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A food-grade self-nanoemulsifying delivery system for enhancing oral bioavailability of ellagic acid
Journal of Functional Foods 34 (2017) 207–215
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A food-grade self-nanoemulsifying delivery system for enhancing oral bioavailability of ellagic acid Shang-Ta Wang a, Chien-Te Chou b, Nan-Wei Su a,⇑ a b
Department of Agricultural Chemistry, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan Genomics Research Center, Academia Sinica, No. 128, Sec. 2, Academia Rd., Taipei 11529, Taiwan
a r t i c l e
i n f o
Article history: Received 5 January 2017 Received in revised form 17 April 2017 Accepted 26 April 2017
Chemical compounds studied in this article: Ellagic acid (PubChem CID: 5281855) Keywords: Ellagic acid Pomegranate Self-nanoemulsifying delivery system Bioavailability
a b s t r a c t Ellagic acid is known of a predominant bioactive component in pomegranate that possesses broad health benefits, but it has low bioavailability. In this work, we developed a food-grade self-nanoemulsifying system to improve the dissolution and absorption of ellagic acid. Solubility assay and pseudo-ternary phase diagrams revealed suitable components for the formulation. The optimal formulation was composed of polyethylene glycol, polysorbate, caprylic/capric triacylglycerol at the ratio of 45/45/10 wt.%. With this optimal formulation and gentle stirring, a fine nanoemulsion was achieved and had mean droplet size of around 120 nm. The dissolution of ellagic acid was significantly elevated with the formulation. Rat pharmacokinetics studies showed that ellagic acid was 6.6- and 3.2-fold more bioavailable with the formulation than with aqueous suspensions and pomegranate extract, respectively. The proposed selfnanoemulsifying system to deliver ellagic acid can be a novel strategy for developing products for dietary supplements and functional foods of ellagic acid. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Pomegranate (Punica granatum L.) has been consumed as a functional food in the Middle East for thousands of years (Johanningsmeier & Harris, 2011). It now enjoys worldwide popularity because of its health benefits, which are attributed to polyphenols such as punicalagin, the major fruit ellagitannin, and ellagic acid (Fig. 1). Ellagic acid is a dilactone of hexahydroxydiphenic acid and the major active component in pomegranate (García-Villalba et al., 2015). Once ellagitannins are orally administered, they undergo spontaneous or microbial hydrolysis and lactonization to ellagic acid at neutral pH condition in the gastrointestinal (GI) tract. Therefore, ellagic acid, via pomegranate Abbreviations: GI tract, gastrointestinal tract; SNEDS, self-nanoemulsifying delivery system; TEM, transmission electron microscopy; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; TPGS, D-a-Tocopherol polyethylene glycol succinate; PEG, polyethylene glycol; PTA, phosphotungstic acid; SDS, sodium dodecyl sulfate; CMC, carboxymethyl cellulose; EA-SNEDS, ellagic acid-based SNEDS; DE, dissolution efficiency; ESI, electrospray ionization; MRM, multiple reaction monitoring; Cmax, the maximum observed peak plasma genistein concentration; Tmax, time point at Cmax; AUC, area under the concentration–time curve; PEO, polyethylene oxide; LCT, long-chain triacylglycerol; MCT, medium-chain triacylglycerol; HLB, hydrophilic-lipophilic balance. ⇑ Corresponding author. E-mail addresses: [email protected] (S.-T. Wang), Cheinderson@gmail. com (C.-T. Chou), [email protected] (N.-W. Su). http://dx.doi.org/10.1016/j.jff.2017.04.033 1756-4646/Ó 2017 Elsevier Ltd. All rights reserved.
consumption, is thought to have significant effects on human health (Aguilera-Carbo, Augur, Prado-Barragan, Favela-Torres, & Aguilar, 2008; Clifford & Scalbert, 2000; Landete, 2011). } mu } s et al., 2011; Ellagic acid has strong antioxidant activity (Gu Ito, 2011; Kunsági-Máté, Stampel, Kollár, & Pour-Nikfardjam, 2008; Priyadarsini, Khopde, Kumar, & Mohan, 2002) and is one of the most potent antioxidants among numerous phytochemicals (Hayes, Allen, Brunton, O’Grady, & Kerry, 2011). In addition, it provides a broad array of health benefits including antioxidation, antiinflammation and anti-tumor benefits, prevention of cardiovascular disease, attenuation of diabetes, and estrogenic/anti-estrogenic activities (Akileshwari et al., 2014; Malik, Afaq, Shahid, Akhtar, & Assiri, 2011; Nuncio-Jáuregui et al., 2015; Papoutsi et al., 2005; Park et al., 2011). Thus, there has been increasing interest in developing ellagic acid-rich pomegranate extract into a functional food product. However, with oral administration, the low water solubility and low intestinal permeability of this polyphenol may result in diminished intestinal absorption, and furthermore contribute to very low oral bioavailability (Doyle & Griffiths, 1980; Lei et al., 2003; Seeram, Lee, & Heber, 2004). Research of humans has shown that ellagic acid has poor bioavailability with oral administration. Healthy volunteers who ingested a standardized extract of pomegranate showed only 0.06–0.1 lM peak plasma level of ellagic acid (Hamad, Al-Momani, Janakat, & Oran, 2009), so this polyphenol may fail to have a biological effect with oral administration.
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Fig. 1. Chemical structures of (A) punicalagin and (B) ellagic acid.
One of the primary factors to the low bioavailability of ellagic acid is due to its poor dissolution profile in aqueous GI fluid and low permeability of the enterocyte epithelial membrane (Whitley, Stoner, Darby, & Walle, 2003). Because of the phenolic backbone and dilactone structure, dissolution in aqueous phase at neutral pH is difficult. As well, the hydroxyl groups on the backbone reduce the lipophilicity, which impedes transportation across the lipid bilayer of enterocyte membranes (Scalbert & Williamson, 2000). Besides showing low plasma level after oral administration, ellagic acid features a very short elimination time in systemic circulation, with a half-life of <1 h (Teel, 1987). First-pass metabolism may play an important role in the elimination process because ellagic acid shows high affinity toward the phase II enzyme, the major first-pass metabolite of ellagic acid, dimethylellagic acid glucuronide, is detected in urine of most humans after ingestion of ellagic acid (Seeram et al., 2006). However, because ellagic acid may not be absorbed well in the upper intestine, the residue would therefore undergo microbial degradation by gut flora to produce urolithins (Larrosa, GonzálezSarrías, García-Conesa, Tomás-Barberán, & Espín, 2006), a subfamily of metabolites from the dibenzopyranone family, which are much better absorbed. Recent research has shown evidence of the biological effects of urolithins supporting their potential contribution to the health effects attributed to pomegranate and ellagitannin-rich foods (Sala et al., 2015). Nevertheless, the metabolism of ellagitannin and ellagic acid to urolithins in the gut is represented by large human interindividual variability in types and quantities of urolithins and was found linked to differences in the colon microbiota (Romo-Vaquero et al., 2015; TomásBarberán, García-Villalba, González-Sarrías, Selma, & Espín, 2014). Therefore, ellagic acid consumption may not have health benefits in people who are not urolithin producers and the extremely low bioavailability of ellagic acid may still be the issue. To improve the oral bioavailability of ellagic acid, research was conducted for new oral formulations by chemical modification and pharmaceutical engineering (Murugan, Mukherjee, Maiti, & Mukherjee, 2009; Ratnam, Chandraiah, Meena, Ramarao, & Kumar, 2009; Sonaje et al., 2007). However, these formulations are not suitable for introduction to nutraceuticals and functional foods because of the acute and chronic toxicity of chemical
reagents and excipients used in the processing, which may be harmful to human health with long-term consumption. In this work, we used self-nanoemulsifying delivery technique with food-grade components to prepare a highly biocompatible formulation for ellagic acid oral administration. A selfnanoemulsifying delivery system (SNEDS) is composed of natural or synthetic oils along with surfactants and co-surfactants that undergo spontaneous emulsification in the aqueous GI tract medium to form an oil-in-water emulsion with globules at nanosize ranges (Rao & Shao, 2008). We aimed to develop and characterize a food-grade ellagic acidbased SNEDS (EA-SNEDS) formulation for use in functional foods. An efficient self-nanoemulsifying vehicle for ellagic acid was developed and optimized by solubility testing and phase diagrams. Optimal ratios of excipient concentrations were selected to develop the delivery system formulations. The formulations were characterized by self-emulsification performance assessment, emulsion droplet size analysis, dissolution test, transmission electron microscopy (TEM) morphology, determination of drug loading, and formulation stability studies. The oral bioavailability of EA-SNEDS formulations was evaluated in vivo in rats and compared with unformulated ellagic acid or pomegranate consumption in terms of plasma levels. 2. Materials and methods 2.1. Chemicals Ellagic acid, oleic acid, castor oil, corn oil, limonene, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) ethyl ether, ethyl oleate, soybean oil, Tween20, Tween80, D-a-Tocopherol polyethylene glycol succinate (TPGS), soy lecithin, polyethylene glycol 200 (PEG200) and 400 (PEG400), ethanol, phosphotungstic acid (PTA), carboxymethyl cellulose (CMC), sodium dodecyl sulfate (SDS), porcine bile extract, sodium bicarbonate, methanol, hydrochloric acid, acetic acid and heparin were from SigmaAldrich (St. Louis, MO, USA). Sucrose ester S1670 was from Mitsubishi-Kagaku Foods (Tokyo). Palmester 3575 (Caprylic/capric triacylglycerols) was from KLK OLEO (Selangor, Malaysia). Pomegranate extract was from the BIOMED herbal research biomedical group (Taichung, Taiwan).
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2.2. Solubility studies of ellagic acid in various vehicles
2.6. TEM morphology study
The solubility of ellagic acid was determined in various vehicles, including oils (oleic acid, castor oil, corn oil, limonene, EPA DHA ether, Palmester 3575, ethyl oleate, soybean oil), surfactants (Tween20, Tween80, soy lecithin, sucrose ester, TPGS), and cosurfactants (PEG400, PEG200, ethanol). An excess amount of ellagic acid was added to each cap vial containing 5 mL of the vehicles. After vials were sealed, the mixture was vortexed with a mixer for 5 min and ultrasonicated for 30 min at 300 Waltzes, shaken in a water-bath shaker maintained at room temperature for another 48 h, then centrifuged at 2415g for 10 min. The supernatant was collected in glass vials and stored at room temperature for HPLC analysis to determine the solubility.
The morphology of the optimal self-nanoemulsifying system was observed on TEM (JEOL-JEM-1400, JEOL, Tokyo). About 0.1 g of the ellagic acid of the self-nanoemulsifying solution was placed in beakers. About 30 mL distilled water was added, and after sonication (100 W for 3 min), the solution was filtered through a microporous membrane (0.22 lm). Samples were dropped on copper grids for 1–2 min, excess was removed by filter paper, then copper grids were placed in 1% PTA for about 30 s. Excess PTA was removed. TEM micrographs of the ellagic acid nanoemulsions were obtained.
2.3. Construction of ternary phase diagrams Pseudo-ternary phase diagrams were constructed by using the water titration method. Mixtures of the oil phase containing oleic acid or Palmester 3575 with the surfactant phase, including Tween20 or Tween80, PEG200 or PEG400, a combination of surfactant and co-surfactant in a ratio of 50% w/w, were prepared at weight ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10 toward oil phase. The mixtures of the oil and surfactant phases of 11 different weight ratios were accurately weighed into 11 glass tubes, and then mixed by using a vortex mixer at room temperature until oily liquid mixtures were obtained. Water was then added drop-by-drop by using a dropper into each oily mixture. During the titration, samples were stirred vigorously for a sufficient length of time for homogenization and visually monitored against a dark background by illuminating the samples with white light. The concentration of water at which turbidity-totransparency and transparency-to-turbidity transition occurred was derived from the weight measurements. These values were then used to determine the boundaries of the nanoemulsion regions corresponding to the selected optimal ratios of combination vehicles for developing the EA-SNEDS.
2.4. Droplet size and polydispersity index of nanoemulsions formed on dilution The droplet sizes of nanoemulsions of formulations were determined by photon correlation spectroscopy (Zetasizer Nano ZS, Malvern, Worcestershire, UK) with dynamic light scattering. EA-SNEDS were mixed with distilled water (200 mL) and underwent mild agitation with a magnetic stirrer for 5 min at room temperature, which resulted in formation of the nanoemulsions. Aliquots of nanoemulsions were loaded into cuvettes and size-measured after dilution to produce the required count rate (100–500 kcps) to enable accurate measurement. The sample viscosity (0.890 cP) and the water refractive index (1.330) were factored in particlesize measurement by using the instrument software. Light scattering was monitored at a 90 °C angle and temperature 25 °C. Distilled water filtered through a 0.45-mm filter was used as the dilution medium. Three replicate analyses were performed for each formulation, and data are presented as mean ± SD.
2.7. Dissolution study Dissolution tests involved use of an USP 32 apparatus II (paddle method) in an RC806 dissolution tester (Shishin Technology Co., Taipei) according to the US Pharmacopoeia XXII general method. An equal micromolar amount of ellagic acid powder and EASNEDS were measured in 900 mL of 0.05 M phosphate buffer, pH 6.8, with and without the solubilizer, 0.2% SDS, at 37 °C under rotation speed 50 rpm. To evaluate the effect of bile salt on ellagic acid dissolution, bile extract (containing bile salts and phospholipids) solution was prepared by dissolving 12 g porcine bile extract and 8.4 g sodium bicarbonate in 1 L deionized water as the dissolution media. After the sample was placed in the dissolution media, an aliquot of 1 mL dissolution medium was obtained and filtered through a 0.2-mm polyethersulfone filter (Pall Corp., New York, NY, USA) at 5, 10, 20, 30, 45 and 60 min incubation. Then, 500 mL of each sample was acidified with 5 mL of 2 M acetic acid and stored at 20 °C for HPLC analysis. Dissolution efficiency (DE), the area under a dissolution curve between defined time points compared for characterizing dissolution profiles, was determined according to the following Eq. (1). t
DE ¼
f t21 y dt y100 ðt2 t1 Þ
100%
ð1Þ
where y is the percentage of dissolved product. DE is the area under the dissolution curve between time points t1 and t2 expressed as a percentage of the curve at maximum dissolution, y100, over the same time period (Anderson et al., 1998). 2.8. HPLC analysis HPLC involved use of an analytical model 584 solvent delivery gradient HPLC module (ESA Biosciences, Chelmsford, MA, USA), a YMC-Pack ODS-AM C18 column (4.6 250 mm, 5 mm) protected by a guard cartridge (Hichrom 5C18, Berkshire, UK) and a Hitachi L-7455 photodiode array detector (Hitachi Ltd., Tokyo). Chromatographic separation performed at 25 °C involved use of a linear mobile phase gradient including 0.2% acetic acid in H2O (solvent A) and 0.2% acetic acid in methanol (solvent B). After sample aliquots (20 mL) were introduced into the HPLC system, solvent B was increased from 0 to 50% over 20 min, to 60% within the next 10 min, isocratic at 60% over 6 min and finally to 0% within 5 min and held for the next 5 min. The eluted components were detected at 254 nm, and the UV spectrum between 200 nm to 400 nm was the reference.
2.5. Loading capacity assessment 2.9. Animals EA-SNEDS were diluted with water to various concentrations of ellagic acid in a definite volume in a flask. The flask was inverted and shaken gently to mix thoroughly. The particle size of the formed nanoemulsions was determined by photon correlation spectroscopy at 25 °C.
The experimental protocol was approved by the Institutional Animal Care and Use Committee of NTU (project no. NTU-100EL-114). Male Wistar rats (250 ± 30 g) were purchased from BioLASCO Taiwan Co. (Taipei) and housed in cages, with free access
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to water and food, at 25 °C with air-conditioning, constant humidity and a controlled 12-h light–dark cycle in the animal house of the Institute of Food Science and Technology, NTU. Rats were acclimatized for at least 7 days before experiments. 2.10. Oral bioavailability in rats Rats were deprived of food but had free access to water for 24 h before experiments and were divided randomly into 3 groups (n = 4) for oral gavage administration of ellagic acid aqueous suspension with 0.5% CMC, EA-SNEDS or pomegranate extract. The dosage of ellagic acid used in the tests was a constant 17.6 mmol/ kg body weight, which was translated from recommended daily intake of ellagic acid by humans, 200 mg/day, by body-surfacearea normalization (Reagan-Shaw, Nihal, & Ahmad, 2008). The dosage of pomegranate extract was determined as ellagic-acid equivalents according to the method described by Päivärinta et. al. (Päivärinta, Pajari, Törrönen, & Mutanen, 2006). Briefly, polymeric ellagitannins were converted to ellagic acid in 20-h acid hydrolysis with 1.2 M HCl at 85 °C and quantified as ellagic-acid equivalents. After oral administration of ellagic acid to rats, 500 mL blood sample was collected from the tail vein into heparinized microcentrifuge tubes at various times, then centrifuged at 4 °C under 5000g for 10 min to obtain plasma. The content of ellagic acid in plasma was determined by an HPLC-MS/MS system (Acquity UPLCQuattro Premier XE MS system, Waters Co., Milford, MA, USA). Analytical samples were prepared by mixing 50 mL plasma and 150 mL methanol, which was centrifuged to remove insoluble matter, and the supernatant underwent HPLC-MS/MS. The calibration curve was prepared by using blank plasma spiked with ellagic acid at 5–500 ng/mL. For HPLC-MS/MS, chromatographic separation involved a Biosil ODS column (4.6 150 mm, 5 mm) with an isocratic elution mobile phase of 60% methanol and 40% H2O at flow rate 1 mL/min with a postcolumn split volume ratio of 2/10 to the mass detector. The MS analysis involved ESI MRM analyses: for peak areas of m/z 301 ? 185.1, the operating parameters were capillary voltage, 3 kV; cone voltage, 37 V; collision voltage, 30 V; ion source temperature, 120 °C; and dissolvation temperature, 400 °C. The pharmacokinetic parameters were determined by using the pharmacokinetic software WinNonlin Standard Edition v1.1 (Pharsight Corp., Mountain View, CA, USA). Maximum concentration (Cmax) and time to reach maximum concentration (Tmax) were obtained directly from the concentration–time curve for each of the corresponding results. Area under the plasma ellagic acid concentration–time curve (AUC0a and AUC01) was determined. 2.11. Statistical analysis Independent Student’s t test was used to compare the means of two groups. The level of significance was set at p < 0.05. Statistical analyses involved use of SigmaPlot 10.0 (SPSS, Inc., Chicago, IL, USA). 3. Results 3.1. Solubility studies In self-nanoemulsifying systems, analytes are solubilized in the oily core and/or on the interface of the nanoemulsion structures (Setthacheewakul, Mahattanadul, Phadoongsombut, Pichayakorn, & Wiwattanapatapee, 2010). Hydrophobicity of the analytes, and presence of surfactants, co-surfactants and oils, affects the analytes solubility. The results from our solubility test (Table 1) revealed that ellagic acid has reached the highest solubility (4.18 and
3.59 mg/mL) in PEG200 and PEG400, which were therefore chosen as co-surfactants for further tests. The greater solubility in PEG may be due to the ability of ellagic acid to form hydrogen bonding with the polyethylene oxide (PEO) groups. Similarly, ellagic acid showed high solubilization capacity with surfactants composed of PEO groups such as Tween20 and Tween80. Oleic acid and Palmester 3575, conferring relatively high solubility of ellagic acid (0.012 and 0.03 mg/mL, respectively), were selected as oil phases. 3.2. Construction of pseudo-ternary phase diagrams Pseudo-ternary phase diagrams were constructed by progressive titration of the component mixtures to identify the nanoemulsion regions and optimize the concentration of the selected vehicles, including PEG200, PEG400, Tween20, Tween80, oleic acid and Palmester 3575. To develop a SNEDS formulation, the optimal ratios of excipient concentrations were established by phase diagram studies, presenting the area of the monophasic region. Moreover, this area must be determined because successful aqueous dilution without a nanoemulsion phase transition is important in oral delivery (Spernath, Aserin, & Garti, 2006). Fig. 2 depict the phase diagrams for eight different oil–surfac tant–water systems. These phase diagrams contained different areas of clear nanoemulsions (grey region), and coarse emulsions (white region). Diagrams containing larger nanoemulsion areas corresponded to formulations with Tween80 as a surfactant phase, PEG200 and 400 as a co-surfactant phase and Palmester 3575 as an oil phase. Formulation with polyethylene glycol, polysorbate, caprylic/ capric triacylglycerol at the ratio of 45/45/10 wt.%, can be fully diluted with water along the dilution line without phase separation, may be suitable for oral administration. Furthermore, the formulation with PEG400 showed higher isotropic stability than those with PEG200 as a co-surfactant (Suppl. 1) and was therefore chosen as components of the optimal SNEDS formulation. 3.3. Nanoemulsion droplet size analysis and morphology observation by TEM The effect of ellagic acid loading on particle size in distilled water is presented in Fig. 3 A. The mean particle size increased slightly with increased ellagic acid loading concentration from 0.5 to 2.5 mg mL. With further increase in ellagic acid loading, particle size increased greatly, as high as 120 nm or more, beyond the range of the colloidal system and no longer presented the properties of nanoemulsion. Undissolved ellagic acid in the formulation could have altered the mean droplet size to increase. The morphol-
Table 1 Solubility of ellagic acid in various vehicles. Vehicles Oils Castor oil Corn oil Limonene Cottonseed oil Oleic acid EPA DHA ethyl esters Palmester 3575a Ethyl oleate Soybean oil
Solubility (mg/mL) N/A* N/A N/A 0.005 0.012 N/A 0.03 N/A N/A
Vehicles Surfactants Lecithin Sucrose esters TPGS Tween20 Tween80 Co-solvents PEG200 PEG400 Ethanol
Solubility (mg/mL) 0.264 0.115 N/A 1.605 2.739 4.178 3.588 0.668
TPGS, D-a-Tocopherol polyethylene glycol succinate; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; PEG, polyethylene glycol. a Caprylic/capric triacylglycerols * N/A, solubility lower than the detection limit.
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Fig. 2. Pseudo-ternary phase diagram of self-nanoemulsifying delivery system (SNEDS) with water as a dilution medium, oleic acid as an oil phase, and (A) Tween20 as a surfactant and PEG200 as a co-surfactant, (B) Tween20 as a surfactant and PEG400 as a co-surfactant, (C) Tween80 as a surfactant and PEG200 as a co-surfactant and (D) Tween80 as a surfactant and PEG400 as a co-surfactant, or Palmester 3575 as an oil phase, and (E) Tween20 as a surfactant and PEG200 as a co-surfactant, (F) Tween20 as a surfactant and PEG400 as a co-surfactant, (G) Tween80 as a surfactant and PEG200 as a co-surfactant and (H) Tween80 as a surfactant and PEG400 as a co-surfactant.
Fig. 3. (A) Effect of ellagic acid concentration on droplet size of nanoemulsions. Photographs represent the corresponding outward appearance of the self-nanoemulsifying delivery system (SNEDS) with various ellagic acid-loading amounts. (B) 25,000 and (C) 5000 of microphotographs of ellagic acid-loaded self-nanoemulsifying delivery system (EA-SNEDS) by transmission electron microscopy. Data are mean ± SD (n = 3).
ogy of ellagic acid nanoemulsion was characterized by TEM (Fig. 3B and C), showing the spherical shape and uniform droplet size of nanoemulsions. In addition, the mean droplet size of nanoemulsion might have increased with the amount of ellagic acid because ella-
gic acid might embed in the interfacial film. Samples were diluted with distilled water before testing to avoid the multiscattering phenomena. The droplet size of the diluted nanoemulsion was not significantly changed.
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3.4. In vitro dissolution study The dissolution of ellagic acid from the SNEDS and the unformulated powder of ellagic acid were evaluated in pH 6.8 phosphate buffer, phosphate buffer containing 0.2% SDS and bile salt solution. The dissolution proportion was significantly higher with ellagic acid in the SNEDS form than the unformulated powder in all dissolution media studied (Fig. 4). Only about 16% of tested ellagic acid was dissolved in dissolution medium with 60-min incubation (Fig. 4A), whereas EA-SNEDS was dissolved at 50% with about 2min incubation and 88% with 20-min incubation. Both 0.2% SDS and bile extract solution improved the dissolution of ellagic acid powder. The dissolution with 0.2% SDS and bile extract solution was increased to about 31% (Fig. 4B) and 23% (Fig. 4C) with 60min incubation. Even so, ellagic acid powder still showed poor dissolution profiles, whereas EA-SNEDS showed highly improved solubility in all dissolution media. Mean DE values for EA-SNEDS were 80.93% to 97.55%, significantly higher than for ellagic acid powder with various dissolution buffers (Table 2). 3.5. Oral bioavailability studies To further evaluate the efficacy of EA-SNEDS for the absorption of ellagic acid by oral administration, we used the optimal formulation of EA-SNEDS and ellagic acid aqueous suspension with 0.5% CMC at 17.6 lmol/kg body weight in rats. In addition, because pomegranate and its products are the most common source of ellagic acid in the functional food market, we introduced pomegranate extract with a same molar ratio of the aforementioned tested group to determine the plasma level of ellagic acid after pomegra-
Table 2 Dissolution efficiency (%) of ellagic acid unformulated powder and ellagic acid-loaded self-nanoemulsifying delivery system (EA-SNEDS) in various dissolution buffers.
a *
Dissolution efficiency (%)a
Phosphate buffer pH 6.8
Phosphate buffer with 0.2% SDS
Bile salt solution
Ellagic acid EA-SNEDS
11.03 ± 0.32 80.93 ± 1.37*
24.49 ± 1.06 97.55 ± 3.08*
17.86 ± 0.43 84.68 ± 3.08*
Data are mean ± SD (n = 3). p < 0.05 comparing EA-SNEDS to ellagic acid group, unpaired t test.
nate consumption. The ellagic acid molar ratio of 1 g pomegranate extract was determined as 0.159 mmol and we used 111 mg/kg body weight of that. Oral absorption of ellagic acid in suspension was the least absorptive and that in SNEDS group was the highest (Fig. 5). The plasma profiles for EA-SNEDS showed two peaks, which indicated that ellagic acid was absorbed rapidly, with Tmax at 30 min after oral administration, and reabsorption via enteralhepatic circulation. The plasma profile for ellagic acid was similar to that from a previous study (Hamad et al., 2009). The Cmax for EA-SNEDS was 527.95 nM (Table 3), about 5 times higher than that for pomegranate extract, whereas the Cmax for ellagic acid suspension was 54.37 nM. The AUC0-1 reflects the exposure of plasma to ellagic acid from time 0 to the time when the plasma ellagic acid concentration returns to baseline. After oral administration, EA-SNEDS showed the highest AUC0-1 for ellagic acid: 6.6 and 3.2 times higher than with aqueous ellagic acid suspension and pomegranate extract, respectively. Thus, orally administered EA-SNEDS could be a strategy for developing novel products of dietary supplements and functional foods with ellagic acid.
Fig. 4. Dissolutions of ellagic acid unformulated powder and ellagic acid-loaded self-nanoemulsifying delivery system (EA-SNEDS) in different solutions. (A) pH 6.8, phosphate buffer alone; (B) phosphate buffer containing 0.2% SDS; (C) bile salt solution. Data are mean ± SD (n = 3).
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Fig. 5. Mean plasma concentration/time profiles of ellagic acid in rats with oral administration of ellagic acid suspension, ellagic acid-loaded self-nanoemulsifying delivery system (EA-SNEDS) or pomegranate extract at 17.6 lmol/kg body weight ellagic acid equivalent. Data are mean ± SE (n = 4).
Table 3 Pharmacokinetic parameters of plasma ellagic acid with oral administration of ellagic acid suspension, pomegranate extract and ellagic acid-loaded self-nanoemulsifying delivery system (EA-SNEDS). Parametersa
Ellagic acid
Pomegranate extract
EA-SNEDS
Cmax (nM) Tmax (min) AUC0-a (nM h) AUC0-1 (nM h)
54.37 ± 2.35 30 41.06 ± 14.40 51.72 ± 14.97
106.09 ± 11.66 60 77.68 ± 21.19 103.08 ± 12.98
527.95 ± 54.83* 30 264.77 ± 28.25* 342.58 ± 43.28*
a
Data are mean ± SE (n = 4). p < 0.05 comparing EA-SNEDS to ellagic acid and pomegranate extract groups, unpaired t test. *
4. Discussion In this work, we developed a food-grade self-nanoemulsifying system to improve the dissolution and absorption in delivering ellagic acid. With this system, the oral bioavailability of ellagic acid was significantly superior to aqueous suspensions of ellagic acid and pomegranate extract. in rats. Pomegranate is one of the world’s oldest known ancient fruits and has gained commercial significance recently with the verification of its nutraceutical and pharmacological properties. Pomegranate extract has been reported to have various health effects such as antioxidation, antiproliferation and inhibition of adipogenesis (Nuncio-Jáuregui et al., 2015; Orgil, Spector, Holland, Mahajna, & Amir, 2016; Wu, Ma, & Tian, 2013). Convincing evidence revealed that the health benefits were due to the production of ellagic acid in the GI tract after oral ingestion by humans (Akileshwari et al., 2014; NuncioJáuregui et al., 2015; Wu et al., 2013), which involved spontaneous hydrolysis from ellagitannins at neutral pH environment. Even though ellagic acid features remarkable bioactivity, it is extremely low aqueous soluble and has been classified under the Biopharmaceutical Classification System as a class IV substance with low solubility (<10 mg/mL in phosphate buffer, pH 7.4) and low permeability (0.13 106 cm/s) (Waldmann et al., 2012). Moreover, ellagic acid has been found to bind irreversibly to cellular DNA and proteins and metabolized by first-pass enzymes or microbes, which may also explain its limited transcellular absorption (Whitley et al., 2003). The poor absorption of ellagic acid has been reported to affect its in vivo bioactivity because sufficient concentrations may not be in plasma or target organs after oral administration and therefore limit its use in functional foods (Seeram et al., 2004). For our EA-SNEDS formulation, the choice of excipients to prepare SNEDS depends on the ellagic acid dissolving capacity. Ellagic
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acid showed extremely low solubility in various oil phases, including long-chain triacylglycerol (LCT), medium-chain triacylglycerol (MCT), terpene, long chain fatty acids and fatty acid esters (Table 1), which may be due to the polarity donated by its hydroxyl groups. Ellagic acid can only be slightly dissolved in oils showing relatively high polarity, such as MCT and fatty acids. MCT are commonly used in SNEDS formulations (Shah, Carvajal, Patel, Infeld, & Malick, 1994). They passively diffuse from the GI tract to the portal system (longer fatty acids are absorbed into the lymphatic system) without the need for modification as for long-chain or very longchain fatty acids. MCT possess higher ester content per gram than LCT that means MCT has better capability of dissolving ellagic acid than LCT (Cao, Marra, & Anderson, 2004). In this study, we select oleic acid and Palmester 3575 for the oil phase of the delivery system. The selection of surfactant and cosurfactant was governed by emulsification efficiency rather than the ability to solubilize substances. The efficiency of selfnanoemulsification is related more to the hydrophilic–lipophilic balance (HLB) value of the surfactant. Surfactants with HLB > 10 are good at providing fine, uniform nanoemulsion droplets. Surfactants increase the permeability by interfering with the lipid bilayer of the epithelial cell membrane (Gursoy & Benita, 2004). We chose two high-value surfactants, Tween20 (HLB = 15, 1.6 mg/mL) and Tween80 (HLB = 15, 2.7 mg/mL), as surfactants for EA-SNEDS. Cosurfactants increase the interfacial fluidity by penetrating the surfactant film and creating a disordered film due to the void space among surfactant molecules. The emulsification efficiency of the SNEDS formulation changes with the chain length of the cosurfactants. PEG200 (4.2 mg/mL) and PEG400 (3.6 mg/mL) solubilize ellagic acid in good amount. Hence, we chose these two cosurfactants for the EA-SNEDS formulation. The ternary phase diagrams (Fig. 2) show a high concentration of surfactant and high self-nanoemulsifying region. The relatively high concentration of surfactant needed to form our nanoemulsions agrees with other studies: high concentrations of surfactants were needed to achieve fast and efficient self-nanoemulsication (Ting, Jiang, Ho, & Huang, 2014). Among all tested formulations, the delivery system of Tween80, PEG400 and Palmester 3575 conferred the highest nanoemulsion ability and stability and could undergo aqueous phase dilution without phase transition and so was chosen as the optimal formulation. The optimal formulation performed a high loading capacity of ellagic acid which is 250 time higher than its aqueous solubility. With the optimal EA-SNEDS, ellagic acid could be dissolved easily because of the small droplet size, which permitted a faster rate of substance dissolution into the aqueous phase, faster than unformulated powder, which may greatly affect the bioavailability. In addition, because phosphate buffer may not mimic the effect of bile salt on solubility of ellagic acid, we used bile extract solution to represent the condition in the intestinal medium (Tzoumaki, Moschakis, Scholten, & Biliaderis, 2013). Phenolic compounds were found to be more bioavailable with solubilizer effect of bile salts when being consumed in fed state. Nevertheless, the results with ellagic acid powder still showed poor dissolution in bile extract solution, indicating extremely low solubility of ellagic acid in GI tract. Ellagic acid with SNEDS showed almost complete dissolution within 60 min, so this formulation may dissolve well in intestinal conditions. The pharmacokinetics profiles agreed highly with the dissolution profiles. With the SNEDS formulation, the plasma concentration of ellagic acid in rats indicated significantly greater improvement of substance absorption than with ellagic acid suspension (Fig. 5). We observed a double peak profile with plasma exposure of EA-SNEDS that may have been due to gastric motility after administration or may represent an active and proficient entero-hepatic (or enterocytic) recycling of ellagic acid. Unexpect-
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edly, pomegranate extract showed delayed Tmax at 60 min, which could be attributed to the release process of ellagic acid from ellagitannins in the extracts. Research indicated that the in vivo hydrolysis of ellagitannins to release ellagic acid can be a critical limiting step in ellagic acid bioavailability (Daniel, Ratnayake, Kinstle, & Stoner, 1991). However, experimental data did not support the concept. González-Sarrías et al. conducted a pharmacokinetic study using pomegranate extract with different ellagitannins to free ellagic acid ratio as oral gavage substance and the results demonstrated that intake of a higher dose of free ellagic acid did not significantly change plasma ellagic acid values, which was agreed with our results (González-Sarrías et al., 2015). In our study, we found that the plasma level of pomegranate extract was even higher than that of free ellagic acid, indicating the food matrix from pomegranate extract may play a role in the bioavailability. Phenolic compounds were reported to be reduced in crystallinity and improved in dissolution by food matrix such as lipids and carbohydrate, and may therefore affect its bioavailability (Chen, Yang, Uang, & Lin, 2013). Even so, SNEDS formulation offered significant improvement of dissolution efficiency and bioavailability of ellagic acid, with almost three-fold higher oral bioavailability than pomegranate extract consumption. Previous research aimed at improving the bioavailability of ellagic acid by performing ellagic acid-phospholipids complex, and may increase both the bioavailability and peak plasma concentration to almost 2.5-fold over free ellagic acid (Murugan et al., 2009). The complex also showed better hepatoprotective activity compared with free ellgic acid. However, the biological effects were based on relative high dosage that would be difficult to consume. In our study, EA-SNEDS formulation performed 6-fold better in bioavailability and almost 10-fold in Cmax, respectively. The amount of ellagic acid we administrated to experimental rats was translated from a reasonable human dosage that may have been seen commonly in ellagic acid based dietary supplement products. The remarkable absorption enhancing ability of EASNEDS may be advantageous for delivering ellagic acid. Literally, this delivery system offers several advantages including high solvent power, improved permeability across the GI tract membrane, decreased or diminished food effect, and improved substance bioavailability (Ghai & Sinha, 2012). The bioavailability-enhancing property of the self-emulsifying formulations results from improved compound dissolution and also several mechanisms including the reduction of first-pass drug metabolism in the liver (Sun et al., 2011). SNEDS may increase the contact of ellagic acid to the epithelial membrane by improving aqueous solubility and thus intestinal absorption (Zou et al., 2015). The SNEDS formulation should result in higher bioavailability because of more rapid and uniform distribution of the substance in the GI. The reduced first-pass effect may be attributed to the association of lipid components with selective compound engulfing into the lymphatic transport system. It may also result from the ability of lipid excipients and their metabolites to cause changes in the absorption of GI fluid-promoting compounds (Khan et al., 2015). In addition, the delivery system was found to inhibit intestinal efflux such as p-glycoprotein and multidrug resistance-associated protein 2, thus enhancing transepithelial flux and increasing systemic exposure (Borhade, Nair, & Hegde, 2008; Li, Yi, & Lam, 2014). These advantages and the delivery strategy may help enhance the absorption and plasma level of ellagic acid with consumption. Hence, for oral administration of ellagic acid, SNEDS might be a promising approach for effective absorption and could be used to increase the bioavailability for other poorly water-soluble substances. In addition, it was reported that the no observed-adverse-effect level (NOAEL) of ellagic acid was 3254 mg/kg/day in rats (Tasaki et al., 2008). In this study, the dosage was set at 5.3 mg/kg, which was far away from the NOAEL.
Thus, it may still be harmless to subjects even though the plasma exposure of ellagic acid was enhanced by the formulation. Moreover, the physiological and toxicological effects of improving absorption are unclear, and therefore, further research is absolutely necessary. The market for pomegranate products has grown in recent years and pomegranate extracts was approved in the health food market (Fischer, Jaksch, Carle, & Kammerer, 2012). According to our results, EA-SNEDS may be an alternative to pomegranate extract and offer improved bioavailability of ellagic acid. Conflict of interest statement The authors have no conflicts of interest for this work. Acknowledgements This work was supported by the Ministry of Science and Technology, Executive Yuan, Taiwan [grant number: MOST 101-2313B-002-068-MY3]. The authors would like to thank Dr. Shu-Chen Hsieh, Institute of Food Science and Technology, National Taiwan University for help with animal housing. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jff.2017.04.033. References Aguilera-Carbo, A., Augur, C., Prado-Barragan, L. A., Favela-Torres, E., & Aguilar, C. N. (2008). Microbial production of ellagic acid and biodegradation of ellagitannins. Applied Microbiology and Biotechnology, 78, 189–199. Akileshwari, C., Raghu, G., Muthenna, P., Mueller, N. H., Suryanaryana, P., Petrash, J. M., & Reddy, G. B. (2014). Bioflavonoid ellagic acid inhibits aldose reductase: Implications for prevention of diabetic complications. Journal of Functional Foods, 6, 373–383. Anderson, N. H., Bauer, M., Boussac, N., Khan-Malek, R., Munden, P., & Sardaro, M. (1998). An evaluation of fit factors and dissolution efficiency for the comparison of in vitro dissolution profiles. Journal of Pharmaceutical and Biomedical Analysis, 17, 811–822. Borhade, V., Nair, H., & Hegde, D. (2008). Design and evaluation of selfmicroemulsifying drug delivery system (SMEDDS) of tacrolimus. An Official Journal of the American Association of Pharmaceutical Scientists, 9, 13–21. Cao, Y., Marra, A. Y., & Anderson, B. D. (2004). Predictive relationships for the effects of triglyceride ester concentration and water uptake on solubility and partitioning of small molecules into lipid vehicles. Journal of Pharmaceutical Sciences, 93, 2768–2779. Chen, C. H., Yang, J. C., Uang, Y. S., & Lin, C. J. (2013). Improved dissolution rate and oral bioavailability of lovastatin in red yeast rice products. International Journal of Pharmaceutics, 444, 18–24. Clifford, M. N., & Scalbert, A. (2000). Ellagitannins – Nature, occurrence and dietary burden. Journal of the Science of Food and Agriculture, 80, 1118–1125. Daniel, E. M., Ratnayake, S., Kinstle, T., & Stoner, G. D. (1991). The effects of pH and rat intestinal contents on the liberation of ellagic acid from purified and crude ellagitannins. Journal of Natural Products, 54, 946–952. Doyle, B., & Griffiths, L. A. (1980). The metabolism of ellagic acid in the rat. Xenobiotica, 10, 247–256. Fischer, U. A., Jaksch, A. V., Carle, R., & Kammerer, D. R. (2012). Determination of lignans in edible and nonedible parts of pomegranate (Punica granatum L.) and products derived therefrom, particularly focusing on the quantitation of isolariciresinol using HPLC-DAD-ESI/MSn. Journal of Agricultural and Food Chemistry, 60, 283–292. García-Villalba, R., Espín, J. C., Aaby, K., Alasalvar, C., Heinonen, M., Jacobs, G., ... Tomás-Barberán, F. A. (2015). Validated method for the characterization and quantification of extractable and nonextractable ellagitannins after acid hydrolysis in pomegranate fruits, juices, and extracts. Journal of Agricultural and Food Chemistry, 63, 6555–6566. Ghai, D., & Sinha, V. R. (2012). Nanoemulsions as self-emulsified drug delivery carriers for enhanced permeability of the poorly water-soluble selective b1adrenoreceptor blocker Talinolol. Nanomedicine, 8, 618–626. González-Sarrías, A., Rocío García-Villalba, R., Núñez-Sánchez, M. A., ToméCarneiro, J., Zafrilla, P., Mulero, J., ... Espín, J. C. (2015). Identifying the limits for ellagic acid bioavailability: A crossover pharmacokinetic study in healthy volunteers after consumption of pomegranate extracts. Journal of Functional Foods, 19, 225–235.
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A food-grade self-nanoemulsifying delivery system for enhancing oral bioavailability of ellagic acid Shang-Ta Wang a, Chien-Te Chou b, Nan-Wei Su a,⇑ a b
Department of Agricultural Chemistry, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan Genomics Research Center, Academia Sinica, No. 128, Sec. 2, Academia Rd., Taipei 11529, Taiwan
a r t i c l e
i n f o
Article history: Received 5 January 2017 Received in revised form 17 April 2017 Accepted 26 April 2017
Chemical compounds studied in this article: Ellagic acid (PubChem CID: 5281855) Keywords: Ellagic acid Pomegranate Self-nanoemulsifying delivery system Bioavailability
a b s t r a c t Ellagic acid is known of a predominant bioactive component in pomegranate that possesses broad health benefits, but it has low bioavailability. In this work, we developed a food-grade self-nanoemulsifying system to improve the dissolution and absorption of ellagic acid. Solubility assay and pseudo-ternary phase diagrams revealed suitable components for the formulation. The optimal formulation was composed of polyethylene glycol, polysorbate, caprylic/capric triacylglycerol at the ratio of 45/45/10 wt.%. With this optimal formulation and gentle stirring, a fine nanoemulsion was achieved and had mean droplet size of around 120 nm. The dissolution of ellagic acid was significantly elevated with the formulation. Rat pharmacokinetics studies showed that ellagic acid was 6.6- and 3.2-fold more bioavailable with the formulation than with aqueous suspensions and pomegranate extract, respectively. The proposed selfnanoemulsifying system to deliver ellagic acid can be a novel strategy for developing products for dietary supplements and functional foods of ellagic acid. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Pomegranate (Punica granatum L.) has been consumed as a functional food in the Middle East for thousands of years (Johanningsmeier & Harris, 2011). It now enjoys worldwide popularity because of its health benefits, which are attributed to polyphenols such as punicalagin, the major fruit ellagitannin, and ellagic acid (Fig. 1). Ellagic acid is a dilactone of hexahydroxydiphenic acid and the major active component in pomegranate (García-Villalba et al., 2015). Once ellagitannins are orally administered, they undergo spontaneous or microbial hydrolysis and lactonization to ellagic acid at neutral pH condition in the gastrointestinal (GI) tract. Therefore, ellagic acid, via pomegranate Abbreviations: GI tract, gastrointestinal tract; SNEDS, self-nanoemulsifying delivery system; TEM, transmission electron microscopy; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; TPGS, D-a-Tocopherol polyethylene glycol succinate; PEG, polyethylene glycol; PTA, phosphotungstic acid; SDS, sodium dodecyl sulfate; CMC, carboxymethyl cellulose; EA-SNEDS, ellagic acid-based SNEDS; DE, dissolution efficiency; ESI, electrospray ionization; MRM, multiple reaction monitoring; Cmax, the maximum observed peak plasma genistein concentration; Tmax, time point at Cmax; AUC, area under the concentration–time curve; PEO, polyethylene oxide; LCT, long-chain triacylglycerol; MCT, medium-chain triacylglycerol; HLB, hydrophilic-lipophilic balance. ⇑ Corresponding author. E-mail addresses: [email protected] (S.-T. Wang), Cheinderson@gmail. com (C.-T. Chou), [email protected] (N.-W. Su). http://dx.doi.org/10.1016/j.jff.2017.04.033 1756-4646/Ó 2017 Elsevier Ltd. All rights reserved.
consumption, is thought to have significant effects on human health (Aguilera-Carbo, Augur, Prado-Barragan, Favela-Torres, & Aguilar, 2008; Clifford & Scalbert, 2000; Landete, 2011). } mu } s et al., 2011; Ellagic acid has strong antioxidant activity (Gu Ito, 2011; Kunsági-Máté, Stampel, Kollár, & Pour-Nikfardjam, 2008; Priyadarsini, Khopde, Kumar, & Mohan, 2002) and is one of the most potent antioxidants among numerous phytochemicals (Hayes, Allen, Brunton, O’Grady, & Kerry, 2011). In addition, it provides a broad array of health benefits including antioxidation, antiinflammation and anti-tumor benefits, prevention of cardiovascular disease, attenuation of diabetes, and estrogenic/anti-estrogenic activities (Akileshwari et al., 2014; Malik, Afaq, Shahid, Akhtar, & Assiri, 2011; Nuncio-Jáuregui et al., 2015; Papoutsi et al., 2005; Park et al., 2011). Thus, there has been increasing interest in developing ellagic acid-rich pomegranate extract into a functional food product. However, with oral administration, the low water solubility and low intestinal permeability of this polyphenol may result in diminished intestinal absorption, and furthermore contribute to very low oral bioavailability (Doyle & Griffiths, 1980; Lei et al., 2003; Seeram, Lee, & Heber, 2004). Research of humans has shown that ellagic acid has poor bioavailability with oral administration. Healthy volunteers who ingested a standardized extract of pomegranate showed only 0.06–0.1 lM peak plasma level of ellagic acid (Hamad, Al-Momani, Janakat, & Oran, 2009), so this polyphenol may fail to have a biological effect with oral administration.
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Fig. 1. Chemical structures of (A) punicalagin and (B) ellagic acid.
One of the primary factors to the low bioavailability of ellagic acid is due to its poor dissolution profile in aqueous GI fluid and low permeability of the enterocyte epithelial membrane (Whitley, Stoner, Darby, & Walle, 2003). Because of the phenolic backbone and dilactone structure, dissolution in aqueous phase at neutral pH is difficult. As well, the hydroxyl groups on the backbone reduce the lipophilicity, which impedes transportation across the lipid bilayer of enterocyte membranes (Scalbert & Williamson, 2000). Besides showing low plasma level after oral administration, ellagic acid features a very short elimination time in systemic circulation, with a half-life of <1 h (Teel, 1987). First-pass metabolism may play an important role in the elimination process because ellagic acid shows high affinity toward the phase II enzyme, the major first-pass metabolite of ellagic acid, dimethylellagic acid glucuronide, is detected in urine of most humans after ingestion of ellagic acid (Seeram et al., 2006). However, because ellagic acid may not be absorbed well in the upper intestine, the residue would therefore undergo microbial degradation by gut flora to produce urolithins (Larrosa, GonzálezSarrías, García-Conesa, Tomás-Barberán, & Espín, 2006), a subfamily of metabolites from the dibenzopyranone family, which are much better absorbed. Recent research has shown evidence of the biological effects of urolithins supporting their potential contribution to the health effects attributed to pomegranate and ellagitannin-rich foods (Sala et al., 2015). Nevertheless, the metabolism of ellagitannin and ellagic acid to urolithins in the gut is represented by large human interindividual variability in types and quantities of urolithins and was found linked to differences in the colon microbiota (Romo-Vaquero et al., 2015; TomásBarberán, García-Villalba, González-Sarrías, Selma, & Espín, 2014). Therefore, ellagic acid consumption may not have health benefits in people who are not urolithin producers and the extremely low bioavailability of ellagic acid may still be the issue. To improve the oral bioavailability of ellagic acid, research was conducted for new oral formulations by chemical modification and pharmaceutical engineering (Murugan, Mukherjee, Maiti, & Mukherjee, 2009; Ratnam, Chandraiah, Meena, Ramarao, & Kumar, 2009; Sonaje et al., 2007). However, these formulations are not suitable for introduction to nutraceuticals and functional foods because of the acute and chronic toxicity of chemical
reagents and excipients used in the processing, which may be harmful to human health with long-term consumption. In this work, we used self-nanoemulsifying delivery technique with food-grade components to prepare a highly biocompatible formulation for ellagic acid oral administration. A selfnanoemulsifying delivery system (SNEDS) is composed of natural or synthetic oils along with surfactants and co-surfactants that undergo spontaneous emulsification in the aqueous GI tract medium to form an oil-in-water emulsion with globules at nanosize ranges (Rao & Shao, 2008). We aimed to develop and characterize a food-grade ellagic acidbased SNEDS (EA-SNEDS) formulation for use in functional foods. An efficient self-nanoemulsifying vehicle for ellagic acid was developed and optimized by solubility testing and phase diagrams. Optimal ratios of excipient concentrations were selected to develop the delivery system formulations. The formulations were characterized by self-emulsification performance assessment, emulsion droplet size analysis, dissolution test, transmission electron microscopy (TEM) morphology, determination of drug loading, and formulation stability studies. The oral bioavailability of EA-SNEDS formulations was evaluated in vivo in rats and compared with unformulated ellagic acid or pomegranate consumption in terms of plasma levels. 2. Materials and methods 2.1. Chemicals Ellagic acid, oleic acid, castor oil, corn oil, limonene, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) ethyl ether, ethyl oleate, soybean oil, Tween20, Tween80, D-a-Tocopherol polyethylene glycol succinate (TPGS), soy lecithin, polyethylene glycol 200 (PEG200) and 400 (PEG400), ethanol, phosphotungstic acid (PTA), carboxymethyl cellulose (CMC), sodium dodecyl sulfate (SDS), porcine bile extract, sodium bicarbonate, methanol, hydrochloric acid, acetic acid and heparin were from SigmaAldrich (St. Louis, MO, USA). Sucrose ester S1670 was from Mitsubishi-Kagaku Foods (Tokyo). Palmester 3575 (Caprylic/capric triacylglycerols) was from KLK OLEO (Selangor, Malaysia). Pomegranate extract was from the BIOMED herbal research biomedical group (Taichung, Taiwan).
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2.2. Solubility studies of ellagic acid in various vehicles
2.6. TEM morphology study
The solubility of ellagic acid was determined in various vehicles, including oils (oleic acid, castor oil, corn oil, limonene, EPA DHA ether, Palmester 3575, ethyl oleate, soybean oil), surfactants (Tween20, Tween80, soy lecithin, sucrose ester, TPGS), and cosurfactants (PEG400, PEG200, ethanol). An excess amount of ellagic acid was added to each cap vial containing 5 mL of the vehicles. After vials were sealed, the mixture was vortexed with a mixer for 5 min and ultrasonicated for 30 min at 300 Waltzes, shaken in a water-bath shaker maintained at room temperature for another 48 h, then centrifuged at 2415g for 10 min. The supernatant was collected in glass vials and stored at room temperature for HPLC analysis to determine the solubility.
The morphology of the optimal self-nanoemulsifying system was observed on TEM (JEOL-JEM-1400, JEOL, Tokyo). About 0.1 g of the ellagic acid of the self-nanoemulsifying solution was placed in beakers. About 30 mL distilled water was added, and after sonication (100 W for 3 min), the solution was filtered through a microporous membrane (0.22 lm). Samples were dropped on copper grids for 1–2 min, excess was removed by filter paper, then copper grids were placed in 1% PTA for about 30 s. Excess PTA was removed. TEM micrographs of the ellagic acid nanoemulsions were obtained.
2.3. Construction of ternary phase diagrams Pseudo-ternary phase diagrams were constructed by using the water titration method. Mixtures of the oil phase containing oleic acid or Palmester 3575 with the surfactant phase, including Tween20 or Tween80, PEG200 or PEG400, a combination of surfactant and co-surfactant in a ratio of 50% w/w, were prepared at weight ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10 toward oil phase. The mixtures of the oil and surfactant phases of 11 different weight ratios were accurately weighed into 11 glass tubes, and then mixed by using a vortex mixer at room temperature until oily liquid mixtures were obtained. Water was then added drop-by-drop by using a dropper into each oily mixture. During the titration, samples were stirred vigorously for a sufficient length of time for homogenization and visually monitored against a dark background by illuminating the samples with white light. The concentration of water at which turbidity-totransparency and transparency-to-turbidity transition occurred was derived from the weight measurements. These values were then used to determine the boundaries of the nanoemulsion regions corresponding to the selected optimal ratios of combination vehicles for developing the EA-SNEDS.
2.4. Droplet size and polydispersity index of nanoemulsions formed on dilution The droplet sizes of nanoemulsions of formulations were determined by photon correlation spectroscopy (Zetasizer Nano ZS, Malvern, Worcestershire, UK) with dynamic light scattering. EA-SNEDS were mixed with distilled water (200 mL) and underwent mild agitation with a magnetic stirrer for 5 min at room temperature, which resulted in formation of the nanoemulsions. Aliquots of nanoemulsions were loaded into cuvettes and size-measured after dilution to produce the required count rate (100–500 kcps) to enable accurate measurement. The sample viscosity (0.890 cP) and the water refractive index (1.330) were factored in particlesize measurement by using the instrument software. Light scattering was monitored at a 90 °C angle and temperature 25 °C. Distilled water filtered through a 0.45-mm filter was used as the dilution medium. Three replicate analyses were performed for each formulation, and data are presented as mean ± SD.
2.7. Dissolution study Dissolution tests involved use of an USP 32 apparatus II (paddle method) in an RC806 dissolution tester (Shishin Technology Co., Taipei) according to the US Pharmacopoeia XXII general method. An equal micromolar amount of ellagic acid powder and EASNEDS were measured in 900 mL of 0.05 M phosphate buffer, pH 6.8, with and without the solubilizer, 0.2% SDS, at 37 °C under rotation speed 50 rpm. To evaluate the effect of bile salt on ellagic acid dissolution, bile extract (containing bile salts and phospholipids) solution was prepared by dissolving 12 g porcine bile extract and 8.4 g sodium bicarbonate in 1 L deionized water as the dissolution media. After the sample was placed in the dissolution media, an aliquot of 1 mL dissolution medium was obtained and filtered through a 0.2-mm polyethersulfone filter (Pall Corp., New York, NY, USA) at 5, 10, 20, 30, 45 and 60 min incubation. Then, 500 mL of each sample was acidified with 5 mL of 2 M acetic acid and stored at 20 °C for HPLC analysis. Dissolution efficiency (DE), the area under a dissolution curve between defined time points compared for characterizing dissolution profiles, was determined according to the following Eq. (1). t
DE ¼
f t21 y dt y100 ðt2 t1 Þ
100%
ð1Þ
where y is the percentage of dissolved product. DE is the area under the dissolution curve between time points t1 and t2 expressed as a percentage of the curve at maximum dissolution, y100, over the same time period (Anderson et al., 1998). 2.8. HPLC analysis HPLC involved use of an analytical model 584 solvent delivery gradient HPLC module (ESA Biosciences, Chelmsford, MA, USA), a YMC-Pack ODS-AM C18 column (4.6 250 mm, 5 mm) protected by a guard cartridge (Hichrom 5C18, Berkshire, UK) and a Hitachi L-7455 photodiode array detector (Hitachi Ltd., Tokyo). Chromatographic separation performed at 25 °C involved use of a linear mobile phase gradient including 0.2% acetic acid in H2O (solvent A) and 0.2% acetic acid in methanol (solvent B). After sample aliquots (20 mL) were introduced into the HPLC system, solvent B was increased from 0 to 50% over 20 min, to 60% within the next 10 min, isocratic at 60% over 6 min and finally to 0% within 5 min and held for the next 5 min. The eluted components were detected at 254 nm, and the UV spectrum between 200 nm to 400 nm was the reference.
2.5. Loading capacity assessment 2.9. Animals EA-SNEDS were diluted with water to various concentrations of ellagic acid in a definite volume in a flask. The flask was inverted and shaken gently to mix thoroughly. The particle size of the formed nanoemulsions was determined by photon correlation spectroscopy at 25 °C.
The experimental protocol was approved by the Institutional Animal Care and Use Committee of NTU (project no. NTU-100EL-114). Male Wistar rats (250 ± 30 g) were purchased from BioLASCO Taiwan Co. (Taipei) and housed in cages, with free access
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to water and food, at 25 °C with air-conditioning, constant humidity and a controlled 12-h light–dark cycle in the animal house of the Institute of Food Science and Technology, NTU. Rats were acclimatized for at least 7 days before experiments. 2.10. Oral bioavailability in rats Rats were deprived of food but had free access to water for 24 h before experiments and were divided randomly into 3 groups (n = 4) for oral gavage administration of ellagic acid aqueous suspension with 0.5% CMC, EA-SNEDS or pomegranate extract. The dosage of ellagic acid used in the tests was a constant 17.6 mmol/ kg body weight, which was translated from recommended daily intake of ellagic acid by humans, 200 mg/day, by body-surfacearea normalization (Reagan-Shaw, Nihal, & Ahmad, 2008). The dosage of pomegranate extract was determined as ellagic-acid equivalents according to the method described by Päivärinta et. al. (Päivärinta, Pajari, Törrönen, & Mutanen, 2006). Briefly, polymeric ellagitannins were converted to ellagic acid in 20-h acid hydrolysis with 1.2 M HCl at 85 °C and quantified as ellagic-acid equivalents. After oral administration of ellagic acid to rats, 500 mL blood sample was collected from the tail vein into heparinized microcentrifuge tubes at various times, then centrifuged at 4 °C under 5000g for 10 min to obtain plasma. The content of ellagic acid in plasma was determined by an HPLC-MS/MS system (Acquity UPLCQuattro Premier XE MS system, Waters Co., Milford, MA, USA). Analytical samples were prepared by mixing 50 mL plasma and 150 mL methanol, which was centrifuged to remove insoluble matter, and the supernatant underwent HPLC-MS/MS. The calibration curve was prepared by using blank plasma spiked with ellagic acid at 5–500 ng/mL. For HPLC-MS/MS, chromatographic separation involved a Biosil ODS column (4.6 150 mm, 5 mm) with an isocratic elution mobile phase of 60% methanol and 40% H2O at flow rate 1 mL/min with a postcolumn split volume ratio of 2/10 to the mass detector. The MS analysis involved ESI MRM analyses: for peak areas of m/z 301 ? 185.1, the operating parameters were capillary voltage, 3 kV; cone voltage, 37 V; collision voltage, 30 V; ion source temperature, 120 °C; and dissolvation temperature, 400 °C. The pharmacokinetic parameters were determined by using the pharmacokinetic software WinNonlin Standard Edition v1.1 (Pharsight Corp., Mountain View, CA, USA). Maximum concentration (Cmax) and time to reach maximum concentration (Tmax) were obtained directly from the concentration–time curve for each of the corresponding results. Area under the plasma ellagic acid concentration–time curve (AUC0a and AUC01) was determined. 2.11. Statistical analysis Independent Student’s t test was used to compare the means of two groups. The level of significance was set at p < 0.05. Statistical analyses involved use of SigmaPlot 10.0 (SPSS, Inc., Chicago, IL, USA). 3. Results 3.1. Solubility studies In self-nanoemulsifying systems, analytes are solubilized in the oily core and/or on the interface of the nanoemulsion structures (Setthacheewakul, Mahattanadul, Phadoongsombut, Pichayakorn, & Wiwattanapatapee, 2010). Hydrophobicity of the analytes, and presence of surfactants, co-surfactants and oils, affects the analytes solubility. The results from our solubility test (Table 1) revealed that ellagic acid has reached the highest solubility (4.18 and
3.59 mg/mL) in PEG200 and PEG400, which were therefore chosen as co-surfactants for further tests. The greater solubility in PEG may be due to the ability of ellagic acid to form hydrogen bonding with the polyethylene oxide (PEO) groups. Similarly, ellagic acid showed high solubilization capacity with surfactants composed of PEO groups such as Tween20 and Tween80. Oleic acid and Palmester 3575, conferring relatively high solubility of ellagic acid (0.012 and 0.03 mg/mL, respectively), were selected as oil phases. 3.2. Construction of pseudo-ternary phase diagrams Pseudo-ternary phase diagrams were constructed by progressive titration of the component mixtures to identify the nanoemulsion regions and optimize the concentration of the selected vehicles, including PEG200, PEG400, Tween20, Tween80, oleic acid and Palmester 3575. To develop a SNEDS formulation, the optimal ratios of excipient concentrations were established by phase diagram studies, presenting the area of the monophasic region. Moreover, this area must be determined because successful aqueous dilution without a nanoemulsion phase transition is important in oral delivery (Spernath, Aserin, & Garti, 2006). Fig. 2 depict the phase diagrams for eight different oil–surfac tant–water systems. These phase diagrams contained different areas of clear nanoemulsions (grey region), and coarse emulsions (white region). Diagrams containing larger nanoemulsion areas corresponded to formulations with Tween80 as a surfactant phase, PEG200 and 400 as a co-surfactant phase and Palmester 3575 as an oil phase. Formulation with polyethylene glycol, polysorbate, caprylic/ capric triacylglycerol at the ratio of 45/45/10 wt.%, can be fully diluted with water along the dilution line without phase separation, may be suitable for oral administration. Furthermore, the formulation with PEG400 showed higher isotropic stability than those with PEG200 as a co-surfactant (Suppl. 1) and was therefore chosen as components of the optimal SNEDS formulation. 3.3. Nanoemulsion droplet size analysis and morphology observation by TEM The effect of ellagic acid loading on particle size in distilled water is presented in Fig. 3 A. The mean particle size increased slightly with increased ellagic acid loading concentration from 0.5 to 2.5 mg mL. With further increase in ellagic acid loading, particle size increased greatly, as high as 120 nm or more, beyond the range of the colloidal system and no longer presented the properties of nanoemulsion. Undissolved ellagic acid in the formulation could have altered the mean droplet size to increase. The morphol-
Table 1 Solubility of ellagic acid in various vehicles. Vehicles Oils Castor oil Corn oil Limonene Cottonseed oil Oleic acid EPA DHA ethyl esters Palmester 3575a Ethyl oleate Soybean oil
Solubility (mg/mL) N/A* N/A N/A 0.005 0.012 N/A 0.03 N/A N/A
Vehicles Surfactants Lecithin Sucrose esters TPGS Tween20 Tween80 Co-solvents PEG200 PEG400 Ethanol
Solubility (mg/mL) 0.264 0.115 N/A 1.605 2.739 4.178 3.588 0.668
TPGS, D-a-Tocopherol polyethylene glycol succinate; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; PEG, polyethylene glycol. a Caprylic/capric triacylglycerols * N/A, solubility lower than the detection limit.
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Fig. 2. Pseudo-ternary phase diagram of self-nanoemulsifying delivery system (SNEDS) with water as a dilution medium, oleic acid as an oil phase, and (A) Tween20 as a surfactant and PEG200 as a co-surfactant, (B) Tween20 as a surfactant and PEG400 as a co-surfactant, (C) Tween80 as a surfactant and PEG200 as a co-surfactant and (D) Tween80 as a surfactant and PEG400 as a co-surfactant, or Palmester 3575 as an oil phase, and (E) Tween20 as a surfactant and PEG200 as a co-surfactant, (F) Tween20 as a surfactant and PEG400 as a co-surfactant, (G) Tween80 as a surfactant and PEG200 as a co-surfactant and (H) Tween80 as a surfactant and PEG400 as a co-surfactant.
Fig. 3. (A) Effect of ellagic acid concentration on droplet size of nanoemulsions. Photographs represent the corresponding outward appearance of the self-nanoemulsifying delivery system (SNEDS) with various ellagic acid-loading amounts. (B) 25,000 and (C) 5000 of microphotographs of ellagic acid-loaded self-nanoemulsifying delivery system (EA-SNEDS) by transmission electron microscopy. Data are mean ± SD (n = 3).
ogy of ellagic acid nanoemulsion was characterized by TEM (Fig. 3B and C), showing the spherical shape and uniform droplet size of nanoemulsions. In addition, the mean droplet size of nanoemulsion might have increased with the amount of ellagic acid because ella-
gic acid might embed in the interfacial film. Samples were diluted with distilled water before testing to avoid the multiscattering phenomena. The droplet size of the diluted nanoemulsion was not significantly changed.
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3.4. In vitro dissolution study The dissolution of ellagic acid from the SNEDS and the unformulated powder of ellagic acid were evaluated in pH 6.8 phosphate buffer, phosphate buffer containing 0.2% SDS and bile salt solution. The dissolution proportion was significantly higher with ellagic acid in the SNEDS form than the unformulated powder in all dissolution media studied (Fig. 4). Only about 16% of tested ellagic acid was dissolved in dissolution medium with 60-min incubation (Fig. 4A), whereas EA-SNEDS was dissolved at 50% with about 2min incubation and 88% with 20-min incubation. Both 0.2% SDS and bile extract solution improved the dissolution of ellagic acid powder. The dissolution with 0.2% SDS and bile extract solution was increased to about 31% (Fig. 4B) and 23% (Fig. 4C) with 60min incubation. Even so, ellagic acid powder still showed poor dissolution profiles, whereas EA-SNEDS showed highly improved solubility in all dissolution media. Mean DE values for EA-SNEDS were 80.93% to 97.55%, significantly higher than for ellagic acid powder with various dissolution buffers (Table 2). 3.5. Oral bioavailability studies To further evaluate the efficacy of EA-SNEDS for the absorption of ellagic acid by oral administration, we used the optimal formulation of EA-SNEDS and ellagic acid aqueous suspension with 0.5% CMC at 17.6 lmol/kg body weight in rats. In addition, because pomegranate and its products are the most common source of ellagic acid in the functional food market, we introduced pomegranate extract with a same molar ratio of the aforementioned tested group to determine the plasma level of ellagic acid after pomegra-
Table 2 Dissolution efficiency (%) of ellagic acid unformulated powder and ellagic acid-loaded self-nanoemulsifying delivery system (EA-SNEDS) in various dissolution buffers.
a *
Dissolution efficiency (%)a
Phosphate buffer pH 6.8
Phosphate buffer with 0.2% SDS
Bile salt solution
Ellagic acid EA-SNEDS
11.03 ± 0.32 80.93 ± 1.37*
24.49 ± 1.06 97.55 ± 3.08*
17.86 ± 0.43 84.68 ± 3.08*
Data are mean ± SD (n = 3). p < 0.05 comparing EA-SNEDS to ellagic acid group, unpaired t test.
nate consumption. The ellagic acid molar ratio of 1 g pomegranate extract was determined as 0.159 mmol and we used 111 mg/kg body weight of that. Oral absorption of ellagic acid in suspension was the least absorptive and that in SNEDS group was the highest (Fig. 5). The plasma profiles for EA-SNEDS showed two peaks, which indicated that ellagic acid was absorbed rapidly, with Tmax at 30 min after oral administration, and reabsorption via enteralhepatic circulation. The plasma profile for ellagic acid was similar to that from a previous study (Hamad et al., 2009). The Cmax for EA-SNEDS was 527.95 nM (Table 3), about 5 times higher than that for pomegranate extract, whereas the Cmax for ellagic acid suspension was 54.37 nM. The AUC0-1 reflects the exposure of plasma to ellagic acid from time 0 to the time when the plasma ellagic acid concentration returns to baseline. After oral administration, EA-SNEDS showed the highest AUC0-1 for ellagic acid: 6.6 and 3.2 times higher than with aqueous ellagic acid suspension and pomegranate extract, respectively. Thus, orally administered EA-SNEDS could be a strategy for developing novel products of dietary supplements and functional foods with ellagic acid.
Fig. 4. Dissolutions of ellagic acid unformulated powder and ellagic acid-loaded self-nanoemulsifying delivery system (EA-SNEDS) in different solutions. (A) pH 6.8, phosphate buffer alone; (B) phosphate buffer containing 0.2% SDS; (C) bile salt solution. Data are mean ± SD (n = 3).
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Fig. 5. Mean plasma concentration/time profiles of ellagic acid in rats with oral administration of ellagic acid suspension, ellagic acid-loaded self-nanoemulsifying delivery system (EA-SNEDS) or pomegranate extract at 17.6 lmol/kg body weight ellagic acid equivalent. Data are mean ± SE (n = 4).
Table 3 Pharmacokinetic parameters of plasma ellagic acid with oral administration of ellagic acid suspension, pomegranate extract and ellagic acid-loaded self-nanoemulsifying delivery system (EA-SNEDS). Parametersa
Ellagic acid
Pomegranate extract
EA-SNEDS
Cmax (nM) Tmax (min) AUC0-a (nM h) AUC0-1 (nM h)
54.37 ± 2.35 30 41.06 ± 14.40 51.72 ± 14.97
106.09 ± 11.66 60 77.68 ± 21.19 103.08 ± 12.98
527.95 ± 54.83* 30 264.77 ± 28.25* 342.58 ± 43.28*
a
Data are mean ± SE (n = 4). p < 0.05 comparing EA-SNEDS to ellagic acid and pomegranate extract groups, unpaired t test. *
4. Discussion In this work, we developed a food-grade self-nanoemulsifying system to improve the dissolution and absorption in delivering ellagic acid. With this system, the oral bioavailability of ellagic acid was significantly superior to aqueous suspensions of ellagic acid and pomegranate extract. in rats. Pomegranate is one of the world’s oldest known ancient fruits and has gained commercial significance recently with the verification of its nutraceutical and pharmacological properties. Pomegranate extract has been reported to have various health effects such as antioxidation, antiproliferation and inhibition of adipogenesis (Nuncio-Jáuregui et al., 2015; Orgil, Spector, Holland, Mahajna, & Amir, 2016; Wu, Ma, & Tian, 2013). Convincing evidence revealed that the health benefits were due to the production of ellagic acid in the GI tract after oral ingestion by humans (Akileshwari et al., 2014; NuncioJáuregui et al., 2015; Wu et al., 2013), which involved spontaneous hydrolysis from ellagitannins at neutral pH environment. Even though ellagic acid features remarkable bioactivity, it is extremely low aqueous soluble and has been classified under the Biopharmaceutical Classification System as a class IV substance with low solubility (<10 mg/mL in phosphate buffer, pH 7.4) and low permeability (0.13 106 cm/s) (Waldmann et al., 2012). Moreover, ellagic acid has been found to bind irreversibly to cellular DNA and proteins and metabolized by first-pass enzymes or microbes, which may also explain its limited transcellular absorption (Whitley et al., 2003). The poor absorption of ellagic acid has been reported to affect its in vivo bioactivity because sufficient concentrations may not be in plasma or target organs after oral administration and therefore limit its use in functional foods (Seeram et al., 2004). For our EA-SNEDS formulation, the choice of excipients to prepare SNEDS depends on the ellagic acid dissolving capacity. Ellagic
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acid showed extremely low solubility in various oil phases, including long-chain triacylglycerol (LCT), medium-chain triacylglycerol (MCT), terpene, long chain fatty acids and fatty acid esters (Table 1), which may be due to the polarity donated by its hydroxyl groups. Ellagic acid can only be slightly dissolved in oils showing relatively high polarity, such as MCT and fatty acids. MCT are commonly used in SNEDS formulations (Shah, Carvajal, Patel, Infeld, & Malick, 1994). They passively diffuse from the GI tract to the portal system (longer fatty acids are absorbed into the lymphatic system) without the need for modification as for long-chain or very longchain fatty acids. MCT possess higher ester content per gram than LCT that means MCT has better capability of dissolving ellagic acid than LCT (Cao, Marra, & Anderson, 2004). In this study, we select oleic acid and Palmester 3575 for the oil phase of the delivery system. The selection of surfactant and cosurfactant was governed by emulsification efficiency rather than the ability to solubilize substances. The efficiency of selfnanoemulsification is related more to the hydrophilic–lipophilic balance (HLB) value of the surfactant. Surfactants with HLB > 10 are good at providing fine, uniform nanoemulsion droplets. Surfactants increase the permeability by interfering with the lipid bilayer of the epithelial cell membrane (Gursoy & Benita, 2004). We chose two high-value surfactants, Tween20 (HLB = 15, 1.6 mg/mL) and Tween80 (HLB = 15, 2.7 mg/mL), as surfactants for EA-SNEDS. Cosurfactants increase the interfacial fluidity by penetrating the surfactant film and creating a disordered film due to the void space among surfactant molecules. The emulsification efficiency of the SNEDS formulation changes with the chain length of the cosurfactants. PEG200 (4.2 mg/mL) and PEG400 (3.6 mg/mL) solubilize ellagic acid in good amount. Hence, we chose these two cosurfactants for the EA-SNEDS formulation. The ternary phase diagrams (Fig. 2) show a high concentration of surfactant and high self-nanoemulsifying region. The relatively high concentration of surfactant needed to form our nanoemulsions agrees with other studies: high concentrations of surfactants were needed to achieve fast and efficient self-nanoemulsication (Ting, Jiang, Ho, & Huang, 2014). Among all tested formulations, the delivery system of Tween80, PEG400 and Palmester 3575 conferred the highest nanoemulsion ability and stability and could undergo aqueous phase dilution without phase transition and so was chosen as the optimal formulation. The optimal formulation performed a high loading capacity of ellagic acid which is 250 time higher than its aqueous solubility. With the optimal EA-SNEDS, ellagic acid could be dissolved easily because of the small droplet size, which permitted a faster rate of substance dissolution into the aqueous phase, faster than unformulated powder, which may greatly affect the bioavailability. In addition, because phosphate buffer may not mimic the effect of bile salt on solubility of ellagic acid, we used bile extract solution to represent the condition in the intestinal medium (Tzoumaki, Moschakis, Scholten, & Biliaderis, 2013). Phenolic compounds were found to be more bioavailable with solubilizer effect of bile salts when being consumed in fed state. Nevertheless, the results with ellagic acid powder still showed poor dissolution in bile extract solution, indicating extremely low solubility of ellagic acid in GI tract. Ellagic acid with SNEDS showed almost complete dissolution within 60 min, so this formulation may dissolve well in intestinal conditions. The pharmacokinetics profiles agreed highly with the dissolution profiles. With the SNEDS formulation, the plasma concentration of ellagic acid in rats indicated significantly greater improvement of substance absorption than with ellagic acid suspension (Fig. 5). We observed a double peak profile with plasma exposure of EA-SNEDS that may have been due to gastric motility after administration or may represent an active and proficient entero-hepatic (or enterocytic) recycling of ellagic acid. Unexpect-
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edly, pomegranate extract showed delayed Tmax at 60 min, which could be attributed to the release process of ellagic acid from ellagitannins in the extracts. Research indicated that the in vivo hydrolysis of ellagitannins to release ellagic acid can be a critical limiting step in ellagic acid bioavailability (Daniel, Ratnayake, Kinstle, & Stoner, 1991). However, experimental data did not support the concept. González-Sarrías et al. conducted a pharmacokinetic study using pomegranate extract with different ellagitannins to free ellagic acid ratio as oral gavage substance and the results demonstrated that intake of a higher dose of free ellagic acid did not significantly change plasma ellagic acid values, which was agreed with our results (González-Sarrías et al., 2015). In our study, we found that the plasma level of pomegranate extract was even higher than that of free ellagic acid, indicating the food matrix from pomegranate extract may play a role in the bioavailability. Phenolic compounds were reported to be reduced in crystallinity and improved in dissolution by food matrix such as lipids and carbohydrate, and may therefore affect its bioavailability (Chen, Yang, Uang, & Lin, 2013). Even so, SNEDS formulation offered significant improvement of dissolution efficiency and bioavailability of ellagic acid, with almost three-fold higher oral bioavailability than pomegranate extract consumption. Previous research aimed at improving the bioavailability of ellagic acid by performing ellagic acid-phospholipids complex, and may increase both the bioavailability and peak plasma concentration to almost 2.5-fold over free ellagic acid (Murugan et al., 2009). The complex also showed better hepatoprotective activity compared with free ellgic acid. However, the biological effects were based on relative high dosage that would be difficult to consume. In our study, EA-SNEDS formulation performed 6-fold better in bioavailability and almost 10-fold in Cmax, respectively. The amount of ellagic acid we administrated to experimental rats was translated from a reasonable human dosage that may have been seen commonly in ellagic acid based dietary supplement products. The remarkable absorption enhancing ability of EASNEDS may be advantageous for delivering ellagic acid. Literally, this delivery system offers several advantages including high solvent power, improved permeability across the GI tract membrane, decreased or diminished food effect, and improved substance bioavailability (Ghai & Sinha, 2012). The bioavailability-enhancing property of the self-emulsifying formulations results from improved compound dissolution and also several mechanisms including the reduction of first-pass drug metabolism in the liver (Sun et al., 2011). SNEDS may increase the contact of ellagic acid to the epithelial membrane by improving aqueous solubility and thus intestinal absorption (Zou et al., 2015). The SNEDS formulation should result in higher bioavailability because of more rapid and uniform distribution of the substance in the GI. The reduced first-pass effect may be attributed to the association of lipid components with selective compound engulfing into the lymphatic transport system. It may also result from the ability of lipid excipients and their metabolites to cause changes in the absorption of GI fluid-promoting compounds (Khan et al., 2015). In addition, the delivery system was found to inhibit intestinal efflux such as p-glycoprotein and multidrug resistance-associated protein 2, thus enhancing transepithelial flux and increasing systemic exposure (Borhade, Nair, & Hegde, 2008; Li, Yi, & Lam, 2014). These advantages and the delivery strategy may help enhance the absorption and plasma level of ellagic acid with consumption. Hence, for oral administration of ellagic acid, SNEDS might be a promising approach for effective absorption and could be used to increase the bioavailability for other poorly water-soluble substances. In addition, it was reported that the no observed-adverse-effect level (NOAEL) of ellagic acid was 3254 mg/kg/day in rats (Tasaki et al., 2008). In this study, the dosage was set at 5.3 mg/kg, which was far away from the NOAEL.
Thus, it may still be harmless to subjects even though the plasma exposure of ellagic acid was enhanced by the formulation. Moreover, the physiological and toxicological effects of improving absorption are unclear, and therefore, further research is absolutely necessary. The market for pomegranate products has grown in recent years and pomegranate extracts was approved in the health food market (Fischer, Jaksch, Carle, & Kammerer, 2012). According to our results, EA-SNEDS may be an alternative to pomegranate extract and offer improved bioavailability of ellagic acid. Conflict of interest statement The authors have no conflicts of interest for this work. Acknowledgements This work was supported by the Ministry of Science and Technology, Executive Yuan, Taiwan [grant number: MOST 101-2313B-002-068-MY3]. The authors would like to thank Dr. Shu-Chen Hsieh, Institute of Food Science and Technology, National Taiwan University for help with animal housing. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jff.2017.04.033. References Aguilera-Carbo, A., Augur, C., Prado-Barragan, L. A., Favela-Torres, E., & Aguilar, C. N. (2008). Microbial production of ellagic acid and biodegradation of ellagitannins. Applied Microbiology and Biotechnology, 78, 189–199. Akileshwari, C., Raghu, G., Muthenna, P., Mueller, N. H., Suryanaryana, P., Petrash, J. M., & Reddy, G. B. (2014). Bioflavonoid ellagic acid inhibits aldose reductase: Implications for prevention of diabetic complications. Journal of Functional Foods, 6, 373–383. Anderson, N. H., Bauer, M., Boussac, N., Khan-Malek, R., Munden, P., & Sardaro, M. (1998). An evaluation of fit factors and dissolution efficiency for the comparison of in vitro dissolution profiles. Journal of Pharmaceutical and Biomedical Analysis, 17, 811–822. Borhade, V., Nair, H., & Hegde, D. (2008). Design and evaluation of selfmicroemulsifying drug delivery system (SMEDDS) of tacrolimus. An Official Journal of the American Association of Pharmaceutical Scientists, 9, 13–21. Cao, Y., Marra, A. Y., & Anderson, B. D. (2004). Predictive relationships for the effects of triglyceride ester concentration and water uptake on solubility and partitioning of small molecules into lipid vehicles. Journal of Pharmaceutical Sciences, 93, 2768–2779. Chen, C. H., Yang, J. C., Uang, Y. S., & Lin, C. J. (2013). Improved dissolution rate and oral bioavailability of lovastatin in red yeast rice products. International Journal of Pharmaceutics, 444, 18–24. Clifford, M. N., & Scalbert, A. (2000). Ellagitannins – Nature, occurrence and dietary burden. Journal of the Science of Food and Agriculture, 80, 1118–1125. Daniel, E. M., Ratnayake, S., Kinstle, T., & Stoner, G. D. (1991). The effects of pH and rat intestinal contents on the liberation of ellagic acid from purified and crude ellagitannins. Journal of Natural Products, 54, 946–952. Doyle, B., & Griffiths, L. A. (1980). The metabolism of ellagic acid in the rat. Xenobiotica, 10, 247–256. Fischer, U. A., Jaksch, A. V., Carle, R., & Kammerer, D. R. (2012). Determination of lignans in edible and nonedible parts of pomegranate (Punica granatum L.) and products derived therefrom, particularly focusing on the quantitation of isolariciresinol using HPLC-DAD-ESI/MSn. Journal of Agricultural and Food Chemistry, 60, 283–292. García-Villalba, R., Espín, J. C., Aaby, K., Alasalvar, C., Heinonen, M., Jacobs, G., ... Tomás-Barberán, F. A. (2015). Validated method for the characterization and quantification of extractable and nonextractable ellagitannins after acid hydrolysis in pomegranate fruits, juices, and extracts. Journal of Agricultural and Food Chemistry, 63, 6555–6566. Ghai, D., & Sinha, V. R. (2012). Nanoemulsions as self-emulsified drug delivery carriers for enhanced permeability of the poorly water-soluble selective b1adrenoreceptor blocker Talinolol. Nanomedicine, 8, 618–626. González-Sarrías, A., Rocío García-Villalba, R., Núñez-Sánchez, M. A., ToméCarneiro, J., Zafrilla, P., Mulero, J., ... Espín, J. C. (2015). Identifying the limits for ellagic acid bioavailability: A crossover pharmacokinetic study in healthy volunteers after consumption of pomegranate extracts. Journal of Functional Foods, 19, 225–235.
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