Oral delivery of quercetin in oil-in-water nanoemulsion: In vitro characterization and in vivo anti-obesity efficacy in mice

Journal of Functional Foods 38 (2017) 571–581

Contents lists available at ScienceDirect

Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff

Oral delivery of quercetin in oil-in-water nanoemulsion: In vitro characterization and in vivo anti-obesity efficacy in mice Rudra Pangeni 1, Si-Won Kang 1, Minho Oak, Eun Young Park ⇑, Jin Woo Park ⇑ Department of Pharmacy, College of Pharmacy and Natural Medicine Research Institute, Mokpo National University, Muan-gun, Jeonnam 58554, Republic of Korea

a r t i c l e

i n f o

Article history: Received 2 July 2017 Received in revised form 17 September 2017 Accepted 26 September 2017

Keywords: Quercetin Obesity Adiposity Nanoemulsion Oral delivery Oral absorption

a b s t r a c t We sought to design an effective oral delivery system for quercetin (QCN) to enhance its solubility and bioavailability and thus improve its anti-obesity effects. QCN-loaded oil-in-water nanoemulsion was prepared by an aqueous phase titration method. The optimized formulation had a mean particle size of 19.3 ± 0.17 nm with a zeta potential of
0.34 ± 0.13 mV. In vitro permeabilities of QCN from the nanoemulsion through an artificial intestinal membrane and Caco-2 cell monolayer were 188- and 3.37-fold higher than those of an aqueous dispersion of QCN, respectively, and the resulting in vivo oral bioavailability was 33.51-fold greater than that of free QCN. Furthermore, high-fat-diet (HFD)-treated mice given daily the oral QCN-loaded nanoemulsion had a maximal reduced weight gain of 23.5% compared with the HFD control group. These findings suggest that a QCN-loaded nanoemulsion may be a promising oral therapy for the treatment of obesity. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4Hchromen-4-one, QCN) is one of the most potent dietary flavonoids, found commonly in onions, apples, berries, many nuts, seeds, barks, flowers, tea, brassica vegetables, and leaves (Albishi, John, Al-Khalifa, & Shahidi, 2013; Mukhopadhyay & Prajapati, 2015). It has attracted attention due to its diverse biological activities, Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; AUCinf, area under the plasma concentration-time curve from zero to infinity; AUClast, area under the plasma concentration-time curve from zero to the time of the last measurable plasma concentration; Cmax, maximum plasma concentration; GI, gastrointestinal; GLUT, glucose transporter; H&E, hematoxylin and eosin; HFD, high-fat-diet; HLB, hydrophilic-lipophilic balance; HPLC, high-performance liquid chromatography; LC/MS, liquid chromatography/tandem mass spectrometry; NaCMC, sodium carboxymethyl cellulose; NC, normal chow; O/W, oil-in-water; PAMPA, parallel artificial membrane permeability assay; Papp, apparent permeability coefficient; PDI, polydispersity index; Pe, effective permeability coefficient; PO, per oral; QCN, quercetin; SLNs, solid lipid nanoparticles; Smix, surfactant and cosurfactant combination; SNEDDS, self-nanoemulsifying drug delivery systems; T½, half-life of plasma concentration; TEM, transmission electron microscopy; Tmax, time to reach maximum plasma concentration. ⇑ Corresponding authors at: Department of Pharmacy, College of Pharmacy, Mokpo National University, 1666 Youngsan-ro, Muan-gun, Jeonnam 58554, Republic of Korea. E-mail addresses: [email protected] (E.Y. Park), [email protected] (J.W. Park). 1 These authors are equally contributed as first authors to this work. https://doi.org/10.1016/j.jff.2017.09.059 1756-4646/Ó 2017 Elsevier Ltd. All rights reserved.

including anti-cancer, anti-inflammatory, anti-ulcer, anti-allergic, wound-healing, and cataract-preventing effects (Bronner & Landry, 1985; Kaul, Middleton, & Ogra, 1985; Mukhopadhyay & Prajapati, 2015). Furthermore, QCN has been reported to inhibit lipid peroxidation, platelet aggregation, and capillary permeability due to its potent anti-oxidant activity; it can directly scavenge free radicals and inhibit xanthine oxidase, which alters anti-oxidant defenses and inhibits lipid peroxidation (Erlund, 2004; Formica & Regelson, 1995; Mukhopadhyay & Prajapati, 2015). Several studies have also demonstrated its anti-diabetic activity in the treatment of diabetes and related complications (Bhatt & Flora, 2009; Coskun, Kanter, Korkmaz, & Oter, 2005). QCN reportedly lowers the activities of maltase and the glucose transporter 2 (GLUT 2) in the small intestine, accelerates the function of GLUT 4 and insulin receptors in muscles, resulting in increasing glucose uptake, increases glucokinase activity in liver, and offers a protective effect against b-cell damage (Rifaai, El-Tahawy, Saber, & Ahmed, 2012). QCN enhanced insulin resistance, glucose tolerance, and lipid metabolism in a high-fat-diet (HFD)-induced obesity model (Panchal, Poudyal, & Brown, 2012). Moreover, QCN exhibited an anti-obesity effect, increasing energy expenditure and decreasing inflammation (Pisonero-Vaquero et al., 2015). However, the potential therapeutic applications of QCN in the pharmaceutical field have been limited by its low oral bioavailability (<2% in human) (Cai, Fang, Dou, Yu, & Zhai, 2013). The major

572

R. Pangeni et al. / Journal of Functional Foods 38 (2017) 571–581

reasons for this include its poor solubility in water, with a solubility of 1 mg/mL, and instability in physiological fluid, poor permeability through the intestine, extensive first-pass metabolism before reaching the systemic circulation, and rapid elimination following oral administration (Li et al., 2009; Manach, Scalbert, Morand, Rémésy, & Jiménez, 2004). To overcome these limitations, various delivery strategies have been suggested to improve its oral bioavailability: site-specific delivery via release in the lower intestinal and colon parts to avoid metabolism, prolongation of drug absorption using sustained release or mucoadhesive formulations, and enhancing drug solubility or permeability using nanocarriers or micelles, emulsions, and drug-polymer conjugates (Cai et al., 2013; Mukhopadhyay & Prajapati, 2015). In this regard, various formulations have been investigated, incorporating QCN into polymeric carriers, including hydrogels, micro or nanoparticles, nanoemulsions, lecithin-based nanocarriers, solid lipid nanoparticles (SLNs), QCN-cyclodextrin complexes, and micelles, using natural or synthetic polymers (Caddeo et al., 2014; Gao et al., 2009; Kale, Saraf, Juvekar, & Tayade, 2006; Li et al., 2009; Zhao et al., 2011). Among these, self-nanoemulsifying drug delivery systems (SNEDDS), a low-energy approach relying on the spontaneous formation of emulsions based on the phase behavior of certain oil, surfactant, co-surfactant, and water systems, have been examined to improve the dissolution and/or oral bioavailability of lipophilic flavonoids by protecting them from enzymatic degradation in the gastrointestinal (GI) tract and increasing their solubility in the GI fluid and permeability through the intestinal wall (Fofaria, Qhattal, Liu, & Srivastava, 2016; Vecchione et al., 2016). Such self-nanoemulsifying systems have been shown to cross enterocytes readily via transcellular transport after reaching the apical membrane of the intestinal cells, resulting in entrance into lymphatic vessels or the bloodstream via the process of exocytosis (Trevaskis, Charman, & Porter, 2008). Moreover, particles with diameters less than 500 nm have been reported to be internalized readily through both caveola and clathrin-mediated endocytosis (Lu, Liu, Wang, & Li, 2015). However, in the SNEDDS, the aqueous phase should be as the external phase, so that it can be diluted easily with GI fluid without phase separation or drug precipitation (Shakeel, Haqa, Al-Dhfyan, Alanazi, & Alsarra, 2014). Thus, an exquisite formulation design is required to obtain colloidal stability with high drug loading and minimum use of surfactant and/or co-surfactant, which potentially raise the safety risk of the drug delivery system, especially with long-term use. Currently, no oral formulation of QCN has been studied for an anti-obesity effect. Thus, the objectives of this study were to design an efficient oral delivery system for QCN based on an oil-in-water (o/w) nanoemulsion and to evaluate its activity in reducing body fat. To achieve this, we constructed QCN-loaded o/w nanoemulsions using titration; we characterized the droplet size by transmission electron microscopy (TEM) and dynamic laser light scattering. We found an optimum formulation for a high QCNloaded nanoemulsion system by assessing in vitro permeability of QCN across an artificial intestinal membrane and a Caco-2 cell monolayer. Finally, oral bioavailability in rats and anti-obesity efficacy of the nanoemulsion in HFD-treated mice were evaluated following oral administration.

2. Materials and methods 2.1. Materials Quercetin (purity > 95%), castor oil, oleic acid, polyethylene glycol 400 (PEG 400), polyoxyethylene (20) sorbitan monolaurate (Tween 20), polyoxyethylene (80) sorbitan monolaurate (Tween

80), sorbitan monolaurate (Span 80), sodium carboxymethyl cellulose (NaCMC), and baicalin (internal standard) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Caprylocaproyl macrogol-8-glycerides (Labrasol), diethylene glycol monoethyl ether (Transcutol HP), oleoyl polyoxylglycerides (Labrafil M 1944 CS), propylene glycol monocaprylate (Capryol 90), propylene glycol dicaprylocaprate (Labrafac PG), and polyglyceryl-6-dioleate (Plurol Oleique CC) were obtained from Gattefossé (Saint Priest, France). Polyethoxylated castor oil (Cremophor EL) was provided by BASF (Ludwigshafen, Germany). Solvents for high-performance liquid chromatography (HPLC) and liquid chromatography/tandem mass spectrometry (LC/MS) analyses were obtained from Merck and Thermo Fisher Scientific (Waltham, MA, USA). 2.2. Animals Sprague Dawley rats (males, 200–250 g) and C57BL6 mice (males, 20–25 g) were purchased from Orient Bio (KyeongGi-do, Republic of Korea). The animals were housed under standard housing conditions of temperature (23 ± 2 °C), relative humidity (55 ± 10%), and light (12/12-h light/dark cycle). The animals had ad libitum access to a standard laboratory diet (Nestlé Purina, St. Louis, MO, USA) and ion-sterilized tap water. Ethical approval for this study was obtained from the Institutional Animal Care and Use Committee (IACUC) of Mokpo National University (Jeonnam, Republic of Korea). All animal experiments were performed in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals and the guidelines of the IACUC. 2.3. Preparation and characterization of o/w nanoemulsions 2.3.1. Selection of components for o/w nanoemulsions The solubility of poorly soluble drugs is the most important criterion for the screening of components of nanoemulsions. To determine the solubility of QCN in different oils, surfactants or cosurfactants, an excess amount of QCN was added to 1 g of oils (castor oil, Capryol 90, oleic acid, Labrafil M 1944 CS, and Labrafac PG), surfactants (Tween 20, Tween 80, Labrasol, and Span 80), and cosurfactants (Transcutol HP, Cremophor EL, PEG 400, and Plurol Oleique CC) in stoppered glass vials. Mixtures of QCN and excipients were first vortexed and then held at 25 ± 1.0 °C in an isothermal shaker for 48 h to allow attainment of equilibrium. The resulting mixtures were centrifuged (6700  g, 15 min) and the supernatants filtered through 0.45-mm-pore-diameter membrane filters (Parmar, Singla, Amin, & Kohli, 2011). The concentration of QCN in the filtrates was determined using a microplate reader (PerkinElmer multimode plate reader; PerkinElmer, Waltham, MA, USA) at 370 nm. The oils, surfactants, and co-surfactants with higher solubility were selected and analyzed for miscibility studies. Furthermore, 2 mL of selected excipients were mixed with each another, vortexed for 10 min and left to equilibrate for at least 30 min. The resulting mixture was analyzed visually for transparency and phase separation, and used as a measure to evaluate the miscibility of excipients (Gao et al., 2009). 2.3.2. Construction of pseudo-ternary phase diagrams and selection of nanoemulsions Selection of components was based on solubility studies of QCN and miscibility of the excipients. Pseudo-ternary phase diagrams were constructed by an aqueous phase titration using combinations of Capryol 90 and Labrafil M 1944 CS (1:1, w/w), Labrasol and Tween 80 (1:1, w/w), Cremophor EL and PEG 400 (1:1, w/w), and deionized water as the oil phase, surfactants, co-surfactants, and aqueous phase, respectively, at room temperature. First, surfactant and co-surfactant combination (Smix) was blended in different weight ratios, such as 3:1, 2:1, 1:1, 1:2, and 1:3, based on

R. Pangeni et al. / Journal of Functional Foods 38 (2017) 571–581

increasing concentration of co-surfactant with respect to surfactant. Second, for each phase diagram, oil and specific Smix ratio were mixed thoroughly in different weight ratios, from 1:9 to 9:1, in different tubes, followed by slow addition of the aqueous phase. In the pseudo-ternary diagram, the o/w nanoemulsion was identified as the region where clear and transparent droplets were obtained, based on visual observation (Pangeni, Sharma, Mustafa, Ali, & Baboota, 2014). Phase diagrams were also constructed in the presence of QCN to study the effects of the drug on the nanoemulsion boundary. Next, several nanoemulsions were selected from the clear zone of each phase diagram, and the criterion for selection was the oil and surfactant concentration that solubilized the required dose of QCN (20 mg) with no precipitation. Combinations with a minimum concentration of Smix and maximum concentration of water were selected to form stable nanoemulsions. Additionally, the influence of change in the concentration of oil, surfactants, and co-surfactants on the nanoemulsion was studied. Finally, selected nanoemulsions were subjected to physicochemical characterization and in vitro intestinal membrane permeability studies. 2.3.3. Characterization of the o/w nanoemulsion system QCN loaded o/w nanoemulsions with different combinations of oil (16.67–25.00%), Smix (20.00–50.00%), and deionized water (33.33–63.33%) were characterized for average droplet size, polydispersity index (PDI), and zeta potential, with a dynamic laser light scattering analyzer (Malvern Zetasizer Nano ZS90; Malvern Instruments, Malvern, UK). Each QCN-loaded nanoemulsion was dispersed in deionized water (1:50), followed by sonication for 1 min, to minimize multiple scattering effects, and measured at 25 °C. The surface morphology, structure, and droplet size of the selected QCN nanoemulsion was confirmed using high-resolution TEM. A drop of QCN-loaded o/w nanoemulsion diluted in water (1:100) was deposited onto a film-coated copper grid and stained with a drop of 2% aqueous solution of phosphotungstic acid to enhance contrast and allow negative staining. The dried grid was observed under a TEM (JEM-200; JEOL, Tokyo, Japan) (Pangeni, Choi, Jeon, Byun, & Park, 2016). 2.4. In vitro permeation studies through a parallel artificial intestinal membrane The parallel artificial membrane permeability assay (PAMPA) is a method for predictive measurements of passive intestinal absorption, which involves a phospholipid-coated lipophilic membrane that simulates the intestinal wall. PAMPA (BD Biosciences, San Jose, CA, USA) was performed for o/w nanoemulsions containing QCN to evaluate their intestinal membrane permeability. A 200-mL aqueous QCN dispersion or nanoemulsions diluted to 400 mg/mL of QCN with phosphate-buffered saline (PBS, pH 6.8) was added to each well of the donor plate and 300 mL of PBS (pH 6.8) was added to each well of the acceptor plate. The donor plate was then coupled with the acceptor plate, ensuring the donor membrane was in contact with the buffer in the acceptor plate. The plate was covered and incubated at room temperature for 5 h (Opatrilova, Cernikova, Coufalova, Dohnal, & Jampilek, 2013) and QCN that permeated to the acceptor plate was measured using a HPLC system. A C18 column (4.6  150 mm, 5 mm, 100 Å, 20 mL sample injection) was used at 35 °C with water (2% acetic acid, pH 2.6)-acetonitrile (40:60, v/v) as the mobile phase, at a flow rate of 1 mL/min. QCN was measured using a UV detector at 370 nm. The effective permeability coefficient (Pe) of QCN for each o/w nanoemulsion was calculated using the following equations:

C equilibrium ¼ ½C D ðtÞ  V D þ C A ðtÞ  V A =ðV D þ V A Þ

ð1Þ

Pe ¼
ln½1
C A ðtÞ=C equilibrium =½A  ð1=V D þ 1=V A Þ  t

573

ð2Þ

where Cequilibrium is the concentration of drug at the theoretical equilibrium, t the total time of incubation during the assay in seconds (s), CD(t) the concentration of drug in the donor well at time t, VD the volume of the donor well (0.2 mL), CA(t) the concentration of drug in the acceptor well at time t, VA the volume of the acceptor well (0.3 mL), Pe the effective permeability (cm/s), and A the effective filter area (0.228 cm2). 2.5. In vitro permeation studies through a Caco-2 cell monolayer The permeability of QCN nanoemulsions was also investigated across a Caco-2 cell monolayer. Caco-2 cells were seeded onto 12-well Transwell filter inserts (pore size 0.4 lm, surface area 1.12 cm2; Corning, NY, USA) at a density of 3  105 cells/well, and grown at 37 °C in an atmosphere of 5% CO2. The culture medium, Dulbecco’s modified Eagle’s medium (DMEM; Lonza, Basel, Switzerland) containing 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific) and 1% penicillin/streptomycin (Gibco), was changed every 48 h for 21–29 days. Inserts with transepithelial electrical resistance (TEER) of >350 X cm2 were selected for transport experiments. Initially, the inserts were pre-incubated with 1.5 mL of Hanks’ balanced salt solution (HBSS) for 20 min at 37 °C and then 0.5 mL of 100 mM of aqueous dispersion of QCN or QCN in 0.3% NaCMC or nanoemulsion E diluted with HBSS, and 1.5 mL HBSS was added to each apical and basolateral compartment, respectively (Walgren, Walle, & Walle, 1998). Transwell plates were incubated further at 37 °C and QCN permeation was evaluated at 0.5, 1, 2, 3, 4, and 5 h. Withdrawn samples were immediately combined with same volume of 10 mM acetic acid to acidify and stabilize the QCN in aqueous solution. The samples were filtered through a membrane filter (0.45 lm, PVDF) and stored at
20 °C until analysis. Then, 100 mL of thawed sample was mixed sequentially with 20 mL internal standard (IS, 12.5 mg/ mL baicalin) and 2 mL ethyl acetate, and vortexed for 2 min. The organic phase was evaporated to dryness in a glass vial using a centrifugal evaporator (Genevac Ltd., Ipswich, UK). The residue obtained was dissolved in 100 mL of methanol and vortexed for 4 min. The concentration of QCN permeating though the monolayer was determined using a LC/MS system. Chromatographic separation was achieved on a C18 column (4.6  150 mm, 5 mm, 100 Å) at 30 °C using acetonitrile–water (0.1% formic acid) (40:60 v/v) as the mobile phase, delivered at 0.8 mL/min. Ionization of QCN and IS were performed using ESI source in negative mode with selected ion monitoring (SIM) under the following conditions: capillary voltage ±3500 V, drying gas flow 3.1 mL/min, and drying gas temperature 300 °C. Quantitative analysis was performed at m/z 301.3 for QCN and m/z 445.4 for baicalin. Under optimized LC/MS conditions, a calibration curve was constructed from a blank HBSS with an IS and seven calibrators covering the entire range (10–10,000 ng/mL), including the lower limit of quantitation (LLOQ, 50 ng/mL). The calibration curves were linear over the concentration range of 50–10,000 ng/mL (r2 > 0.999). The retention times of QCN and IS were about 6.2 and 4.7 min, respectively. The extraction efficiency from HBSS was >90.1% and 87.5% for QCN and IS, respectively. The intraday accuracy for QCN was 94.7–105.3%, with a precision of 2.3–4.3%. The interday accuracy for QCN was 93.5–106.7%, with a precision of 3.2–5.7%. The apical-to-basolateral apparent permeability coefficient (Papp) of QCN was calculated using the following equation:

Papp ¼ dQ =dt  1=ðA  C 0 Þ

ð3Þ

where Papp is the apparent permeability (cm/s), dQ/dt the rate of linear appearance of mass on the apical side (mmoL/s), C0 the initial

574

R. Pangeni et al. / Journal of Functional Foods 38 (2017) 571–581

concentration of QCN on the apical side (mmoL/mL), and A the surface area of the monolayer (cm2). 2.6. In vitro dissolution study Dissolution tests were performed in 500 mL of medium containing 0.1 N HCl solution (pH 1.2) or PBS (pH 6.8) at 37 °C ± 0.2 °C using a USP type 1 apparatus (basket) rotating at 100 rpm. First, 20 mg of QCN in 800 mg of 0.3% NaCMC or nanoemulsion E, comprising oil and Smix, were filled in a hard gelatin capsule, size 00. Each capsule was subjected to a dissolution test, and 1-mL samples were withdrawn at 15, 30, 45, 60, 90, and 120 min. After filtration, the amount of QCN in the samples was quantified by HPLC using a UV detector, as described in Section 2.4. 2.7. In vivo oral absorption in rats The improvement in intestinal absorption of QCN from the nanoemulsion was evaluated by oral administration to rats. Each rat was orally administered 400 lL of aqueous dispersion of QCN in 0.3% NaCMC or 400 lL of nanoemulsion E diluted with water to comprise QCN (40 mg/kg). Further, 200-mL blood samples were collected from a capillary in the retro-orbital plexus at predetermined time intervals, placed into heparinized tubes, and immediately centrifuged (2500  g, 15 min, 4 °C). The plasma was separated and kept frozen at
70 °C until analysis. The QCN concentration in plasma was determined by liquid phase extraction; 100 mL of each standard and plasma samples were mixed with 100 mL of 2 moL/mL hydrochloric acid and vortexed for 2 min. Then, the samples were hydrolyzed for 30 min in a water bath at 80 °C. After hydrolysis, the samples were cooled at room temperature and 20 mL IS (1 mg/mL) was added sequentially with 2 mL ethyl acetate and vortexed for 4 min. Furthermore, the samples were centrifuged (3300  g, 10 min, 4 °C). The organic phase was transferred to a clean glass vial and evaporated to dryness using a centrifugal evaporator (Genevac Ltd.). The residue was reconstituted with 100 mL of methanol and vortexed for 1 min. Finally, the samples were subjected to LC/MS system, as described in Section 2.5. Under the LC/MS conditions, a calibration curve was prepared using blank plasma with an IS and seven calibrators covering the entire range (10–10,000 ng/mL), including the LLOQ (50 ng/mL). The calibration curves were linear over the concentration range of 50–10,000 ng/mL (r2 > 0.998). The retention times of QCN and IS were about 6.2 and 4.7 min, respectively. The extraction efficiency from rat plasma was >85.6% and 84.3% for QCN and IS, respectively. The intraday accuracy for QCN was 93.8– 106.2%, with a precision of 3.5–5.2%. The interday accuracy for QCN was 92.2–107.8%, with a precision of 4.2–6.7%. 2.8. In vivo anti-obesity effect of QCN-loaded nanoemulsion At 6 weeks of age, mice were provided with either a HFD (60% fat) or normal chow (5.4% fat). After 1 week, mice were randomly divided into four groups (n = 10 each): the normal chow group (NC), the PBS (pH 7.4)-treated HFD group (PBS-HFD), the QCN dispersed in 0.3% NaCMC-treated HFD group (Q-HFD), and the QCNloaded nanoemulsion E-treated HFD group (Q-NE-HFD). First, 150 lL of the QCN dispersed in 0.3% NaCMC or QCN-loaded nanoemulsion E diluted with PBS (pH 7.4) to comprise QCN (150 mg/kg) was given by oral gavage, daily, for 10 weeks. The PBS-HFD group was given the same volume of PBS by oral gavage. Body weight and food consumption were measured weekly. At the end of 10 weeks of treatment, animals were sacrificed and fat pads (epididymal, subcutaneous, and perirenal) were collected. At the same time, blood samples were collected from the orbital sinus under anesthesia after 3 h of food deprivation and centrifuged at

(3000  g, 20 min). Then, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using a Beckman Coulter AU 480 (Beckman Coulter, CA, USA). Liver tissues were also isolated and fixed in 10% neutralbuffered formalin after 10 weeks of administration. The liver tissues were then embedded in paraffin wax, sectioned at a thickness of 4 mm, and stained with hematoxylin and eosin (H&E) for histological evaluation. 2.9. Pharmacokinetic and statistical analyses Pharmacokinetic parameters were estimated using a nonÒ compartmental method with the WinNonlin software (ver. 5.3; Pharsight Corporation, Mountain View, CA, USA). All data are expressed as means ± standard deviations. A p value of <0.05 was considered to indicate statistical significance, using a t-test between two mean values for unpaired data or one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test among more than three mean values for unpaired data. 3. Results and discussion 3.1. Preparation and characterization of QCN-loaded o/w nanoemulsion Appropriate selection of oil, surfactant, and co-surfactant has a strong influence on maximizing solubility, achieving high drug loading, and avoiding precipitation of drug throughout the shelf life of a nanoemulsion. Thus, solubility studies of QCN were carried out to identify suitable excipients for the development of the QCNloaded nanoemulsion. Moreover, all excipients selected for the nanoemulsion were among the ‘generally recognized as safe’ (GRAS) category. The solubility of QCN in various surfactant oils is shown in Supplementary Fig. 1A. QCN had high solubility in Capryol 90 (23.7 ± 1.47 mg/g), followed by castor oil (17.3 ± 1.03 mg/g), and Labrafil M 1944 CS (4.69 ± 0.26 mg/g). However, a combination of Capryol 90 and Labrafil M 1944 CS (1:1, w/w) was selected as the oil phase, based on comparable solubility (13.1 ± 1.58 mg/g) and good miscibility with the selected surfactants and co-surfactants. The solubilities of QCN in various surfactants, co-surfactants, and combinations are also shown in Supplementary Fig. 1A. QCN had high solubility in most of the tested surfactants, such as Labrasol (138 ± 6.84 mg/g), Tween 80 (110 ± 6.12 mg/g), and Tween 20 (83.3 ± 3.69 mg/g), but not Span 80 (5.56 ± 0.31 mg/g), as well as co-surfactants, such as Transcutol HP (150 ± 2.86 mg/g), PEG 400 (149 ± 1.76 mg/g), and Cremophor EL (105 ± 1.5 mg/g), but not Plurol Oleique CC (10.1 ± 0.69 mg/g). Moreover, combinations of Labrasol and Tween 80 (1:1, w/w) as the surfactant, and Cremophor EL and PEG 400 (1:1, w/w) as the co-surfactant, yielded higher solubility, 126 ± 7.94 mg/g and 120 ± 7.17 mg/g, respectively, with good miscibility with oil. The combinations that provided the best solubility with appropriate miscibility were further assessed to develop pseudo-ternary phase diagrams. In this study, semi-synthetic oils, such as Capryol 90 and Labrafil M 1944 CS, which exhibited relatively higher emulsification efficiency, and nonionic surfactants, like Labrasol, Tween 80, and PEG 400, which are less irritating and less cytotoxic than ionic surfactants, unaffected by changes in pH, and have a lower critical micellar concentration, were used to form a stable nanoemulsion (Borhade, Pathak, Sharma, & Patravale, 2012; Czajkowska-Kosnik, Szekalska, Amelian, Szymanska, & Winnicka, 2015; Parmar et al., 2011). Moreover, Labrasol, Cremophor EL, and Tween 80, with a high hydrophilic-lipophilic balance (HLB) value of 14, have been reported to enhance the intestinal absorption of drugs, and were

R. Pangeni et al. / Journal of Functional Foods 38 (2017) 571–581

thus selected for the nanoemulsion formulation (Tran, Guo, Song, Bruno, & Lu, 2014). However, a study investigating different solubilizing agents found that Cremophor EL may cause intestinal membrane toxicity, with no absorption-promoting effect (Hamid, Katsumi, Sakane, & Yamamoto, 2009). Thus, the use of Chremophor EL was minimized and further studies are required of it. Pseudo-ternary phase diagrams for o/w nanoemulsion were constructed with the objective of identifying nanoemulsifying regions, optimizing the concentrations of the oil, surfactant, and co-surfactant, and investigating the relationship between phase behavior and components of the nanoemulsion. The phase diagrams for o/w nanoemulsion were studied for three different Smix ratios (3:1, 1:1, and 1:3) containing Capryol 90:Labrafil M1944 CS (1:1, w/w) as the oil, Labrasol:Tween 80 (1:1, w/w) as the surfactant, and Cremophor EL:PEG 400 (1:1, w/w) as the cosurfactant. Moreover, combinations of surfactant and cosurfactant play an important role in reducing the interfacial energy and formation of a mechanical barrier to coalescence, respectively (Mahdi et al., 2011). In Supplementary Fig. 1B–D, the area of isotropic nanoemulsion differed with each Smix ratio. In the Smix ratio 3:1, when the concentration of surfactant was higher than the cosurfactant, the area for nanoemulsion was high compared with Smix 1:1 and Smix 1:3, indicating that the emulsification of the oil phase by Smix 3:1 was higher. This may be attributed to the fact that a higher concentration of surfactant combination with high HLB values has high emulsification efficiency, leading to greater penetration of the oil phase in the hydrophobic region of the surfactant monomers, thereby reducing the interfacial tension and ultimately improving the thermodynamic stability of the nanoemulsion (Gao et al., 2009; Mahdi et al., 2011). The maximum concentration of the oil phase that could be emulsified in the aqueous phase was 29.5% with the addition of 25% Smix 3:1. Moreover, when the concentration of surfactant was decreased, with an increase in the cosurfactant concentration (Smix 1:3), there was small decrease in nanoemulsion area, indicating that no further increase in cosurfactant concentration and no more emulsification occurred. The phase diagram for Smix 1:3 showed that a maximum concentration of 20% oil phase could be emulsified in the aqueous phase by addition of 20% Smix 1:3. In this study, the results showed that the Gibbs free energy of the nanoemulsion formation might depend on the extent to which the Smix lowers the interfacial surface tension of the o/w and changes the dispersion entropy, resulting in a thermodynamically stable o/w nanoemulsion (Bali, Ali, & Ali, 2010). Additionally, the effects of surfactant, co-surfactant, and oil on nanoemulsifying area can be observed readily. Thus, from each phase diagram, several nanoemulsions that were clear, transparent, and stable with differing concentrations of surfactant and co-surfactant were selected and studied further for particle size, PDI, and in vitro membrane permeability (data not shown). Moreover, the effects of change in concentration of surfactant and co-surfactant (oil and aqueous phase kept constant) on particle size, PDI, and zeta potential were studied. Finally, the optimum o/ w nanoemulsion was composed of 16.67% Capryol 90:Labrafil M 1944 CS (1:1, w/w) as the oil phase, 37.50% mixture of Labrasol and Tween 80 (1:1, w/w) as the surfactant, 12.50% Cremophor EL:PEG 400 (1:1, w/w) as the co-surfactant, and 33.33% as the aqueous phase. The selected nanoemulsion prepared by a conventional lowenergy emulsification technique was transparent and monophasic, and was characterized in terms of droplet size, PDI, and zeta potential (Table 1 and Supplementary Table 1). The average droplet sizes of all nanoemulsion samples were in the range of 18.7 ± 0.04 to 126 ± 11.5 nm, with an average PDI lower than 0.41 ± 0.06. When the amount of Smix in the nanoemulsion was constant, at 50%, with different ratios of surfactant to co-surfactant (nanoemulsions A–E), no significant changes were observed in droplet size or PDI. How-

575

ever, from nanoemulsions E–H, with a decrease in Smix concentration, droplet size increased, from 19.3 ± 0.17 nm (nanoemulsion E) to 126 ± 11.5 nm (nanoemulsion H), and PDI increased, from 0.20 ± 0.01 to 0.41 ± 0.06, respectively. This may be attributed to the fact that higher Smix concentration resulted in the formation of a closely packed film at the o/w interface, thereby providing stronger stabilization (Bali et al., 2010). Moreover, an increase in the concentration of oil in the nanoemulsion, with a constant concentration of Smix, led to an increased droplet size with similar PDI (nanoemulsion F: 21.7 ± 0.13 nm, nanoemulsion I: 83.7 ± 3.96 nm, and nanoemulsion J: 114 ± 3.44 nm), which may be due to interfacial disruption and the coalescence of oil droplets. However, decreased solubility was observed with an increase in oil concentration, indicating that QCN was dispersed primarily in the Smix layer (Gao et al., 2009). Zeta potential values for all nanoemulsions were observed in the range of
3.12 to 1.10 mV (close to neutral), indicating that the surfactant and co-surfactant cover the oil phase to form a stable o/w nanoemulsion. Moreover, a negative zeta potential is an indication of a negatively charged, stable nanoemulsion system. A TEM image of the optimum nanoemulsion containing QCN is shown in Supplementary Fig. 2. The QCN-loaded nanoemulsion droplets were spherical and uniformly dispersed, having a narrow size distribution, with a diameter within 50 nm, consistent with the results obtained from the droplet size analyzer. Furthermore, no sign of precipitation was observed that would interfere with the stability of the nanoemulsion. 3.2. In vitro parallel artificial intestinal membrane permeability The artificial intestinal membrane permeability assay showed significant increases in the permeability of a series of QCN nanoemulsions when compared with QCN solution used as control (Supplementary Fig. 3). The permeability of QCN was increased from 110-fold for nanoemulsion A [(12.6 ± 2.27)  10
6 cm/s] to 180-fold for nanoemulsion E [(21.2 ± 1.07)  10
6 cm/s], compared with an aqueous dispersion of QCN [(0.11 ± 0.13)  10
6 cm/s], which may have been caused by the increase in surfactant concentration, from 12.5% to 37.5%. Moreover, decreasing the concentration of Smix by 10% significantly decreased the permeability in nanoemulsion F [(15.9 ± 2.31)  10
6 cm/s] and nanoemulsion G [(6.56 ± 0.93)  10
6 cm/s]. This is likely due to the low solubilizing effect of the reduced Smix as well as the increase in droplet size of the nanoemulsion. The permeability was also decreased significantly with an increase in oil concentration for nanoemulsion I [(12.8 ± 2.06)  10
6 cm/s] and nanoemulsion J [(12.1 ± 1.39)  10
6 cm/s], as compared with nanoemulsion F. Thus, the formulation of nanoemulsion E showed a significant increase in passive permeability of QCN, and this depended on the concentration of surfactant in the preparation (Piazzini et al., 2017). Based on the results of the in vitro artificial membrane permeability analyses, we performed further tests using nanoemulsion E to assess in vitro permeability across a Caco-2 monolayer and in vivo intestinal absorption in rats as well as an anti-obesity study in mice. 3.3. In vitro Caco-2 cell monolayer permeability Transepithelial permeability of a drug across a human Caco-2 cell monolayer is a well-accepted method of assessing intestinal absorption. The in vitro permeability of QCN in aqueous dispersion, 0.3% NaCMC, and nanoemulsion E across a Caco-2 cell monolayer is shown in Table 2. To avoid drug precipitation on the cell monolayer, a concentration of 100 mM was optimized. The aqueous dispersion of QCN had low permeability [(3.59 ± 0.37)  10
6 cm/s] compared with that of QCN dispersed in 0.3% NaCMC [(5.62 ± 1.41)  10
6 cm/s]. This characteristic may be due to the

576

R. Pangeni et al. / Journal of Functional Foods 38 (2017) 571–581

Table 1 Composition and mean droplet size of the selected QCN-loaded o/w nanoemulsions. Formulation code

Composition of o/w nanoemulsions (%) Oil phase

A B C D E F G H I J a b c

a

16.67 16.67 16.67 16.67 16.67 16.67 16.67 16.67 20.00 25.00

Droplet size (nm)

Smix

Aqueous phase

Surfactantb

Co-surfactantc

12.50 16.67 25.00 33.33 37.50 30.00 22.50 15.00 30.00 30.00

37.50 33.33 25.00 16.67 12.50 10.00 7.50 5.00 10.00 10.00

33.33 33.33 33.33 33.33 33.33 43.33 53.33 63.33 40.00 35.00

20.3 ± 0.16 19.8 ± 0.25 18.8 ± 0.28 18.7 ± 0.04 19.3 ± 0.17 21.7 ± 0.13 42.5 ± 2.27 126 ± 11.5 83.7 ± 3.96 114 ± 3.44

The oil phase was composed of Capryol 90 and Labrafil M 1944 CS (1:1, w/w). The surfactant was Labrasol and Tween 80 (1:1, w/w). The co-surfactant was Cremophor EL and PEG 400 (1:1, w/w). Each value represents the mean ± standard deviation (n = 6 for each group).

Table 2 In vitro permeabilities of QCN from aqueous dispersion or nanoemulsion across a Caco-2 cell monolayer.

QCN in 0.3% NaCMC (pH 1.2) QCN in 0.3% NaCMC (pH 6.8) QCN-loaded nanoemulsion E (pH 1.2) QCN-loaded nanoemulsion E (pH 6.8)

Apparent permeability (Papp  10
6 cm/s)a

110

Aqueous dispersion of QCN QCN in 0.3% NaCMC QCN-loaded nanoemulsion E

3.59 ± 0.37 5.62 ± 1.41 12.1 ± 2.95***,###

100

Apparent permeability coefficient (Papp) of quercetin across a Caco-2 cell monolayer. Each value represents the mean ± standard deviation (n = 6 for each group). *** P < 0.001, vs. the aqueous dispersion of QCN. ### P < 0.001, vs. QCN in 0.3% NaCMC.

increased mucoadhesive property of QCN in NaCMC solution, along with the enlargement of tight junctions by adherence to the intracellular portion (Perez, Urista, Martinez, Nava, & Rodriguez, 2016). Furthermore, the permeability of QCN from nanoemulsion E was significantly greater, by 3.37-fold, than that of aqueous dispersion of QCN [(12.1 ± 2.95)  10
6 cm/s vs. (3.59 ± 0.37)  10
6 cm/s]. QCN in an aqueous dispersion may precipitate from solution, which can limit its transport compared with QCN solubilized in the nanoemulsion, resulting in a decreased concentration gradient across a Caco-2 cell monolayer. Furthermore, QCN can enhance the barrier function of Caco-2 cells by increasing levels of tight junction proteins, such as claudin-1, claudin-4, occludin, and zonula occludens-2 (ZO-2), resulting in promotion of the association of these proteins with the actin cytoskeleton, as well as their assembly at the tight junction (Suzuki & Hara, 2009). On the other hand, the presence of a surfactant and co-surfactant combination in the nanoemulsion may increase membrane permeability by disrupting cell membranes and partitioning into the cell membrane (Gao et al., 2009; Tran et al., 2014). Labrasol is known to open intestinal epithelial tight junctions through interaction with ZO-1 and filamentous actin (F-actin) (Sha, Yan, Wu, Li, & Fang, 2005). Cremophor EL has also been shown to act as an enhancer, loosening tight junctions and increasing cell membrane fluidity (Buyukozturk, Benneyan, & Carrier, 2010; Lu, Qi, & Wu, 2012). It can also bind with the hydrophobic domain of P-glycoprotein (Pgp), resulting in a conformational change and decreased drug efflux (Yin et al., 2009). 3.4. In vitro drug dissolution study The prepared nanoemulsion was also tested for in vitro dissolution. At pH 1.2, more than 90% of QCN was released from nanoemulsion E within 60 min (Fig. 1). However, less than 15% of QCN was dissolved from the 0.3% NaCMC up to 120 min. In a

90 80 70 60 50 40 30 20 10 0 0

10

20

30

40

50

60

70

80

90

100 110 120

Time (min) Fig. 1. In vitro cumulative percentage release profiles of QCN dispersed in 0.3% NaCMC or QCN-loaded nanoemulsion E in pH 1.2 or pH 6.8 media. Each value represents the mean ± standard deviation (n = 4 for each group).

Plasma concentration of quercetin (μg/mL)

a

Cumulative release (%)

Formulation

QCN in 0.3% NaCMC QCN-loaded nanoemulsion E

14 12 10 8 6 4 2 0 0

1

2

3

4

5

6

7

8

Time (h) Fig. 2. Venous plasma concentration-time profiles of QCN after a single oral administration of QCN (40 mg/kg) in 0.3% NaCMC or nanoemulsion E to rats. Each value represents the mean ± standard deviation (n = 4 for each group).

577

R. Pangeni et al. / Journal of Functional Foods 38 (2017) 571–581 Table 3 Pharmacokinetic parameters after oral administration of an aqueous dispersion of QCN or QCN-loaded nanoemulsion. Test variable

QCN in 0.3% NaCMC

QCN-loaded nanoemulsion E

Oral dose (mg/kg) Tmax (h)a T½ (h)b Cmax (lg/mL)c AUClast (lg h/mL)d AUCinf (lg h/mL)e Relative bioavailabilityf

40 2.01 ± 0.82 2.67 ± 0.92 0.350 ± 0.106 1.353 ± 0.563 1.763 ± 0.414 1

40 2.51 ± 0.55 7.25 ± 2.97*** 10.04 ± 2.010*** 45.35 ± 13.61*** 93.98 ± 32.81*** 33.51 ± 8.213***

a

Tmax, time to reach Cmax. T½, half-life of plasma concentration. c Cmax, maximum plasma concentration. d AUClast, area under the plasma concentration–time curve from zero to the time of the last measurable plasma concentration. e AUCinf, area under the plasma concentration–time curve from zero to infinity. f Relative bioavailability, (AUClast, QCN nanoemulsion/DoseQCN in nanoemulsion)/(AUClast, QCN in 0.3% NaCMC/DoseQCN in 0.3% NaCMC). Each value represents the mean ± standard deviation (n = 4 per group). *** P < 0.001, vs. QCN in 0.3% NaCMC. b

Fig. 3. Effects of QCN or QCN-loaded nanoemulsion treatment on body weight and food intake. At 1 week after beginning a high-fat diet, C57BL6 mice were orally administered 150 mg/kg of QCN dispersed in 0.3% NaCMC or encapsulated in nanoemulsion E or PBS daily: (A) a diagram of experimental procedure, 10 weeks after treatment, (B) body weights and (C) weight gain, and (D) food intake and (E) weight gain per food intake were measured weekly. NC: untreated, normal chow diet; PBS: PBStreated, high-fat diet (HFD); Q-HFD: QCN dispersed in 0.3% NaCMC-treated, HFD; and Q-NE-HFD: QCN-loaded nanoemulsion E-treated, HFD. Each value represents the mean ± standard deviation (n = 10 for each group). **P < 0.01, vs. the NC group. #P < 0.05, ##P < 0.01, vs. the PBS-HFD group. PO indicates per oral.

578

R. Pangeni et al. / Journal of Functional Foods 38 (2017) 571–581

A

4 3

∗∗

∗∗

∗∗

2 1 0

3.2 ∗∗

Epididymal fat (g)

Food intake (g/day/mouse)

D

2.4

PBS

Q

0.8

Q-NE

NC

PBS

∗∗

∗∗

0.08

∗∗ ##

0.04 0.00

NC

PBS

Q

Subcutaneous fat (g)

Weight gain/food intake

B

0.16 0.12

Q

Q-NE

HFD

HFD

E

∗∗ ##

1.6

0.0

NC

∗∗

2.1 ∗∗

1.4

∗∗ ##

0.7

0.0

Q-NE

NC

PBS

C Subcutaneous fat (g)

medium at pH 6.8, the maximum accumulative release values of QCN from the 0.3% NaCMC and nanoemulsion E were 28.9% and 96.0%, respectively, after 30 min. The dissolution rate of a highly lipophilic QCN (log P = 1.81) was improved significantly after incorporation into the nanoemulsion, indicating that the selfnanoemulsification occurred successfully after dispersing in the medium, without phase separation or drug precipitation. In both cases, the cumulative released QCN was decreased after 30 min. This may be because of the chemically instability of QCN, especially in the aqueous alkaline medium of the GI tract, because QCN is reportedly unstable due to the attack of hydroxyl ions on the C-ring of QCN molecules (Mukhopadhyay & Prajapati, 2015).

Q

Q-NE

HFD

HFD Fig. 3 (continued)

∗∗

2.1 ∗∗

∗∗

1.4

∗∗ ##

0.7

0.0

NC

PBS

Q

Q-NE

HFD 3.5. In vivo oral absorption in rats A pharmacokinetic study of QCN was performed to examine the increase in oral bioavailability, by formulation with a nanoemulsion. Mean plasma QCN concentration versus time profiles and the pharmacokinetic parameters following a single-dose oral administration of 40 mg/kg of QCN in 0.3% NaCMC and nanoemulsion E are shown in Fig. 2 and Table 3. A maximum concentration (Cmax) of 10.04 ± 2.010 mg/mL was achieved at 2.51 ± 0.55 h after oral administration of nanoemulsion E, which was 28.7-fold higher than that of the aqueous dispersion of QCN in 0.3% NaCMC. Although the solubility and permeability of QCN in the nanoemulsion increased significantly, the time to reach Cmax (Tmax) was not changed compared with that of the aqueous dispersion of QCN. QCN undergoes pre- and post-absorptive metabolism through the GI tract, such as efflux due to specific transporters, rapid hydrolysis in the small intestine by intestinal bacteria in the colon, generation of QCN aglycones, and metabolic conversion to

Fig. 4. Effect of Q and FQ treatment on fat tissue weight. At 1 week after beginning a high-fat diet, C57BL6 mice were administered orally QCN dispersed in 0.3% NaCMC and QCN-loaded nanoemulsion E (150 mg/kg of QCN) or PBS daily. After 10 weeks of QCN dispersed in 0.3% NaCMC and QCN-loaded nanoemulsion E treatment, (A) epididymal, (B) subcutaneous, and (C) perirenal fat pads were collected and weighed. NC: untreated, normal chow diet; PBS: PBS-treated, high-fat diet (HFD); Q-HFD: QCN dispersed in 0.3% NaCMC-treated; and Q-NE-HFD: QCN-loaded nanoemulsion E-treated mice. Each value represents the mean ± standard deviation (n = 10 for each group). **P < 0.01, vs. NC group; #P < 0.05, ##P < 0.01, vs. PBS-HFD group.

glucuronidated or sulfated conjugates by phase II enzymes of intestinal and liver cells (Manach et al., 2004; Mukhopadhyay & Prajapati, 2015; Shah, Joshi, & Patravale, 2009). These processes result in the production of glucuronide and metabolites of QCN, such as isorhamnetin and kaempferol, which are found mainly in blood. A small fraction of free QCN can be observed in the systemic circulation (Morand et al., 1998). Therefore, these increases in Cmax and AUClast are indicative of significant improvement in the

R. Pangeni et al. / Journal of Functional Foods 38 (2017) 571–581

intestinal absorption of QCN as nanoemulsion E; a 33.5-fold increase in AUClast (45.35 ± 13.61 mgh/mL) was achieved without affecting its elimination kinetics. A possible mechanism of the enhanced oral absorption of the QCN-loaded nanoemulsion E is through the opening of tight junctions caused by surfactants (paracellular transport) compared with QCN in aqueous dispersion, which precipitated in the cell monolayer (Sha et al., 2005). Additionally, the oil/lipid constituents of the nanoemulsion are digested by pancreatic lipases on the apical sides of enterocytes, and subsequently the QCN solubilized by surfactant may passively diffuse across the intestinal epithelium layer. The intact nanoemulsions may also diffuse directly across the intestinal membrane due to their small sizes (20 nm) or be taken

579

up into the enterocytes via clathrin- or caveola-mediated enterocytosis and macropincytosis (Lu et al., 2015; Rejman, Oberle, Zuhorn, & Hoekstra, 2004). For these reasons, the oral bioavailability of QCN was enhanced after incorporation into the o/w nanoemulsion compared with that of free QCN, and the relative bioavailability of QCN from the optimized nanoemulsion E increased by 3351% compared with that of QCN in 0.3% NaCMC. 3.6. Reduction in body weight and adiposity effects of oral QCN-loaded nanoemulsion To compare the efficacy of the QCN versus QCN-loaded nanoemulsion on obesity improvement, we measured body

A 80

ALT (U/L)

60 #

40 20 0

NC

PBS

Q

Q-NE

HFD

B 200

AST (U/L)

150 100 50 0

NC

PBS

Q

Q-NE

HFD

C

Fig. 5. Effect of QCN dispersed in 0.3% NaCMC and QCN-loaded nanoemulsion E treatments on hepatotoxicity. At 1 week after the initiation of a high-fat diet (HFD), C57BL6 mice were orally administered QCN dispersed in 0.3% NaCMC or QCN-loaded nanoemulsion E (150 mg/kg QCN) or PBS daily. At 10 weeks of treatment, serum ALT (A) and AST (B) levels were measured in mice, and (C) liver histopathology was performed. Each value represents the mean ± standard deviation (n = 10 per group). Scale bar in (C) = 50 lm. NC: untreated, normal chow diet; PBS: PBS-treated, HFD; Q-HFD: QCN dispersed in 0.3% NaCMC-treated; Q-NE-HFD: QCN-loaded nanoemulsion E-treated. # P < 0.05 vs. PBS-HFD group.

580

R. Pangeni et al. / Journal of Functional Foods 38 (2017) 571–581

weights in aqueous dispersion of QCN or QCN-loaded nanoemulsion E-treated HFD mice (Fig. 3A). QCN-loaded nanoemulsion Etreated HFD mice had significantly lower body weights and less weight gain compared with the PBS-HFD group, whereas the QHFD group did not differ from the PBS-HFD group (Fig. 3B and C). At 10 weeks after daily oral administration, the QCN-loaded nanoemulsion E demonstrated 23.5% and 21.0% inhibition of body weight gain compared with the PBS-HFD control and the Q-HFD group, respectively (Fig. 3C). However, the amount of food consumed per day did not differ significantly among the PBS-HFD, Q-HFD, and Q-NE-HFD groups (Fig. 3D). The food efficiency ratio of the Q-NE-HFD mice was significantly lower than that of the PBS-HFD mice (Fig. 3E). At the end of treatment, the epididymal, subcutaneous, and perirenal fat pad masses in the Q-NE-HFD mice were reduced by 21.2%, 37.4%, and 31.7% compared with the PBSHFD control, respectively, but the oral combinational treatment of QCN dispersed in 0.3% NaCMC did not significantly decrease the fat pad masses in HFD-treated mice (Fig. 4). Several reports have suggested that QCN treatment ameliorates obesity by improving fatty acid metabolism and fatty acid oxidation in high-fat-induced-obesity animal models. However, some reports suggest that QCN had no effect in reducing body weight or fat content (Enos et al., 2016; Hoek-van den Hil et al., 2013). Although these discrepancies may be due to differences in animal models, QCN concentrations, and caloric intake, the absorption capacity of QCN may also be an important factor. In our results, QCN treatment did not result in a significant reduction in body weight or fat content. However, we observed a significant antiobesity effect of the QCN-loaded nanoemulsion, as evidenced by a decrease in body weight gain and fat mass, without changing food consumption. These results suggest that improved oral bioavailability of QCN may help it to be developed as a ‘functional food’ component and possibly as an anti-obesity therapeutic agent. To examine whether treatment with QCN or QCN-loaded nanoemulsion caused hepatotoxicity, we performed serum biochemical assays (for the liver toxicity markers ALT and AST) and assessed liver histopathology. Although the differences were not significant, serum ALT and AST levels increased slightly in HFDtreated mice (Fig. 5A and B). In contrast, the ALT level in the QNE-HFD group decreased significantly, by 34.9% and 37.7% compared with the PBS-HFD control and Q-HFD groups, respectively (Fig. 5A). H&E staining analysis of the liver showed no change in histopathology or sign of toxicity in the QCN- or QCN-loaded nanoemulsion-treated mice (Fig. 5C). These results suggest that nanoemulsion treatment has no hepatotoxic effect. However, further toxicity studies in other organs, including those of the GI tract, after repeated oral administration of various doses of the QCNloaded nanoemulsion are required. Excessive fat accumulation in the liver causes hepatocyte damage and changes in lipid metabolism (Imai et al., 2007; Larter et al., 2012). In our study, the HFD increased lipid vacuoles; however, the QCN and QCN-loaded nanoemulsion treatments reduced the lipid vacuole content, with the QCN-loaded nanoemulsion being more effective than QCN. The serum ALT level, a marker of liver injury, increased in the HFD-fed group but decreased in the QCN-loaded nanoemulsion-treated group. These results indicate that the QCN-loaded nanoemulsion E can ameliorate fatty liver more effectively than can free QCN.

4. Conclusions In this study, we demonstrated that an o/w nanoemulsion of QCN facilitated the aqueous solubility and intestinal membrane permeability of QCN. An orally administered QCN-loaded nanoemulsion in rats led to a 33.51-fold increase in oral bioavail-

ability compared with that of QCN dispersed in 0.3% NaCMC, as well as improved pharmacokinetic properties. Moreover, the increase in water solubility and GI absorption of QCN in the oral nanoemulsion system contributed to preventing weight gain and adiposity in HFD-treated mice, resulting in a maximal reduction in body weight gain and epididymal fat mass by 23.5% and 21.2%, compared with the HFD-treated control group, respectively. Thus, the oral nanoemulsion system developed, incorporating QCN, may be a useful candidate for the treatment of obesity. Acknowledgments This research was supported by the Basic Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-2017R1D1 A1B03032283, NRF2017R1D1A1B03032529). Declaration of interests The authors declare that they have no conflict of interest. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.jff.2017.09.059. References Albishi, T., John, J. A., Al-Khalifa, A. S., & Shahidi, F. (2013). Antioxidant, antiinflammatory and DNA scission inhibitory activities of phenolic compounds in selected onion and potato varieties. Journal of Functional Foods, 5, 930–939. Bali, V., Ali, M., & Ali, J. (2010). Study of surfactant combinations and development of a novel nanoemulsion for minimising variations in bioavailability of ezetimibe. Colloids and surfaces. B, Biointerfaces, 76, 410–420. Bhatt, K., & Flora, S. J. (2009). Oral co-administration of a-lipoic acid, quercetin and captopril prevents gallium arsenide toxicity in rats. Environmental Toxicology and Pharmacology, 28, 140–146. Borhade, V., Pathak, S., Sharma, S., & Patravale, V. (2012). Clotrimazole nanoemulsion for malaria chemotherapy. Part I: preformulation studies, formulation design and physicochemical evaluation. International Journal of Pharmaceutics, 431, 138–148. Bronner, C., & Landry, Y. (1985). Kinetics of the inhibitory effect of flavonoids on histamine secretion from mast cells. Agents and Actions, 16, 147–151. Buyukozturk, F., Benneyan, J. C., & Carrier, R. L. (2010). Impact of emulsion-based drug delivery systems on intestinal permeability and drug release kinetics. Journal of Controlled Release, 142, 22–30. Caddeo, C., Nácher, A., Díez-Sales, O., Merino-Sanjuán, M., Fadda, A. M., & Manconi, M. (2014). Chitosan-xanthan gum microparticle-based oral tablet for colontargeted and sustained delivery of quercetin. Journal of Microencapsulation, 31, 694–699. Cai, X., Fang, Z., Dou, J., Yu, A., & Zhai, G. (2013). Bioavailability of quercetin: Problems and promises. Current Medicinal Chemistry, 20, 2572–2582. Coskun, O., Kanter, M., Korkmaz, A., & Oter, S. (2005). Quercetin, a flavonoid antioxidant, prevents and protects streptozotocin-induced oxidative stress and beta-cell damage in rat pancreas. Pharmacological Research, 51, 117–123. Czajkowska-Kosnik, A., Szekalska, M., Amelian, A., Szymanska, E., & Winnicka, K. (2015). Development and evaluation of liquid and solid self-emulsifying drug delivery systems for atorvastatin. Molecules, 20, 21010–21022. Enos, R. T., Velazquez, K. T., Carson, M. S., McClellan, J. L., Nagarkatti, P., Nagarkatti, M., et al. (2016). A low dose of dietary quercetin fails to protect against the development of an obese phenotype in mice. PLoS One, 11, e0167979. Erlund, I. (2004). Review of the flavonoids quercetin, hesperetin, and naringenin. Dietary sources, bioactivities, bioavailability, and epidemiology. Nutrition Research, 24, 851–874. Fofaria, N. M., Qhattal, H. S., Liu, X., & Srivastava, S. K. (2016). Nanoemulsion formulations for anti-cancer agent piplartine-characterization, toxicological, pharmacokinetics and efficacy studies. International Journal of Pharmaceutics, 498, 12–22. Formica, J. V., & Regelson, W. (1995). Review of the biology of quercetin and related bioflavonoids. Food and Chemical Toxicology, 33, 1061–1080. Gao, Y., Wang, Y. Y., Ma, Y. K., Yu, A. H., Cai, F. Q., Shao, W., & Zhai, G. X. (2009). Formulation optimization and in situ absorption in rat intestinal tract of quercetin-loaded microemulsion. Colloids and Surfaces. B, Biointerfaces, 71, 306–314. Hamid, K. A., Katsumi, H., Sakane, T., & Yamamoto, A. (2009). The effects of common solubilizing agents on the intestinal membrane barrier functions and membrane toxicity in rats. International Journal of Pharmaceutics, 379, 100–108.

R. Pangeni et al. / Journal of Functional Foods 38 (2017) 571–581 Hoek-van den Hil, E. F., Keijer, J., Bunschoten, A., Vervoort, J. J., Stankova, B., Bekkenkamp, M., et al. (2013). Quercetin induces hepatic lipid omega-oxidation and lowers serum lipid levels in mice. PLoS One, 8, e51588. Imai, Y., Varela, G. M., Jackson, M. B., Graham, M. J., Crooke, R. M., & Ahima, R. S. (2007). Reduction of hepatosteatosis and lipid levels by an adipose differentiation-related protein antisense oligonucleotide. Gastroenterology, 132, 1947–1954. Kale, R., Saraf, M., Juvekar, A., & Tayade, P. (2006). Decreased B16F10 melanoma growth and impaired tumour vascularization in BDF1 mice with quercetincyclodextrin binary system. The Journal of Pharmacy and Pharmacology, 58, 1351–1358. Kaul, T. N., Middleton, E., Jr., & Ogra, P. L. (1985). Antiviral effect of flavonoids on human viruses. Journal of Medical Virology, 15, 71–79. Larter, C. Z., Yeh, M. M., Van Rooyen Rooyen, D. M., Brooling, J., Ghatora, K., & Farrell, GC. (2012). Peroxisome proliferator-activated receptor-alpha agonist, Wy 14,643, improves metabolic indices, steatosis and ballooning in diabetic mice with non-alcoholic steatohepatitis. Journal of Gastroenterology and Hepatology, 27, 341–350. Li, H. L., Zhao, X. B., Ma, Y. K., Zhai, G. X., Li, L. B., & Lou, H. X. (2009). Enhancement of gastrointestinal absorption of quercetin by solid lipid nanoparticles. Journal of Controlled Release, 133, 238–244. Lu, R., Liu, S., Wang, Q., & Li, X. (2015). Nanoemulsions as novel oral carriers of stiripentol: insights into the protective effect and absorption enhancement. International Journal of Nanomedicine, 10, 4937–4946. Lu, Y., Qi, J., & Wu, W. (2012). Absorption, disposition and pharmacokinetics of nanoemulsions. Current Drug Metabolism, 13, 396–417. Mahdi, E. S., Sakeena, M. H., Abdulkarim, M. F., Abdullah, G. Z., Sattar, M. A., & Noor, A. M. (2011). Effect of surfactant and surfactant blends on pseudoternary phase diagram behavior of newly synthesized palm kernel oil esters. Drug Design, Development and Therapy, 5, 311–323. Manach, C., Scalbert, A., Morand, C., Rémésy, C., & Jiménez, L. (2004). Polyphenols: food sources and bioavailability. The American Journal of Clinical Nutrition, 79, 727–747. Morand, C., Crespy, V., Manach, C., Besson, C., Demigné, C., & Rémésy, C. (1998). Plasma metabolites of quercetin and their antioxidant properties. The American Journal of Physiology, 275, R212–R219. Mukhopadhyay, P., & Prajapati, A. K. (2015). Quercetin in anti-diabetic research and strategies for improved quercetin bioavailability using polymer-based carriers – a review. RSC Advances, 5, 97547–97562. Opatrilova, R., Cernikova, A., Coufalova, L., Dohnal, J., & Jampilek, J. (2013). In vitro permeation of micronized and nanonized alaptide from semisolid formulations. The Scientific World Journal, 2013, 787283. Panchal, S. K., Poudyal, H., & Brown, L. (2012). Quercetin ameliorates cardiovascular, hepatic, and metabolic changes in diet-induced metabolic syndrome in rats. The Journal of Nutrition, 142, 1026–1032. Pangeni, R., Choi, S. W., Jeon, O. C., Byun, Y., & Park, J. W. (2016). Multiple nanoemulsion system for an oral combinational delivery of oxaliplatin and 5fluorouracil: preparation and in vivo evaluation. International Journal of Nanomedicine, 11, 6379–6399. Pangeni, R., Sharma, S., Mustafa, G., Ali, J., & Baboota, S. (2014). Vitamin E loaded resveratrol nanoemulsion for brain targeting for the treatment of Parkinson’s disease by reducing oxidative stress. Nanotechnology, 25, 485102. Parmar, N., Singla, N., Amin, S., & Kohli, K. (2011). Study of cosurfactant effect on nanoemulsifying area and development of lercanidipine loaded (SNEDDS) self

581

nanoemulsifying drug delivery system. Colloids and Surfaces. B, Biointerfaces, 86, 327–338. Perez, Y. A., Urista, C. M., Martinez, J. I., Nava, M. D. C. D., & Rodriguez, F. A. R. (2016). Functionalized polymers for enhance oral bioavailability of sensitive molecules. Polymers, 8, 1–22. Piazzini, V., Monteforte, E., Luceri, C., Bigagli, E., Bilia, A. R., & Bergonzi, M. C. (2017). Nanoemulsion for improving solubility and permeability of Vitex agnus-castus extract: Formulation and in vitro evaluation using PAMPA and Caco-2 approaches. Drug Delivery, 24, 380–390. Pisonero-Vaquero, S., Martínez-Ferreras, Á., García-Mediavilla, M. V., MartínezFlórez, S., Fernández, A., Benet, M., ... Sánchez-Campos, S. (2015). Quercetin ameliorates dysregulation of lipid metabolism genes via the PI3K/AKT pathway in a diet-induced mouse model of nonalcoholic fatty liver disease. Molecular Nutrition & Food Research, 59, 879–893. Rejman, J., Oberle, V., Zuhorn, I. S., & Hoekstra, D. (2004). Size-dependent internalization of particles via the pathways of clathrin- and caveolaemediated endocytosis. The Biochemical Journal, 377, 159–169. Rifaai, R. A., El-Tahawy, N. F., Saber, E. A., & Ahmed, R. (2012). Effect of quercetin on the endocrine pancreas of the experimentally induced diabetes in male albino rats: a histological and immunohistochemical study. Journal of Diabetes & Metabolism, 24, 1–11. Sha, X., Yan, G., Wu, Y., Li, J., & Fang, X. (2005). Effect of self-microemulsifying drug delivery systems containing Labrasol on tight junctions in Caco-2 cells. European Journal of Pharmaceutical Sciences: Official Journal of the European Federation for Pharmaceutical Sciences, 24, 477–486. Shah, K. A., Joshi, M. D., & Patravale, V. B. (2009). Biocompatible microemulsions for fabrication of glyceryl monostearate solid lipid nanoparticles (SLN) of tretinoin. Journal of Biomedical Nanotechnology, 2009, 396–400. Shakeel, F., Haqa, N., Al-Dhfyan, A., Alanazi, F. K., & Alsarra, I. A. (2014). Double w/o/ w nanoemulsion of 5-fluorouracil for self-nanoemulsifying drug delivery system. Journal of Molecular Liquids, 200, 183–190. Suzuki, T., & Hara, H. (2009). Quercetin enhances intestinal barrier function through the assembly of zonula occludens-2, occludin, and claudin-1 and the expression of claudin-4 in Caco-2 cells. The Journal of Nutrition, 139, 965–974. Tran, T. H., Guo, Y., Song, D., Bruno, R. S., & Lu, X. (2014). Quercetin-containing selfnanoemulsifying drug delivery system for improving oral bioavailability. Journal of Pharmaceutical Sciences, 103, 840–852. Trevaskis, N. L., Charman, W. N., & Porter, C. J. (2008). Lipid-based delivery systems and intestinal lymphatic drug transport: a mechanistic update. Advanced Drug Delivery Reviews, 60, 702–716. Vecchione, R., Quagliariello, V., Calabria, D., Calcagno, V., De Luca, E., Iaffaioli, R. V., et al. (2016). Curcumin bioavailability from oil in water nano-emulsions: In vitro and in vivo study on the dimensional, compositional and interactional dependence. Journal of Controlled Release, 233, 88–100. Walgren, R. A., Walle, U. K., & Walle, T. (1998). Transport of quercetin and its glucosides across human intestinal epithelial Caco-2 cells. Biochemical Pharmacology, 55, 1721–1727. Yin, Y. M., Cui, F. D., Mu, C. F., Choi, M. K., Kim, J. S., Chung, S. J., ... Kim, D. D. (2009). Docetaxel microemulsion for enhanced oral bioavailability: preparation and in vitro and in vivo evaluation. Journal of Controlled Release, 140, 86–94. Zhao, L. Y., Shi, Y. K., Zou, S. H., Sun, M., Li, L. B., & Zhai, G. X. (2011). Formulation and in vitro evaluation of quercetin loaded polymeric micelles composed of pluronic P123 and D-a-tocopheryl polyethylene glycol succinate. Journal of Biomedical Nanotechnology, 7, 358–365.