Fabrication of a Soybean Bowman–Birk Inhibitor (BBI) Nanodelivery

*(X.-Q.Y.) Phone: (+86) 020-87114262. ... Here, a novel self-assembly nanoparticle delivery carrier has been successfully developed by using soybean ...
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Fabrication of Soybean Bowman-Birk Inhibitor (BBI) Nanodelivery Carrier to Improve Bioavailability of Curcumin Chun Liu, Fenfen Cheng, and Xiao-Quan Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00097 • Publication Date (Web): 01 Mar 2017 Downloaded from http://pubs.acs.org on March 2, 2017

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Journal of Agricultural and Food Chemistry

Fabrication of Soybean Bowman-Birk Inhibitor (BBI) Nano-delivery Carrier to Improve Bioavailability of Curcumin

Chun Liu a, Fenfen Cheng a, Xiaoquan Yang* a,b

a

Research and Development Center of Food Proteins, School of Food Science and Engineering,

South China University of Technology, Guangzhou 510640, People’s Republic of China b

Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety,

South China University of Technology, Guangzhou 510640, People’s Republic of China

*Corresponding author: Xiao-Quan Yang Tel: (+86) 020-87114262 Fax: (+86) 020-87114263 E-mail addresses: [email protected], [email protected]

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ABSTRACT

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Curcumin is a poorly water-soluble drug and its oral bioavailability is very low. Here, a novel

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self-assembly nanoparticle delivery carrier has been successfully developed by using soybean

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Bowman-Birk inhibitor (BBI) to improve the solubility, bioaccessibility and oral absorption of

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curcumin. BBI is a unique protein, which can resistant to the pH range and proteolytic enzymes in

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the gastrointestinal tract (GIT), bioavailable, and not allergenic. The encapsulation efficiencies (EE)

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and the loading capacities (LC) of curcumin in the curcumin-loaded BBI nanoparticles

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(Cur-BBI-NPs, size: 89.8 nm, PDI: 0.103) were 86.17 and 10.31%, respectively. The in vitro

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bioaccessibility of Cur-BBI-NPs was superior to that of curcumin-loaded sodium caseinate (SC)

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nanoparticles (Cur-SC-NPs) (as control). Moreover, Cur-BBI-NPs significantly enhanced

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bioavailability of curcumin in rats compared with Cur-SC-NPs. And the clathrin-mediated

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endocytosis pathway was probably contributed to the favorable bioavailability of Cur-BBI-NPs, as

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revealed by the cellular uptake inhibition study.

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KEYWORDS: Soybean Bowman-Birk inhibitor, curcumin, nanoparticles, bioaccessibility,

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bioavailability

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■ INTRODUCTION

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With the rapid development of nanotechnology, protein-based nanoparticles have gained increasing

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interest as delivery systems for drugs and nutraceuticals in the past few decades.1 To date, drug-

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loaded nanoparticles have been synthesized successfully from various proteins, including both

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water-soluble (bovine or human serum albumin (BSA or HSA), β-lactoglobulin (βLG)) and insoluble

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proteins (zein, gliadin, barley protein etc.).2,

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advantages, such as improved solubility, controlled release property and enhanced bioavailability of

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encapsulated nutraceuticals.4 Additionally, they exhibit low toxicity due to superior biocompatibility

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and nutritional value.5 Despite these advantages, the application of protein-based nanoparticles has

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been impeded by several drawbacks. First, proteins are sensitive to pH conditions and tend to

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precipitate at a pH around their isoelectric point.6 Second, an ionic effect on proteins can also cause

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the aggregation of proteins.7 More importantly, digestive enzymes in the gastrointestinal tract (GIT)

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can readily hydrolyze protein to polypeptides and amino acids, which cause burst release of bioactive

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compounds and subsequent drug degradation and poor absorption.6,

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above-mentioned drawbacks of protein-based nanoparticles, a unique protein that resistant to

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digestive enzyme hydrolysis and readily absorption is desirable to find.

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These nanoscaled systems exhibited various

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To overcome the

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Soybean-derived Bowman-Birk Inhibitor (BBI), a serine protease inhibitor, is a 71-amino acid

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protein (8 kDa) with seven disulfide bonds which stabilize its active conformation.9 BBI is stable

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within the pH range encountered in most foods, resistant to the pH range and proteolytic enzymes in

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the GIT,10, 11 bioavailable,12, 13 and not allergenic.13, 14 It has been extensively studied for its ability to

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prevent carcinogenesis in a wide variety of in vivo and in vitro model systems.14 It previously was

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believed that very little of BBI would be taken up into the blood stream and distributed to organs 3

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outside the GIT following dietary ingestion; thus, a number of publications from Kennedy and

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colleagues15, 16, 17 address mechanisms to increase the uptake of BBI into the bloodstream so that

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organs outside of the GIT would be exposed to increased concentrations of BBI following delivery

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via the diet. Subsequently, Billings et al. found that reasonable dietary concentrations of BBI result

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in a sufficient amount of BBI being taken up into the bloodstream, with subsequent distribution

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throughout the body, to prevent carcinogenesis at many different organ sites.10, 14 Information about

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the absorption, distribution, and excretion of BBI primarily comes from animal studies, which

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indicate that approximately one-half of the BBI administered orally is excreted in the feces in an

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unaltered form, while the rest enters intestinal epithelial cells or crosses the intestinal lumen.14 At 3 h

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after an oral BBI dose, BBI is widely distributed in the body, and present in an active form in all

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major internal organs examined (except the brain): the percent distributions of the labelled BBI

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found in each organ and in body fluids have been described by Billings et al.10 However, Clemente

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et al.18 reported that the survival rates of BBI from chickpea-based diets at the terminal ileum in pig,

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expressed in terms of trypsin (TIA) and chymotrypsin inhibitory activity (CIA), were 7.3 and 4.4%,

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respectively. The different survival rates of BBI in the colon may be due to the different

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measurement methods between two studies.

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Curcumin, a naturally active constituent extracted from the plants of the Curcuma longa, has a

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variety of biological activities and pharmacological actions, such as anti-tumor, anti-inflammatory,

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anti-virus, anti-oxidation and anti-HIV as well as low toxicity with promising clinical application.19

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However, curcumin is slightly absorbed in the GIT due to its poor solubility in water (the maximum

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solubility was reported to be 11 ng/mL in plain aqueous buffer pH 5.0).20 In humans, due to its fast

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metabolic turnover in the liver and intestinal wall, blood concentrations of curcumin are low and 4

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tissue distribution is limited following oral dosing.21 Maximum plasma curcumin concentrations in

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humans, even upon intake of doses as high as 10 or 12 g curcumin, remain in the low nanomolar

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range (< 160 nmol/L).21 To improve the bioavailability of curcumin, numerous approaches have been

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investigated. These approaches include loading curcumin into polymeric or lipid-based carriers such

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as liposomes, micelles, dendrimers and nanoparticles.22 In the literature, many studies have

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demonstrated the enhancement of solubility and bioavailability of drugs encapsulated in nanocarriers.

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However, recent studies have demonstrated that only a few orally administered particles are taken up

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across the intestinal epithelium,23 thus having a chance to reach the bloodstream and being able to

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attain their pharmacological target while keeping their native structure. Therefore, besides

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bioavailability enhancement, the current challenge is to develop particles that remain intact from the

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mouth to the bloodstream, consequently being real oral nanovectors with similar properties as those

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injected via the intravenous route.

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In this study, efforts have been made from the following aspects: firstly, BBI was efficiently

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extracted from soy whey using a novel strategy based on the principles of salting out and

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coacervation; secondly, a nanoparticulate delivery carrier has been developed by the use of BBI that

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can solubilise curcumin in aqueous media, protect it from hydrolytic degradation; thirdly, the in vitro

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bioaccessibility and the in vivo bioavailability of curcumin in rats have been investigated. Further,

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possible mechanisms of cellular uptake of nanoparticulate curcumin were also studied by using

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various endocytosis pathway inhibitors.

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■ MATERIALS AND METHODS

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Materials. Curcumin, pepsin, pancreatin powder, bile extract, nystatin, chlorpromazine,

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nocodazole, and cytochalasin D were purchased from Sigma-Aldrich Co. (Shanghai, China). Sodium 5

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caseinate (SC) was obtained from Meryer Chemical Technology Co., Ltd (Shanghai, China).

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Dulbecco’s modified Eagle medium (DMEM), Hank’s buffered salt solution (HBSS), fetal bovine

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serum (FBS) and 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were purchased

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from Fisher Scientific (Pittsburgh, PA, USA). Caco-2 cell line was purchased from the American

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Type Culture Collection (ATCC; Manassas, VA). All other chemicals used were of analytical grade.

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The BBI was isolated by our lab described as follows: the protease inhibitors including BBI and

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Kunitz trypsin inhibitor (KTI) were precipitated from soy whey by salting out method (adding 15%

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(w/w) sodium sulfate mass to 12% (w/v) soy whey protein solution at pH 4.5). Subsequently, BBI

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was separated from the KTI and BBI mixture by solid-liquid separation method (two volume of PBS

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(5 mM, pH 4.5) was added to precipitation, and homogenised by using an Ultra-Turrax T10

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(IKA-Werke GMBH & CO., Germany) at 5000 r/min for 15 min. The slurry was kept at pH 4.5.

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Then the slurry was separated to precipitate (KTI) and supernatant (BBI concentrate, BBIC) by

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centrifugation (5000g, 10 min, 25°C). The BBIC solution was ultra-filtrated to remove sodium

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sulfate and other small molecule impurities). Then BBI was further separated from BBIC by

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interactions between proteins with τ-carrageenan based on coacervation principle: BBIC (0.1%, w/v)

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and τ-carrageenan (0.1%, w/v) solutions were adjusted to pH 7.0, respectively; then

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BBIC/τ-carrageenan mixtures were obtained at the protein to τ-carrageenan mixing ratio of 10:1 (pH

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4.3) by addition of 0.5 M HCl with gentle stirring. After centrifugation (5000g, 30 min), the

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supernatant (BBI/τ-carrageenan mixtures) was obtained. To remove τ-carrageenan, the supernatant

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containing BBI was adjusted to pH 7.0 and ultra-filtrated. After that, the BBI solution was dialyzed

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for 48 h at 4 °C against Millipore pure water and freeze-dried. The lyophilized powder of BBI was

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stored at -20 °C. The SDS-PAGE pattern and gel-permeation chromatography (GPC) profile of BBI 6

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are shown in Figure 1A, which indicated that a desired purity of BBI was achieved.

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Determination of Interfacial Activity of BBI by Isotherms (ISO) Curve. The ISO curve was

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determined by using a custom KSV NIMA film balance apparatus (KSV NIMA Instruments,

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Helsinki, Finland) at 25 oC. The subphase was Millipore ultrapure water (18.2 MΩ, pH 7.0).

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Monolayers of BBI were obtained by spreading 30 µL (5 mg/mL) of BBI deionized aqueous solution

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at the surface of the subphase. An equilibration period of 20 min was allowed. The surface

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pressure-area ISO was monitored using a platinum Wilhelmy plate. Surface pressure is obtained as Π

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= γw – γ, where γw is the surface tension of pure water.

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Fabrication and Characterizations of Curcumin-loaded BBI Nanoparticles (Cur-BBI-NPs).

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Preparation of Cur-BBI-NPs. To obtain Cur-BBI-NPs, 0.1 mL stock solution of curcumin (4 mg

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mL-1 in ethanol) was added into 2.9 mL of BBI solutions (1, 2, 3, and 4 mg mL-1) in successive

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titrations with magnetic stirring. The mixtures were centrifuged at 10 000g, 25 oC for 20 min to

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pellet the unbound curcumin, and the supernatants containing curcumin nanocomplexes were

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preserved in a light-resistant container at 4 oC for determination. As contrasts, SC (3 mg/mL) instead

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BBI were also prepared at the same conditions, and curcumin without protein in the same PBS

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solution with homologous concentration were also prepared.

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Determination of Encapsulation Efficiency (EE) and Loading Capacity (LC). The EE (%) of

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curcumin in the Cur-BBI-NP was estimated as the percentage of curcumin encapsulated in the

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proteins by the following equation: EE (%) = 100 - (amount of free curcumin (mg)/total amount of

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added curcumin (mg)) × 100, where the amount of free curcumin is determined from the precipitate

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obtained by centrifugation. The precipitate was extracted in 5 mL of ethanol with mild stirring for 5

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min under magnetically stirred conditions and then centrifuged at 10 000g for 15 min at 25 oC to 7

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remove the protein aggregates. The supernatant was subjected to spectrophotometric analysis at 426

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nm with a GENESYS 10S UV-Vis spectrophotometer (Thermo Scientific, USA), and the curcumin

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concentration was determined using an established standard curve of curcumin (R2 = 0.9958). The

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loading capacity (LC) of the samples was calculated with the following equations: LC (%) = mass of

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encapsulated curcumin/(total mass of encapsulated curcumin + protein).

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Dynamic Light Scattering (DLS). The nanoparticle samples were diluted to 1 mg mL-1 with

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Millipore water, and the pH was adjusted to 7.4 or 2.0; then the particle size and polydispersity index

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(PDI) were measured using a Nano ZS Zetasizer instrument (Malvern Instruments, Worcestershire,

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UK). All measurements were carried out at 25 oC, and the results are reported as averages of three

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readings.

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Transmission Electron Microscopy (TEM). TEM was used to observe the surface morphology

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of nanoparticles and to further confirm particle diameter by DLS. A drop of diluted sample was

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deposited onto a carbon-coated copper grid, and excess of sample was removed after 5 min with a

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filter paper. Then, a droplet of phosphotungstic acid (1%, w/v) was put onto the grid and removed

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after 5 min. Observations were made with JEM-2100F transmission electron microscope operating at

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200 kV (JEOL, Japan).

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Interaction of BBI with Curcumin. UV-Vis Spectrum. UV-spectrum of the mixed BBI-Cur

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dispersion was determined after appropriate dilution using UV-vis spectrometer. Appropriate

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controls of free curcumin in deionized water and BBI without curcumin solution at the same

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concentrations were tested as well. All samples were prepared and determined at room temperature

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(25 oC).

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Fluorescence Spectra. The fluorescence spectra were recorded using an F7000 fluorescence

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spectrophotometer (Hitachi Co., Japan). Protein intrinsic fluorescence was measured at constant BBI

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concentration (0.5 mg/mL) and different curcumin concentrations in 10 mM phosphate buffer (pH 8

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7.4). Emission spectra were recorded from 260 to 420 nm at an excitation wavelength of 280 nm.

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Both the excitation and emission slit widths were set at 5 nm. The fluorescence spectra of the

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phosphate buffer were subtracted from the respective spectra of the samples. Fluorescence quenching

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is described according to the Stern-Volmer equation: F0/F = 1 + k0kqτ0[Q] = 1 + KSV[Q] (eq 1). In this

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equation F0 and F are the fluorescence intensities in the absence and presence of a quencher,

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respectively, [Q] is the quencher concentration, KSV is the Stern-Volmer quenching constant, kq is the

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bimolecular quenching rate constant, and τ0 is the lifetime of fluorescence in the absence of a

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quencher (the fluorescence life time of the biopolymer is 10-8 s practically). Hence, eq 1 was applied

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to determine KSV by linear regression of a plot of F0/F versus [Q]. For the static quenching, the

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binding constant Ka and the number of binding places can be calculated according to a double

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logarithmic eq2: lg((F0-F)/F) = lgKa +nlg[Q] (eq2). The intercept of the double logarithmic

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Stern-Volmer plot provides the binding constant (Ka), and the slope yields the number of binding

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sites (n).24

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X-Ray Diffraction (XRD). The XRD patterns of the samples were characterized by a Bruker

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AXS (Karlsruhe, Germany) D8 Advance diffractometer. The instrument was equipped with a copper

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anode that produced Cu-Ka Xrays (λ = 0.15418 nm) with an accelerating voltage 40 kV and a tube

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current 40 mA. The diffractogram was collected with a monocap collimator of 0.3 mm during 300 s.

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XY amplitude of 2 mm resulted in a 2θ between 3o and 40o after merging the separate recordings.

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Rocking and amplitude oscillation were used to obtain an average diffractogram of the sample and

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minimise a preferred orientation of crystals.

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In Vitro Bioaccessibility Study. An in vitro model that stimulated sequential gastric and

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intestinal digestion was applied to assess the effect of digestion on the in vitro bioaccessibility of

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nanoparticulate curcumin (Cur-BBI-NPs and Cur-SC-NPs) according to the method described

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elsewhere25 with slight modifications. Briefly, 10 mL of freshly prepared samples were well mixed

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with 40 mL of 0.1 mol/L HCl (pH = 1.5), and preincubated in a shaker (at 37 oC) at a rate of 100 9

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r/min for 10 min. If necessary, the pH of the mixtures was adjusted to 1.5 using 1 mol/L HCl.

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Subsequently, 10 mg of pepsin powder was added and well mixed to start the simulated gastric

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digestion (0-60 min). After 60 min, the pH of the pepsin-digests was immediately adjusted to 7.0

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using 4 mol/L NaOH. Then 250 mg of bile extract was added and well dispersed in the shaker for 10

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min. Last, 20 mg of pancreatin powder was added to start the intestinal digestion (60-180 min). After

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180 min, 500 µL of the final digest dispersion was also collected and centrifuged at 55 000g at 4 oC

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for 95 min (Micro Ultracentrifuge CS15ONX, Hitachi, Japan). The aqueous fraction was collected

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from the centrifuge tube and then passed through a filter with 0.22 µm pores (Millipore, Billerica,

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MA, USA) to ensure that the curcumin in the aqueous fraction were actually in nanoparticles or

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mixed micelles. The amounts of curcumin in nanoparticles or mixed micelles and the whole digests

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were extracted and determined according to the method described above. The whole process was

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kept in the dark for purpose of avoid light-induced degradation. Proteins (BBI and SC) before and

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after in vitro digestion were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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(SDS-PAGE) according to Laemmli (1970).26

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In Vivo Bioavailability Study. For in vivo bioavailability study, twelve male Sprague Dawley

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(SD) rats weighing 260-300 g were used. The protocol was approved by the University Animal

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Ethics Committee and performed according to the guiding principles for the use and care of

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experimental animals at the Guangzhou University of Chinese Medicine (Qualification No.

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440059000, experimental licence SYXK, Guangdong, 2008–0001, Guangzhou, China). The animals

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were divided into two groups (n = 6). Group 1 was administered 100 mg/kg body weight (bw)

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Cur-BBI-NPs and Group 2 was administered 100 mg/kg bw Cur-SC-NPs by oral gavage. The blood

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samples (0.5 mL) were collected from the retro-orbital plexus under mild ether anesthesia into

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heparinized microcentrifuge tubes (containing 20 µL of 1000 IU heparin/mL of blood). After each

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sampling, 1 mL of dextrose-normal saline was administered to prevent changes in the central

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compartment volume and electrolytes. Plasma was separated by centrifuging the blood samples at 10

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4000g for 10 min at 4 oC. To 250 µL of plasma, 25 µL of 2.8% of acetic acid was added (for stability

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of curcumin) and 50 µL of Internal Standard (IS) 17β-estradiol acetate was added and vortexed for

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20 s. The extraction was done by adding 1.2 mL of ethyl acetate and vortexed for 10 min. Finally it

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was centrifuged at 10000g for 10 min and organic layer was separated which contained curcumin

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and IS. This was evaporated for 5 h to remove ethyl acetate. The residue left was reconstituted in 125

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µL of methanol and analyzed using HPLC method.27

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Cellular Uptake Inhibition Study. The cellular uptake mechanism of Cur-BBI-NPs and

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Cur-SC-NPs was investigated by using cellular uptake inhibitor. Caco-2 cells were seeded into a

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6-well plate at a density of 4 × 105 cells/well and allowed for attachment for 24 h. Then, the cells

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were pretreated with various inhibitors of cellular uptake for 30 min at the following concentrations:

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nystatin 25 µg/ml, chlorpromazine 10 µg/mL, nocodazole 10 µg/mL, and cytochalasin D 5 µg/mL.28

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Then, cells were incubated with 200 µg/mL Cur-BBI-NPs or Cur-SC-NPs for 2 h in the presence of

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the same inhibitor. Four control samples were set as the untreated cell samples exposed or not

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exposed to Cur-BBI-NPs or Cur-SC-NPs, respectively. The treated cells were collected and fixed for

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fluorescence analysis.

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Statistical Analysis. Results were expressed as mean values + standard deviations. Sample

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comparison, by multivariate analysis of variance (ANOVA), followed by Duncan’s comparison test,

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was used to assess the differences.

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■ RESULTS

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Interfacial Activity of BBI. Proteins are natural amphiphilic molecules with surface activity,

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which can be determined by isotherm of proteins at the air-water interface. The pressure-area

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isotherm of BBI spreading directly at the interface is shown in Figure 1B. This isotherm began at

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893.84 Å2/molecule and reached 106.39 Å2/molecule at a pressure of 35.04 mM/m. No significant

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phase transition was observed. This isotherm revealed for the first time that BBI could form a stable

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monolayer at the air-water interface, even though it is a water-soluble protein. The maximal interface 11

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pressure of BBI (35.04 mN/m) is higher than that of other water-soluble proteins or peptides such as

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cytochrome c (14 mN/m),29 lactoferricin B (19 mN/m)30 and melittin (20.5 mN/m).31 This result

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suggested that BBI possessed good surface activity.

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Fabrication and Characterization of Cur-BBI-NPs. The EE and LC of curcumin in BBI are

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shown in Figure 2. As can be seen, as the concentration of BBI increased from 0.1% to 0.3%, the EE

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of curcumin progressively increased from 38.6% to 86.17%. However, when the concentration of

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BBI was continued to increase to 0.4%, the increase of EE was very small (only 0.86%). While the

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LC of curcumin linearly decreased from 13.37% to 8.01% as the concentration of BBI increased

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from 0.1% to 0.4%. Because the increase of EE was very small with increasing BBI concentration

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from 0.3% to 0.4%, BBI concentration more than 0.4% has not been studied, and 0.3% of BBI

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concentration was selected as the optimum concentration for preparing Cur-BBI-NPs in the

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following assays. The appearances of curcumin in pure PBS and 3 mg/mL BBI solution can be

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observed in Figure 2 (inset). The free curcumin in pure PBS was turbid due to its poor water

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solubility. However, the solubility of curcumin was increased in the BBI solution, and the

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protein-curcumin mixture solutions exhibited yellow and highly transparent appearances.

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Mean particle size and polydispersity index (PDI) as well as size distributions of

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curcumin-loaded nanoparticles are presented in Table 1 and Figure 3, respectively. As can be seen,

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the mean particle size of Cur-BBI-NP was 90.09 nm (pH7.4) or 104.60 nm (pH2.0) with good

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monodispersity (PDI = 0.103 (pH7.4) or 0.148 (pH2.0)), which was evidenced by TEM (the inset in

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Figure 2 and Figure 3B). The particle size only slightly increased from 90.09 nm to 101.00 nm at pH

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7.4 after 30 days storage under ambient conditions, and the PDI was also slightly increased.

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Interestingly, at pH 2.0, the particle size decreased from 104.60 nm to 93.95 nm after 30 days storage

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under ambient conditions, and the PDI was slightly decreased as well. These results indicated that

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pH stability and storage stability of Cur-BBI-NPs were favorable.

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Interaction of BBI with Curcumin. The UV-visible absorption spectroscopy of a free Cur, 12

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BBI alone and Cur-BBI mixture in PBS are shown in Figure 4A. Since poor solubility, free Cur in

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PBS has only a weak absorption peak at 425 nm. However, the absorbance intensity of the Cur-BBI

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mixture is remarkably increased. Additionally, BBI has no absorbance at this wavelength. These

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results indicated that curcumin could be solubilized significantly due to the existence of BBI.

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The fluorescence quenching method was used to study the binding reaction between small

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molecules and proteins. Fluorescence quenching refers to any process that decreases the fluorescence

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intensity of a sample.32 Figure 4B shows the effect of curcumin on the Tyr intrinsic fluorescence

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emission spectra of BBI. As the concentration of curcumin was increased from 0 to 40 µg/mL, the

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addition of curcumin gave rose to a progressive quenching of the fluorescence of BBI, suggesting

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that there was interaction between BBI and curcumin. To further discern the fluorescence quenching

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mechanism, the Stern-Volmer eq 1 was used for the fluorescence data analysis. As shown in the inset

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of Figure 4B, the value of Kq (3.0 × 1015 M-1 s-1) is much higher than the maximal dynamic

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quenching constant (2.0 × 1010 M-1 s-1), indicating that the fluorescence quenching induced by

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curcumin is static quenching. For the static mechanism of quenching, the double logarithmic

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Stern-Volmer equation (eq 2) could be used to analyze the quenching data and calculate the apparent

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binding constants (Ka) of the forming of Cur-BBI complexes. The Ka and the number of binding sites

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per BBI molecule (n) for curcumin with BBI were 1.35 × 109 L·M-1 and 1.25, respectively. The great

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magnitude of Ka revealed a strong binding force between BBI and curcumin. The value of n was 1.25,

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demonstrating that approximately one association site on BBI for curcumin.

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XRD was performed to investigate the crystallinity of curcumin after its complexation with BBI

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and the results are shown in Figure 4C. The XRD patterns of free curcumin exhibited intense

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diffraction peaks between 5o and 30o, indicating its highly crystallized structure. Oppositely, the

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typical amorphous XRD pattern was observed for BBI. However, the diffraction spectrum of

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curcumin-loaded BBI showed complete disappearance of all the characteristic crystalline peaks of

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curcumin, indicating the formation of amorphous curcumin. This observation should be attributed to 13

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the inhibition of its crystallization in the nanoscale confinement and the formation of an amorphous

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complex with BBI in the particle matrix. Additionally, the physical-mixing of curcumin and BBI has

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also been measured by XRD. As we can see from Figure 4C, most of the intense diffraction peaks of

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curcumin between 5o and 30o were not disappeared although the intensities of peaks were decreased

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slightly. This result further verified that the interactions between BBI and curcumin occurred exactly.

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In Vitro Bioaccessibility of Curcumin. During the digestion, the nanoparticulate curcumin

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may undergo dramatic changes in environmental conditions (pH and ionic strength), action of

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proteases against the proteins, and even changes due to the presence of different active surfactants.25

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After sequential processes of in vitro gastric (60 min) and intestinal (120 min) digestion, the

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bioaccessible amount of curcumin transferred to the aqueous phase of the digests and whole digests

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for Cur-BBI-NPs and Cur-SC-NPs is exhibited in Figure 5. For Cur-SC-NPs, only 51.23% of

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curcumin was remained in the aqueous phase after the whole digestion. In contrast, for Cur-BBI-NPs,

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there was no significant loss of curcumin throughout the digestion, and the remaining amount in the

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aqueous phase could reach up to 95.65%, which was near the value in the whole digests (98.21%). In

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the case of Cur-SC-NPs, SC may be precipitated when encountered acidic conditions in gastric, most

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importantly, it could also be readily hydrolyzed to polypeptides and amino acids, which cause burst

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release of curcumin and subsequent drug degradation or recrystallization hence reduction of

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bioaccessibility. These results indicated that BBI could prominently increase the bioaccessibility of

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curcumin. Intestinal absorption of hydrophobic bioactives, such as curcumin, is dependent on their

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solubilization in the aqueous intestinal environment via the emulsifying action of the bile salts.2 The

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bile salts can trap lipophilic compounds in mixed micelles or vehicles, carry them through intestinal

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cells barriers, and transport them into the blood circulation. In addition, the bioaccessibility of

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bioactives is a prerequisite for their bioavailability. It assumes that the solubilized substance may

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have a high potential to be absorbed by the small intestine.33

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In Vivo Bioavailability of Curcumin. The Cur-BBI-NPs were designed to improve the oral 14

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bioavailability of curcumin. Blood levels after oral administration of Cur-BBI-NPs were compared

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with oral Cur-SC-NPs. The mean curcumin concentrations in the plasma after oral administration of

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curcumin nanoparticles (100 mg/kg) at single dose in SD rats are illustrated in Figure 6. The relevant

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pharmacokinetic parameters including Cmax, Tmax and AUC0-∞ are presented in the inset of Figure 6.

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As can be seen, during the 0-15 minutes, the serum available curcumin in Cur-SC-NPs administered

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were higher than those in Cur-BBI-NPs administered, this may be due to there are more free

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curcumin in the case of Cur-SC-NPs administered, while in the case of Cur-BBI-NPs administered,

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curcumin are protected by BBI in Cur-BBI-NPs, the diffusion rate of nanoparticulate curcumin was

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relatively slow than that of free curcumin. However, the serum available curcumin in Cur-BBI-NPs

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administered were significantly higher (p < 0.01) than those in Cur-SC-NPs administered after 30

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min. A sustained release of curcumin over 24 h was observed in the Cur-BBI-NPs form, where as in

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case of Cur-SC-NPs, the level was very low beyond 5 h. Cur-SC-NPs upon oral administration

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resulted in sharp Cmax within 1 h; nevertheless, the plasma concentration of curcumin decreased

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rapidly, indicating rapid metabolism of curcumin. Whereas, relatively slow increase and sustained

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plasma concentration of curcumin for a longer time was observed after administration of

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Cur-BBI-NPs, with significantly delayed Cmax occurring at 2 h, suggesting an obvious sustained

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release of curcumin from Cur-BBI-NPs. There was a prominent difference in the AUC0–∞ between

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Cur-BBI-NPs and Cur-SC-NPs. The AUC0–∞ for curcumin was higher in the animals administered

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with Cur-BBI-NPs, with a relative bioavailability of 3.11 as compared to Cur-SC-NPs. These results

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indicated that Cur-BBI-NPs formulation could improve bioavailability of curcumin.

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Cellular Uptake Inhibition Study. There is agreement that formulating nanostructured

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delivery systems yields an increase in drug uptake, however the mechanisms by which this occurs

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are not well understood. It is reported that endocytosis is the mechanism most relative to the cellular

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uptake of nanoparticles.34 To investigate the endocytosis pathway of Cur-BBI-NPs and Cur-SC-NPs

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entering Caco-2 cells, various pathway inhibitors were selected: nystatin was used for the inhibition 15

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of caveolin-mediated endocytosis; chlorpromazine had the inhibition of clathrin-mediated

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endocytosis; nocodazole and cytochalasin D, can disrupt microtubule and actin, respectively, which

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are fundamental parts of micropinocytosis.23 The effect of different inhibitors in Caco-2 cells was

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evaluated quantitatively using flow cytometry and the result is shown in Figure 7. The 0% uptake and

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100% uptake was set according to the mean fluorescent intensities of two samples. The 0% uptake

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was determined from cell samples with no Cur-BBI-NPs or Cur-SC-NPs incubation, while the 100%

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uptake (control group) from cell samples with Cur-BBI-NPs or Cur-SC-NPs incubation. As exhibited

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in Figure 7, chlorpromazine resulted in a decrease of cellular uptake of Cur-BBI-NPs with a 43.26%

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reduction of uptake relative to the control group, and nystatin resulted in a decrease of cellular uptake

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of Cur-SC-NPs, where the uptake of Cur-SC-NPs reduced by 18.66%. Nevertheless, neither

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nocodazole, nor cytochalasin D had effect on the uptake of Cur-BBI-NPs or Cur-SC-NPs in Caco-2

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cells. The result indicated that the endocytosis pathway of Cur-BBI-NPs is clathrin-mediated

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pathway, while the endocytosis pathway of Cur-SC-NPs is caveolin-mediated pathway at somewhat.

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■ DISCUSSION

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Although BBI is a potent trypsin and chymotrypsin inhibitor that has been extensively studied for its

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ability to prevent carcinogenesis in many different model systems,14 the isolation of BBI from soybean

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is high cost, which is a key limiting factor for their large-scale use. On the other hand, soy whey, a

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by-product from the preparation of soybean food stuffs such as soy protein isolates and tofu, contains

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0.43% (w/v) protein including BBI. Soy whey is characterized by its high chemical oxygen demand

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(COD) (10-20 g/L) and biological oxygen demand (BOD) (5~8 g/L) value derived from the protein

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content. Discarded soy whey is not only accountable for pollution problem, but also represents

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critical issues for creating the economic and nutritional penalty in this era. Therefore, recovery of

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bioactive proteins such as BBI from soy whey by an alternative large scale and economic technique

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is a valuable issue. In the present study, a novel strategy has been developed to isolate BBI from soy

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whey based on the principles of salting out and coacervation. The results indicated that an abundant 16

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of BBI with desired purity was obtained (Figure 1A).

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The field of nanoparticle delivery systems for nutrients and nutraceuticals with poor water

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solubility has been expanding, almost exponentially, over the last decade, and some of these

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technologies are now in the process of being incorporated in food products. The interest in the

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pharmaceutical and food related applications of these technologies has sparked tremendous

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developments in mechanical (top-down) and chemical (bottom-up) processes to obtain such

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nanoparticle systems. Mechanical approaches are capable of producing nanoparticles, typically in the

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100-1000 nm range, whereas chemical methods tend to produce 10-100 nm particles.35 In the present

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study, BBI was recovered effectively from soy whey wastewater by a novel method developed by

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our lab (Figure 1), and Cur-BBI-NPs were prepared successfully by simple anti-solvent process

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(Figure 2). The particle size of Cur-BBI-NPs is 90.09 nm with good monodispersity (PDI: 0.103)

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(Table 1 and Figure 3).

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The EE and LC of bioactives in the biopolymer-based nanoparticles are two important

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parameters for nano-delivery carrier. When the concentration of BBI was 0.3%, the EE (86.17%)

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(Figure 2) is comparable to that of curcumin encapsulated in zein-based colloidal particles

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synthesized by an anti-solvent precipitation (71–87%).36 The LC of curcumin (39.62 µg/mg) is much

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greater than that (1.743-1.784 µg/mg of SPI) in soybean protein isolates (SPI)-curcumin

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nanocomplexes25 and that (19 µg/mg of casein) in the supernatant of curcumin encapsulated in casein

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nanocapsules.37 Generally, the typical LC for hydrophobic compound-loaded protein nanoparticles is

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about 5%.5 In the present study, the LC (8.01-13.37%) (Figure 2) was greater than this value. It

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should be pointed out that a number of literature studies claimed LC values >50%, but these results

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were arguably inaccurate because of improper measuring methods, for example, counting

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precipitated nutraceutical molecules as encapsulated ones or neglecting the weight of a secondary

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coating when calculating the LC.5

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After the food/dose has been partially digested (mainly by mastication) in the oral cavity, the 17

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food goes through a dissolution process in the stomach at acidic conditions (pH~1 to 2) during a

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period of time that ranges from 1 to 3 h. Various enzymes (pepsin and others) are released in the

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stomach to help break down some of the proteins and carbohydrates.35 Dissolution in the stomach of

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the nanoparticle may or may not be desirable depending on the stability of the active ingredients in

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the acidic pH. In the present study, in the case of Cur-SC-NPs, because SC was readily precipitated

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in the acidic environment and could not resist to hydrolysis by pepsin and other enzymes in the

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stomach, most of curcumin in the Cur-SC-NPs might be released in this process. Besides degradation

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and recrystallization of some curcumin, remain curcumin in the digested food (in the form of a

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suspension) leaves the stomach and enters the duodenum, it mixes with the bile salts released by the

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gall bladder and formed mixed micelles. In addition to the release of bile salts, a bicarbonate solution

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containing a cocktail of enzymes (trypsin among others) is also released in the duodenum, increasing

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the pH of the solution to 6-7. The suspension (containing mixed micelles) then enters the small

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intestine where it resides for about 3 to 5 h for absorbing before entering the large intestine. In the

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case of Cur-BBI-NPs, since BBI could protect curcumin against enzymolysis and the acidic

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environment in the stomach, the vast majority of curcumin was probably not released in this process.

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In the duodenum, some Cur-BBI-NPs might be disintegrated by bile salts, and the released curcumin

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could form mixed micelles with bile salts immediately. But some intact Cur-BBI-NPs might still be

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existed, because there are a strong binding force between BBI and curcumin that was evidenced by

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the Ka value (1.35 × 109 L·M-1) (Figure 4B).

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The absorption of nutrients through the small intestine occurs via two main mechanisms, passive

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and active transport. Most hydrophobic compounds are highly permeable through the intestines and

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transport using passive diffusion.35 Yu and Huang38 reported that curcumin permeated across the

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monolayers fairly rapidly and the permeation mechanism was found as passive diffusion.

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Furthermore, they pointed out the permeation rates of curcumin complexed with bovine serum

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albumin and in the bile salts-fatty acids mixed micelles were determined as Papp(the apparent 18

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permeation rate)(mixed micelle) > Papp(DMSO) > Papp(protein complex). These results suggested

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that solubilization agents play an important role in the permeation of solubilized curcumin, and

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stronger binding between the solubilization agents and curcumin may decrease the permeation rate.

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This may be the reason why the serum available curcumin in Cur-SC-NPs administered were higher

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than those in Cur-BBI-NPs administered during the 0-15 minutes (Figure 6). In the other hand,

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because the serum available curcumin in Cur-BBI-NPs administered were significantly higher (p