Article pubs.acs.org/JAFC
Improving Flavonoid Bioaccessibility using an Edible Oil-Based Lipid Nanoparticle for Oral Delivery Choongjin Ban,†,$ So Jeong Park,∥,$ Seokwon Lim,⊥ Seung Jun Choi,# and Young Jin Choi*,†,‡,§ †
Department of Agricultural Biotechnology, ‡Center for Food and Bioconvergence, and §Research Institute of Agriculture and Life Sciences, Seoul National University, 1 Gwanakro, Gwanakgu, Seoul 151-921, Korea ∥ Dongsuh Foods Corporation, 203 Bupyeongbuk-ro, Bupyeong-gu, Incheon 403-858, Korea ⊥ Department of Food and Biotechnology, Hoseo University, 79-20 Hoseoro, Asan, Chungnam 336-795, Korea # Department of Food Science and Technology, Seoul National University of Science and Technology, 232 Gongneungro, Nowongu, Seoul 139-743, Korea S Supporting Information *
ABSTRACT: To enhance the oral bioaccessibility of flavonoids, including quercetin, naringenin, and hesperetin, we prepared an edible oil-based lipid nanoparticle (LNP) system. Flavonoid-loaded LNPs were similar to the blank LNP in physicochemical characteristics (z average 71%, which was otherwise 98%; SQ) was obtained from Alfa Aesar (Heysham, England). Tween 20 (T20; polyoxyethylene sorbitan monolaurate) and soybean lecithin (SL) were purchased from SigmaAldrich Co. (St. Louis, MO, USA) and Fisher Scientific (Pittsburgh, PA, USA), respectively. Quercetin and naringenin were obtained from MP Biomedicals LLC (Solon, OH, USA), and hesperetin was obtained from Sigma-Aldrich Co. All other chemicals were of analytical reagent grade. Lipid Nanoparticle Production. The LNPs were prepared using an oil-in-water emulsion technique with a high-speed blender and sonication probe as reported previously by us,17 with a slight modification. First, the lipid (5 wt %) and aqueous (95 wt %) phases were heated to 85 °C, mixed using a high-speed blender (Ultra-Turrax T25D, Ika Werke GmbH & Co., Staufen, Germany) at 8000 rpm for 1 min and then at 11000 rpm for 1 min, and maintained at 85 °C; the lipid phase of the blank LNPs was a mixture of 5−3.5 wt % FHCO (100−70 wt % of the lipid phase) and 0−1.5 wt % of each liquid oil (0−30 wt % of the lipid phase) among LSO, LCO, and SQ;, and the aqueous phase was prepared by adding an emulsifier mixture (onethird of the lipid phase weight) composed of T20 (100−0 wt % of the emulsifier mixture) and SL (0−100 wt % of the mixture) in doubly
EE (%) =
Wt − Wn × 100 Wt
where Wt is the total flavonoid molecule weight in the entire LNP sample system and Wn is the flavonoid molecule weight in the nbutanol layer after centrifugation. Determining the in Vitro Digestion Patterns of the Lipid Nanoparticles. The simulated in vitro digestion test model was modified from the version described by Hur et al:25 (I) preingestion: LNPs were filtered with a 1 μm pore sized filter (II) mouth (pH 7; 5 min): 5 mL of filtered LNPs was blended with 6 mL simulated salivary medium (III) stomach (pH 3; 2 h): 12 mL of simulated gastric juice was added (IV) small intestine (pH 6.5−7; 2 h): simulated duodenal juice (12 mL), bile juice (6 mL), and NaHCO3 solution (2 mL) were added B
DOI: 10.1021/acs.jafc.5b01495 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Table 1. Formulations and Concentrations of the Various Media and Juices for the Simulated in Vitro Digestion Test of Blank Lipid Nanoparticles saliva medium inorganic solution
20 mL NaHCO3, 84.7 g L−1 10 mL KCl, 89.6 g L−1
gastric juice 15.7 mL NaCl, 175.3 g L
duodenal juice −1
9.2 mL KCl, 89.6 g L−1
10 mL NaH2PO4, 88.8 18 mL CaCl2·2H2O, 22.2 g L−1 g L−1 1.7 mL NaCl, 175.3 g L−1 3.0 mL NaH2PO4, 88.8 g L−1 10 mL Na2SO4 ,57 g L−1 10 mL NH4Cl, 30.6 g L−1 10 mL KSCN, 20 g L−1 6.5 mL HCl, 35% g g−1
40 mL NaCl, 175.3 g L
30 mL NaCl, 175.3 g L−1
40 mL NaHCO3, 84.7 g L−1 6.3 mL KCl, 89.6 g L−1
60.3 mL NaHCO3, 84.7 g L−1 4.2 mL KCl, 89.6 g L−1
9 mL CaCl2·2H2O, 22.2 g L−1 10 mL KH2PO4 ,8 g L−1 10 mL MgCl2, 5 g L−1 180 μL HCl, 35% g g−1 4 mL urea, 25 g L−1
10 mL CaCl2·2H2O, 22.2 g L−1 150 μL HCl, 35% g L−1
organic solution
8 mL urea, 25 g L−1
add to mixture inorganic + organic solution
15 mg uric acid
34 mL urea, 25 g L−1 10 mL glucose, 65 g L−1 10 mL glucosamine hydrochloride, 33 g L−1 10 mL glucuronic acid, 2 g L−1 1 g BSA 1 g BSA
25 mg mucin 290 mg α-amylase 6.8
3 g mucin 2.5 g pepsin 1.3
pH
bile juice −1
10 mL urea, 25 g L−1
1.8 g BSA
9 g pancreatin 1.5 g lipase 8.1
30 g bile 8.2
Figure 1. Creaming pattern of the blank lipid nanoparticle dispersion diluted 10-fold: (a) blank lipid nanoparticle dispersions prepared using 100 wt % fully hydrogenated canola oil (FHCO) as the lipid phase; blank lipid nanoparticle dispersions prepared using a mixture of 90−70 wt % FHCO and 10−30 wt % liquid oil, including (b) liquid soybean oil (LSO), (c) squalene (SQ), or (d) liquid canola oil (LCO) as the lipid phase. The formulations of the simulated saliva medium and gastric, duodenal, and bile juices are shown in Table 1. All samples were stirred (60 rpm) in a shaking water bath (BS-31, JEIO Tech., Seoul, Korea) during the in vitro digestion test and maintained at 37 °C to mimic gastrointestinal tract motility. After each digestion step (I−IV), the size distribution of samples was measured using the Zetasizer. After 4 h 5 min of digestion, the relative bioaccessibility of the flavonoid (quecetin, naringenin, and hesperetin) was determined as described earlier,16 with modifications. Briefly, 2 mL of digesta was centrifuged at 1500 RCF for 30 min, and 1 mL of the supernatant was recentrifuged at 16000 RCF for 20 min. A 0.2 mL aliquot of the supernatant was collected and diluted 10-fold with 1.8 mL of methanol, and the solution was centrifuged at 16000 RCF for 20 min. The concentration of flavonoids in the supernatant was quantified
using a spectrophotometer and calculated using standard curves ranging from 0.625 to 10 μg mL−1 for quercetin (λ 384 nm, R2 = 0.9971), naringenin (λ 325 nm, R2 = 0.9990), and hesperetin (λ 324.5 nm, R2 = 0.9999). The relative bioaccessibility of the flavonoids in the digested micellar fraction was determined using the equation bioaccessibility (%) =
Wt − Ws × 100 Wt
where Ws is the weight of the flavonoid molecules in the supernatant after digestion and centrifugation. In addition to the flavonoidencapsulated LNPs, equivalent quantities (150 μg mL−1) of quercetin, naringenin, and hesperetin in their native forms were also investigated. The protectibility (%) of LNPs for flavonoids against harsh conditions (pH and salt) was determined after each digestion step C
DOI: 10.1021/acs.jafc.5b01495 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Table 2. Nonaggregated Particle Content (Yield), Particle Size, Polydispersity Index (PDI), ζ Potential (ZP), and Entrapment Efficiency (EE) Values of Blank (BLK) and Flavonoid-Loaded Lipid Nanoparticles (LNPs)a flavonoid concn (wt %) code name BLK LNP 0.1Q LNP 0.2Q LNP 0.3Q LNP 0.4Q LNP 0.5Q LNP 0.1N LNP 0.2N LNP 0.3N LNP 0.4N LNP 0.5N LNP 0.1H LNP 0.2H LNP 0.3H LNP 0.4H LNP 0.5H LNP a
Q
N
H
0.1 0.2 0.3 0.4 0.5 0.1 0.2 0.3 0.4 0.5 0.1 0.2 0.3 0.4 0.5
yield (%) 94.4 94.7 94.7 94.5 94.8 95.4 94.1 94.1 94.2 94.2 94.1 94.9 94.8 94.2 94.3 94.9
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.4a 0.4a 0.1a 0.1a 0.2a 0.1a 0.8a 0.3a 1.1a 0.9a 0.2a 0.3a 0.3a 0.4a 0.9a 0.8a
particle size (nm) 155.7 152.7 153.7 154.7 148.9 144.9 156.2 152.1 149.9 148.8 149.1 155.2 154.7 154.8 150.9 149.9
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.4ab 1.7abcd 0.8abc 2.2ab 0.9e 0.8f 0.2a 0.6bcde 0.4de 1.8e 0.2de 2.7ab 0.2ab 0.2ab 0.8cde 1.2de
PDI 0.15 0.16 0.19 0.16 0.17 0.16 0.14 0.16 0.14 0.16 0.17 0.18 0.17 0.17 0.16 0.16
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.01a 0.02a 0.01a 0.02a 0.03a 0.00a 0.02a 0.01a 0.03a 0.02a 0.01a 0.01a 0.01a 0.01a 0.02a 0.02a
ZP (mV) −41.8 −45.1 −44.2 −43.6 −42.8 −43.9 −42.4 −40.6 −42.1 −42.3 −42.9 −40.5 −42.2 −40.8 −41.7 −42.1
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.5abcd 0.8g 0.5fg 0.4defg 0.6cdef 0.6efg 0.3bcde 0.7ab 0.6abcde 0.5abcde 0.3cdef 1.0a 0.5abcde 0.7ab 0.6abc 0.7abcd
EE (%) 69.7 74.6 81.0 71.3 67.9 80.0 89.0 90.0 85.8 88.0 72.5 82.1 90.0 88.7 89.7
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.9ef 1.3de 0.9bc 2.9ef 0.4f 0.4cd 2.0a 0.5a 1.4ab 0.4a 4.0ef 1.5bc 2.5a 1.2a 1.1a
Different letters a−g in a column are significantly different at p < 0.05. Abbreviations: quercetin, Q; naringenin, N; hesperetin, H.
on the surfaces formed micrometer-sized aggregates. 17 According to Stokes’ law, the particle size effect on the creaming rate predominates over the density difference at the same composition. Therefore, the aggregated micrometer-sized particles creamed readily, whereas the stable nanosized LNPs were well-dispersed in aqueous solution.17 The creaming phenomenon of the LNP aggregates was observed when blank LNPs prepared with 100 wt % FHCO as the lipid phase were diluted 10-fold with DDW (Figure 1a). The micrometer-sized LNP aggregates and the nanosized LNPs formed creaming and aqueous layers, respectively. The particle size of all blank LNPs prepared with 100 wt % FHCO was 123.2−165.1 nm, indicating that submicron-sized particles comprised the aqueous layer. Thus, the thickness of the aqueous layer reflected the number of LNPs stably produced on a nanosize scale, which was related to the colloidal stability of the LNP system. The aqueous layers of samples stabilized with a single emulsifier (SL contents: 0 and 100 wt %) were thinner than those emulsified with T20 and SL (SL contents: 25, 50, and 75 wt %). Additionally, the yields of the 25, 50, and 75 wt % SL content samples were larger than those of the 0 and 100 wt % SL content samples (Table S1 in the Supporting Information), suggesting that combined use of T20 and SL enhanced the colloidal stability of the LNP system, which agrees with other studies.26 This tendency was also applied to the LNP samples fabricated using a lipid mixture with FHCO and liquid oil (LSO, SQ, or LCO) as the lipid phase (Figure 1b−d). We observed in our previous study that increasing the liquid oil content in the LNP lipid phase improves the colloidal stability of the system,17 which was confirmed here (Figure 1b− d). Furthermore, the thickness of the aqueous layer differed when the liquid oil type was changed. Therefore, LNP samples with SQ as the liquid oil in the lipid matrix had the thickest aqueous layer at the same liquid oil and SL contents. This result is in agreement with the yield data (Table S1 in the Supporting Information). These results suggest that using SQ as the liquid oil could produce optimum colloidal stability among LSO, SQ, and LCO. Thus, the preparation formula of blank LNP was optimized by using SQ as the liquid oil.
(II−IV) without enzymes. After each step, 0.2 mL of digesta was collected and diluted 10-fold with 1.8 mL of methanol and centrifuged at 16000 RCF for 20 min. The concentration of flavonoids in the supernatant was quantified using a spectrophotometer and calculated using standard curves ranging from 0.625 to 10 μg mL−1: step II, quercetin (λ 389 nm, R2 = 0.9999), naringenin (λ 325.5 nm, R2 = 1.0000), and hesperetin (λ 325.5 nm, R2 = 0.9999); step III, quercetin (λ 379 nm, R2 = 1.0000), naringenin (λ 290 nm, R2 = 0.9998), and hesperetin (λ 287.5 nm, R2 = 0.9998); step IV, quercetin (λ 389 nm, R2 = 0.9999), naringenin (λ 325 nm, R2 = 1.0000), and hesperetin (λ 325 nm, R2 = 1.0000)). The protectibility of the LNPs incorporating the flavonoids against the enzyme-free digestion medium was determined by the equation protectibilty (%) =
Wt − Ws × 100 We
where We is the weight of the flavonoids encapsulated in the LNPs immediately after preparation. The release patterns of the flavonoids from the LNPs were studied using dialysis bags with a 12 kDa molecular weight cutoff. The bags were immersed in DDW for 12 h before use. They were filled with 1 mL of the flavonoid-loaded LNP sample, tightly sealed, and suspended in 49 mL of 50% (v/v) ethanol to produce a sink condition. The enzyme-free simulated in vitro digestion medium mixture was added, and the bags were rotated at 100 rpm in a 37 °C water bath. At predetermined time intervals, a 1 mL aliquot was withdrawn from the medium mixture, and 1 mL of fresh medium was replaced immediately. Next, the absorbance of the aliquot at the wavelengths for quercetin (377.5 nm), naringenin (291.5 nm), and hesperetin (289.5 nm) was measured with a spectrophotometer. The concentrations of quercetin, naringenin, and hesperetin in the aliquots were calculated using standard curves ranging from 3.125 to 250 μg mL−1 for quercetin (R2 = 0.9996), naringenin (R2 = 0.9999), and hesperetin (R2 = 0.9999). Statistical Analysis. All results were analyzed using Tukey’s significant difference test using the IBM SPSS Statistics version 21.0 software (IBM Co., Armonk, NY, USA). Data represent means of at least three independent experiments or measurements. A p value
