polymer was shown to be an amorphous material due to absence of complete stereo regularity and the presence of bulky side group in the polymer. In PMs, peak which was less intense but at same 2q value was observed. This indicates no interaction of CUR has occurred with polymer. A less intense peak was due to less proportion of CUR in PMs. In SDs of CUR broadening of peak was observed. This indicates

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233

the decreasing crystallinity of CUR and formation of amorphous CUR. A less intense peak was seen in CUR03 and CUR04 showed presence of crystallinity of CUR in polymer. The spray dried SDs with organic solvent showed absence of characteristic peaks of CUR reasserting the conversion of crystalline CUR to amorphous form. This result was supported with DSC measurements.

Fig. 4. DSC thermo gram of (A) Plain CUR, (B) CUR03, (C) CUR04, (D) CUR05, (E) Eudragit EPO.

3.8.3. FT-IR spectroscopy (FTIR) FTIR was used to investigate curcuminepolymer matrix interactions. FTIR spectra of plain and solid dispersions are shown (Fig. 6). The sharp band at 3502.73 cm1 and the broad peak centered at 3300 cm1 in the crystalline CUR spectrum are attributed to OH stretching. In solid dispersions of CUR, this peak broadens considerably with a maximum at around 3400 cm1 and a shoulder at 3500 cm1 suggesting a difference in the molecular environment of the hydroxyl groups in solid dispersions relative to plain CUR. The dispersions also shows broad peaks at 3500e3400 cm1, similar to bands seen in plain CUR. Overall, the band shows higher intensity on the high wavenumber side in the presence of the EuD relative to pure CUR [20]. The significant peaks in EuD have disappeared and

Fig. 5. XRPD diagrams (A) Plain CUR, (B) CUR05, (C) CUR03, (D) CUR04, (E) Eudragit EPO.

Fig. 6. FT-IR spectra of (A) Plain CUR, (B) CUR03, (C) CUR04, (D) CUR05, (E) Eudragit EPO.

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Fig. 7. SEM micrographs of (A) Plain CUR, (B) CUR03, (C) CUR04, (D) & (E) CUR05.

broadened indicating the CUR: EuD interactions. The carbonyl group of the carrier, being a stronger electron donor than the hydroxyl group of the CUR, might be favored in H-bonding resulting in the stability and solubility advantages. In the IR of curcumin, the peak at 1728.22 cm1 is due to the carbonyl functionality present in both the tautomeric forms, whereas the peak at 3383.14 cm1 is due to the hydroxyl group in the enolic form. Both these peaks have disappeared or shifted which is indicative of formation of the Curcumin-Eudragit solid dispersions. 3.8.4. Scanning electron microscopy Scanning electron microscopy (SEM) was used to visualize the particle structural and surface morphology of the spray-dried and rota evaporated powders. SEM of CUR showed flat broken needles of different sizes, with well-developed edges (Fig. 7 A). The representative SEM (Fig. 7B and C) indicated that the spray dried SDs prepared by aqueous solvent are irregular shape particles and the SDs prepared using rota evaporation are irregular shape with rough and hard surface particles. The spray dried SDs prepared using organic solvents are regular spherical particles with a diameter of less than 10 mm (Fig. 7D). The unevenness on the surface may form when the internal pressure rapidly increases while the moisture cannot be released [30]. The morphology of the product obtained from the higher solid concentration and the feed rate (Fig. 7 E) showed the fractured shell and bigger size. Therefore, the solid concentration and feed rate should be carefully selected for producing desirable microspheres. 3.9. Stability of SD The dissolution stability was also evaluated for both initial and

stored samples. Spray dried SDs (CUR05) with organic solvent formulations after storage were analogous to the preliminary formulations and did not show any melting endotherm as shown in the DSC thermogram. This indicated decreased crystallinity of the drug in formulations, indicating the amorphous nature of the CUR. Both DSC and XRD results on aged samples confirmed that there was no recrystallization of the amorphous drug in the SD formulations, suggesting good physical stability. The dissolution profiles of stored samples relative to fresh spray dried formulations further proved that the amorphous state of the drug was maintained in the aged formulations. The enhanced physical stability of the spray dried formulations CUR05 upon storage is attributed to drugepolymer interactions and solubilizing effect of the polymer. The formulations were stable during 6 months period analyzed by UV. The formulations CUR03 and CUR04 after storage were also analogous to the preliminary formulations but they showed broadening of melting endotherm at lower temperature as shown in the DSC thermogram. This indicated presence of crystallinity of CUR in the formulations. The accelerated stability studies showed that there was no considerable change in drug content during study duration. Drug content was found to be almost same as initial i.e. 34e43%. No significant change in dissolution profile observed after stability study. 3.10. Antioxidant assay The free radical scavenging activity of each sample was evaluated through the change of absorbance produced by the reduction of DPPH. Antioxidant DPPH radical scavenging ability shown in (Fig. 8) as decrease in absorbance with concentration of antioxidant

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CUR01

0.7 0.6

Abs. = -0.0525 onc. (CUR05) + 0.5891 R² = 0.9946

0.5 % inhibition

235

CUR02 CUR03 CUR04

0.4

CUR05

0.3 Abs. = -0.0926 conc.(asc. acid) + 0.5914 0.2 R² = 0.9964

Plain CUR Asc. Acid

Abs. = -0.0832 conc. (plain CUR)+ 0.5916 R² = 0.9958

0.1

Linear (CUR05)

0 0

1

2 3 4 Concentration (μg/ml)

5

Linear (Plain CUR) Linear (Asc. Acid)

6

Fig. 8. Reduction of oxidizing ability of DPPH by CUR-SDs (as function of decrease in absorbance).

90 80

% inhibition

70 60

CUR01 (%)

50

CUR02 (%)

40 30

CUR03 (%)

20

CUR04 (%)

10 0

CUR01 (%) CUR02 (%) CUR03 (%) CUR04 (%) CUR05 (%) Pure CUR (%) Asc. Acid (%)

CUR05 (%) 1 2.43 5.91 1.565 5.56 7.3 9.73 11.82

2 12.86 13.73 10.95 13.39 14.96 27.82 30.95

3 20.69 22.08 18.78 21.56 24.17 41.73 46.78

4 33.04 33.73 30.78 33.56 35.65 54.6 59.82

5 40.34 41.04 37.91 40.69 42 68.69 77.91

Pure CUR (%) Asc. Acid (%)

concentration (μg/ml) Fig. 9. % Inhibition of anti-oxidant activity of DPPH by CUR-SDs and ascorbic acid.

at 517 nm. In this study DPPH a stable radical at room temperature accepts an electron or hydrogen radical to become stable DPPH-H molecule. The decrease in absorbance of DPPH radical was caused by blocking this reaction with the radical through donation of hydrogen (Hþ) [1]. This was observed visually as change in color from violet to yellow. CUR-SDs exhibited a comparable antioxidant activity (p˂ 0.5) with ascorbic acid as the standard at various concentrations (1, 2, 3, 4 and 5 mg/ml). Results showed that the CUR-SDs has a significant free radical scavenging activity owning to its hydrogen-donating ability. The comparison of the antioxidant activity of CUR-SDs and ascorbic acid as % inhibition of DPPH scavenging activity (IC50) shown in (Fig. 9). The % inhibition activity of ascorbic acid and CUR-SDs at concentration range of 1e5 mg/ml showed a moderate antiradical activity against DPPH radical. Results suggested that CUR retains its antioxidant activity as solid dispersion complex with EuD.All, formulations showed significant in vitro free radical scavenging activity. 3.11. Molecular modeling interaction studies The structure of CUR exhibits two tautomeric forms, the diketo and keto-enol forms. Hence both these forms were used in docking studies on monomer and dimer forms of EuD, and further in

molecular simulations. The docking study showed the conformation of diketo curcumin gave better binding (in terms of Glide gscores) as compared to the keto-enol form, while the emodel values are almost the same as shown in (Table 9). The energy contributions due to van der Waals forces, coulombic and hydrogen bonding interactions were calculated during docking process and represented in (Table 10). The table shows that highest contribution to the interactions was due to the hydrophobic van der Waals type of interactions, in both diketo and keto-enol form. Lesser magnitude of the coulombic interactions implies lesser contributions from the electrostatic interactions in keto-enol form as compared to diketo form. The diketo form showed a higher hydrogen bonding propensity compared to ketoenol form. The docking and MD simulation studies showed that hydrophobic interactions were important in the binding of CUR with EuD Table 9 Docking study scores of diketo and keto-enol conformation for monomer and dimer. Ligand

Glide g-score

Glide emodel

CUR-Diketo with EuD monomer CUR-Keto-enol with EuD monomer CUR-Diketo with EuD dimer CUR-Keto-enol with EuD dimer

1.467 1.064 1.958 1.784

21.821 22.467 21.031 19.483

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Table 10 Calculation of energy contribution due to van der Waals forces, coulombic and hydrogen bonding interactions. Ligand

van der Waals contribution

Coulombic contribution

Hydrogen bonding contribution

CUR-Diketo with EuD monomer CUR-Keto-enol with EuD monomer CUR-Diketo with EuD dimer CUR-Keto-enol with EuD dimer

18.050 19.372 16.612 17.842

1.942 0.257 1.503 2.123

1.000 0.000 1.903 1.882

Fig. 10. Various dock poses of Curcumin on EuD. A. Dock-pose of diketo form of CUR on monomer B. Dock-pose of keto-enol form of CUR on monomer C. Dock-pose of diketo form of CUR on dimer D. Dock-pose of diketo form of CUR on dimer (Thick tubes represent monomer unit of polymer, and thin tubes represent CUR; pink dotted lines indicate most important interactions, and the distances are mentioned in Å units).

in both the tautomeric forms of CUR and hydrogen bonding was important for the diketo form in comparison to the keto-enol form as shown in (Figs. 10A and B). The stable lowest energy complexes obtained from docking studies were minimized and subjected to MD simulations. MD simulations concluded that hydrogen bonding interactions were not important in the binding of drug with polymer with only 3% and 2% hydrogen bonding propensity observed for the diketo and keto-enol form CUR with EuD respectively as depicted in (Fig. 11). The insilico studies are in line with the FTIR spectra, where the carbonyl group of CUR were not found to be involved in the interactions. Similarly the terminal phenolic groups were seen to be involved in the binding as indicated by the shift in FTIR spectra. Overall, the drugepolymer combination demonstrates strong hydrophobic interactions along with lesser contribution from hydrogen bonding interactions. Thus it can be concluded that the increase in the solubility and dissolution rate can be attributed to the use of hydrophilic polymer and the technique used for preparing the SDs. 4. Conclusion

Fig. 11. 2D representation of structures of A. diketo form of curcumin on monomer showing hydrogen bonding of phenolic OH group for 3% of the simulation time. B. keto-enol form of curcumin on monomer shows hydrogen bonding interaction of enolic OH for 2% of the total simulation time. UNK900 indicates the polymer Eudragit EPO.

EuD showed a significant increase in solubility and in-vitro release performance of CUR by spray drying and rota evaporation technique. The solubility and dissolution rate of CUR prepared by rota evaporation technique (solubility 224.12e459.46 mg/ml &

dissolution 8.00e18.00% in 2.00 h) was found to be low as compared to SDs prepared by spray drying technique (solubility 412.32e730.78 mg/ml & 32.00e46.00% in 2.00 h). A molecular

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complex of CUR was formed with polymer, which significantly enhanced its acidic pH and aqueous solubility. The insilico studies were in line with the FTIR spectra, showing the involvement of the terminal phenolic groups in the binding with polymer. Overall, the drugepolymer combination demonstrates strong hydrophobic interactions along with lesser contribution from hydrogen bonding interactions, and it can be concluded that the increase in the solubility and dissolution rates of CUR can be attributed to the use of hydrophilic polymer and to the technique used for preparing the SDs. Acknowledgment This article does not contain any studies with human and animal subjects performed by any of the authors. All authors (AB Gangurde, HS Kundaikar, SD Javeer, DR. Jaiswar, MS Degani and PD. Amin) declare that they have no conflict of interest. The authors are thankful to Patel Phyto Extractions Pvt Ltd., India for providing the gift sample of Curcumin. The authors are also thankful to UGC-SAP2801-PH for providing the research fellowship. References [1] M. Abayomi, A.S. Adebayo, D. Bennett, R. Porter, J. Shelly-Campbell, In vitro antioxidant activity of Bixa orellana (Annatto) Seed Extract, J. App. Pharm. Sci. 4 (2014). [2] M.A. Alam, R. Ali, F.I. Al-Jenoobi, A.M. Al-Mohizea, Solid dispersions: a strategy for poorly aqueous soluble drugs and technology updates, Expert Opin. Drug. Deliv. 9 (2012) 1419e1440. [3] A. Almeida, S. Possemiers, M. Boone, T. De Beer, T. Quinten, L. Van Hoorebeke, J.P. Remon, C. Vervaet, Ethylene vinyl acetate as matrix for oral sustained release dosage forms produced via hot-melt extrusion, Eur. J. Pharm. Biopharm. 77 (2011) 297e305. [4] P. Anand, A.B. Kunnumakkara, R.A. Newman, B.B. Aggarwal, Bioavailability of curcumin: problems and promises, Mol. Pharm. 4 (2007) 807e818. [5] G.P. Andrews, O. Abu-Diak, F. Kusmanto, P. Hornsby, Z. Hui, D.S. Jones, Physicochemical characterization and drug-release properties of celecoxib hot-melt extruded glass solutions, J. Pharm. Pharmacol. 62 (2010) 1580e1590. [6] R. Araujo, C. Teixeira, L. Freitas, The preparation of ternary solid dispersions of an herbal drug via spray drying of liquid feed, Dry. Techno. 28 (2010) 412e421. [7] P. Basnet, H. Hussain, I. Tho, N. Skalko-Basnet, Liposomal delivery system enhances anti-inflammatory properties of curcumin, J. Pharm. Sci. 101 (2012) 598e609. [8] R. Chang, L. Sun, T.J. Webster, Short communication: selective cytotoxicity of curcumin on osteosarcoma cells compared to healthy osteoblasts, Int. J. Nanomedicine 9 (2014) 461. [9] A.M. Chuah, B. Jacob, Z. Jie, S. Ramesh, S. Mandal, J.K. Puthan, S. Shreeram, Enhanced bioavailability and bioefficacy of an amorphous solid dispersion of curcumin, Food Chem. 156 (2014) 227e233. [10] M.M. Crowley, F. Zhang, M.A. Repka, S. Thumma, S.B. Upadhye, S. Kumar Battu, J.W. McGinity, C. Martin, Pharmaceutical applications of hot-melt extrusion: part I, Drug Dev. Ind. Pharm. 33 (2007) 909e926.

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