Fabrication of a Soybean BowmanâBirk Inhibitor (BBI) Nanodelivery

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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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ACS Paragon Plus Environment


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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

231

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

243

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

246

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

251

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

252

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

255

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

259

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

260

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

261

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

263

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

264

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

265

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

266

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

267

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

268

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

269

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

270

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

271

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

272

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

273

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

274

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

275

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

276

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

278

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

279

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

280

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

281

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

282

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



283

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

284

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

285

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

286

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

287

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

288

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

289

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

290

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

291

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

292

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

293

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

294

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

295

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

296

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

297

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

298

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

299

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

300

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

301

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

302

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

303

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

304

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

305

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

306

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

307

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

309

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

310

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

311

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

312

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

313

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

314

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

315

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

316

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

317

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

318

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

319

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

320

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

321

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

322

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

323

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

324

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

325

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

326

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

327

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

328

Cellular Uptake Inhibition Study. There is agreement that formulating nanostructured

329

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

330

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

331

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

332

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



333

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

334

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

335

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

336

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

337

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

338

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

339

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

340

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

341

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

342

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

343

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

344

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

345

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

346

■ DISCUSSION

347

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

348

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

349

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

350

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

351

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

352

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

353

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

354

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

355

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

356

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

357

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).

359

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

360

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

361

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

362

pharmaceutical and food related applications of these technologies has sparked tremendous

363

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

364

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

365

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

366

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

367

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

368

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

369

(Table 1 and Figure 3).

370

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

371

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

372

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

373

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

374

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

375

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

376

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

377

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

378

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

379

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

380

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

381

coating when calculating the LC.5

382

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



383

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

384

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

385

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

386

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

387

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

388

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

389

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

390

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

391

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

392

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

393

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

394

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

395

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

396

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

397

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

398

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

399

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

400

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

401

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

402

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

403

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

404

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

405

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

406

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

407

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

409

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

410

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

411

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

412

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

413

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

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