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Enhancement of curcumin bioavailability by encapsulation in Sophorolipid-coated nanoparticles: An in vitro and in vivo study David Julian McClements, Shengfeng Peng, Ziling Li, Liqiang Zou, Wei Liu, and Chengmei Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05478 • Publication Date (Web): 29 Jan 2018 Downloaded from http://pubs.acs.org on January 29, 2018
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Journal of Agricultural and Food Chemistry
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Enhancement of curcumin bioavailability by encapsulation in
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Sophorolipid-coated nanoparticles: An in vitro and in vivo study
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Shengfeng Peng a, Ziling Liab, Liqiang Zou a, Wei Liu *a, Chengmei Liu a, David Julian
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McClements *c.
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a
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330047, Jiangxi, PR China
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b
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang
School of Life Science, Jiangxi Science and Technology Normal University, Nanchang,
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330013, Jiangxi, PR China
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c
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Massachusetts, Amherst, MA 01003, USA
Biopolymers and Colloids Laboratory, Department of Food Science, University of
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*Corresponding authors
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E-mail:
[email protected]; Fax: +86 791 88334509; Tel: + 86 791 88305872x8106.
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E-mail:
[email protected]; Fax: +1 413 545 1262; Tel: +1 413 545 1019.
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ABSTRACT
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There is great interest in developing colloidal delivery systems to enhance the water-
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solubility and oral bioavailability of curcumin, which is a hydrophobic nutraceutical
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claimed to have several health benefits. In this study, a natural emulsifier was used to form
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sophorolipid-coated curcumin nanoparticles. The curcumin was loaded into sophorolipid
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micelles using a pH-driven mechanism based on the decrease in curcumin solubility at
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lower pH values. The sophorolipid-coated curcumin nanoparticles formed using this
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mechanism were relatively small (61 nm) and negatively charged (-41 mV). The
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nanoparticles also had a relatively high encapsulation efficiency (82%) and loading
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capacity (14%) for curcumin, which was present in an amorphous state. Both in vitro and
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in vivo studies showed that the curcumin nanoparticles had an appreciably higher
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bioavailability than free curcumin crystals (2.7-3.6-fold), which was mainly attributed to
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their higher bioaccessibility. These results may facilitate the development of natural
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colloidal systems that enhance the oral bioavailability and bioactivity of curcumin in food,
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dietary supplement, and pharmaceutical products.
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Keywords:
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biosurfactant; sophorolipid
curcumin;
bioavailability;
nanoparticles;
bioaccessibility;
stability;
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INTRODUCTION
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Nutraceuticals are bioactive components found in foods, which are claimed to have
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certain health benefits when ingested orally . Curcumin is a polyphenolic nutraceutical
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typically isolated from the herb turmeric, which is a member of the ginger family . It has
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been reported to exhibit a broad range of biological activities that may be beneficial to
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human health, including anticancer, anti-inflammatory, antimicrobial, and antioxidant
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activities . However, the utilization of curcumin as a nutraceutical ingredient in functional
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food and beverage products is currently limited by its poor water-solubility, chemical
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stability, and oral bioavailability characteristics . These challenges can often be overcome
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by encapsulating curcumin within colloidal delivery systems that contain small particles
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dispersed within an aqueous medium, such as emulsions , nanoemulsions , micelles ,
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liposomes
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performance of each colloidal delivery system depends on the composition and structure
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of the particles it contains. Consequently, they must be carefully designed to exhibit the
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functional attributes required for a specific application. These functional attributes include:
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physical and chemical stability under different environmental conditions; rheological
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characteristics; optical properties; flavor profile; and gastrointestinal fate (including
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bioavailability) .
56 57
1
2
2
3
4-5
8-10
, biopolymer microgels , and polymer nanoparticles 4
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11-14
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. The functional
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The food industry is increasingly moving towards formulating its products from natural ingredients, rather than synthetic ones
16-17
. Consequently, it would be beneficial to 3
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create curcumin delivery systems from natural components. Synthetic surfactants are often
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used to facilitate the formation and/or increase the stability of the particles in colloidal
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delivery systems. They do this by adsorbing to particle surfaces and generating strong
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repulsive forces between the particles, such as steric or electrostatic repulsion . Synthetic
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surfactants can often be replaced with natural alternatives (“biosurfactants”), which can be
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isolated from animal, plant, or microbial sources
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potential application of sophorolipids as biosurfactants for forming curcumin-loaded
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colloidal delivery systems. Sophorolpids are surface-active glycolipids composed of a
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polar sophorose group and a non-polar fatty acid group, which can be produced by
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microbial fermentation . In addition, a recently developed method of forming curcumin-
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loaded nanoparticles is utilized, which is based on changes in the solubility of curcumin
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with pH
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molecule with a low water-solubility . However, at a sufficiently high pH, some of the
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hydroxyl groups on curcumin become deprotonated, leading to a negative charge that
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increases the water-solubility. Consequently, curcumin can be loaded into the hydrophobic
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core of surfactant micelles by mixing an alkaline solution of curcumin with an acidic
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solution of surfactant micelles. When the two solutions are mixed, the pH is reduced, which
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causes the curcumin to become more hydrophobic and move into the surfactant micelles
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(Fig. 1). Previous studies have shown that polyphenols such as curcumin and rutin can be
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loaded into synthetic and natural surfactant micelles using the pH-driven method
18
16-17
. In this research, we focus on the
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9, 20-21
. At relatively low pH, the curcumin has no charge and is a highly hydrophobic 15
9, 20-21
. Some 4
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of the advantages of this loading method are that it simple to perform, does not require
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heating or organic solvents. To the best of our knowledge, this is the first study of the
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preparation of sophorolipid-coated curcumin nanoparticles using the pH-driven method.
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The main objectives of the current study are to determine whether curcumin
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nanoparticles could be successfully produced from sophorolipids using the pH-driven
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method, to characterize the physicochemical properties of the nanoparticles formed, and to
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assess their potential for increasing curcumin bioavailability using both in vitro and in vivo
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methods. The results of this study may lead to the development of novel all natural
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curcumin delivery systems that can be incorporated into functional foods, dietary
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supplements, and pharmaceutical preparations.
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MATERIALS AND METHODS
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Materials. Powdered curcumin (composed of 76.4% curcumin, 17.3% demethoxycurcumin
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and 3.8% bisdemethoxycurcumin) was purchased from the Aladdin Industrial Corporation
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(Shanghai, China). Sophorolipid was bought from the Boliante Chemical Company (Xian, China).
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Ethanol, phosphoric acid, sodium hydroxide and other reagent chemicals were all of analytical
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grade.
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Nanoparticle preparation. Curcumin nanoparticles were prepared using the pH-
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driven method described in our previous study with some slight modifications . Briefly, a
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series of solutions were prepared by dissolving different amounts of the biosurfactant in 20
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mM phosphoric acid to obtain sophorolipid concentrations of 2, 4, 8, 12 and 16 mg/mL.
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Powdered curcumin was then weighed into a 30 mM sodium hydroxide solution and stirred
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for 5 min to reach a final curcumin concentration of 2.0 mg/mL. This curcumin level was
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used because it was fully soluble within the initial alkaline solution, as well as within the
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sophorolipid micelles formed after the following step. The alkaline curcumin solutions
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were then added to similar volumes of acidic sophorolipid solutions while stirring
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continuously at 500 rpm on a magnetic stir-plate. The resulting mixtures were incubated
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for 30 min at room temperature and then centrifuged at 10,000 g for 10 min to remove any
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insoluble curcumin particles. The curcumin samples used for the X-ray Diffraction and
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storage stability studies were in a powdered form, which was produced by lyophilization
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using a freeze-drier (Alpha 2–4 LD plus, Martin Christ Gefriertrocknungsanlagen GmbH,
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Osterode am Harz, Germany).
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Nanoparticle characterization. The particle size distribution and electrical
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characteristics (z-potential) of the curcumin nanoparticles were measured at 25°C using a
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combined dynamic light scattering (DLS) – electrophoresis instrument (Nicomp 380 ZLS,
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Santa Barbara, CA, USA).
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measured at an angle of 90º after the nanoparticle suspensions had been diluted 4-fold with
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water to avoid multiple scattering effects.
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standard deviation based on at least three samples with each sample being measured in
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triplicate.
The time-dependent fluctuations in light intensity were
All data were calculated as the mean and
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Microstructure images of the nanoparticle suspensions were obtained using an Atomic
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Force Microscope (AFM). An aliquot of nanoparticle suspension was placed on to a freshly
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cleaved mica substrate, and then images of the samples were acquired using an AFM
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(Agilent 5500, Agilent Technologies, Santa Clara, CA, USA) with a silicon cantilever of
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force constant of 0.58 N m operated in tapping mode at room temperature. -1
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The X-Ray Diffraction (XRD) patterns of pure curcumin powder and powdered
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sophorolipid-coated curcumin nanoparticles were recorded using a D8 Advance X-ray
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diffractometer (Bruker, Germany). The divergence slit was set at 1°, and the receiving
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slit was set at 0.1 mm for the incident beam. The scan rate was 2° per min over a 2q angle
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range of 5° to 40°.
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The encapsulation efficiency (EE) and loading capacity (LC) of the curcumin
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nanoparticles were determined according to the procedures described in our previous study
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non-encapsulated curcumin. The supernatant was then removed and diluted with
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anhydrous ethanol. The absorbance at 420 nm was determined using a UV-Vis
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spectrophotometer (Pgeneral T6, China) and the concentration of loaded curcumin was
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calculated from a calibration curve. The EE and LC were calculated using the following
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expressions:
. Briefly, nanoparticle suspensions were centrifuged at 10,000 g for 10 min to remove any
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EE (%) = mL,C / mIC × 100
(1)
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LC (%) = mL,C / mP × 100
(2) 7
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Here mL,C is the mass of curcumin trapped within the nanoparticles, mI,C is the initial mass of
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curcumin present in the system, and mP is the total mass of the nanoparticles (curcumin +
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sophorolipids). The total mass of the nanoparticles was determined by freeze drying the
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centrifuged nanoparticle suspension and then weighing the resulting powder. The mass of
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curcumin loaded in the nanoparticles was determined by rehydrating the nanoparticles in
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an aqueous solution and then measuring the absorbance using a UV-vis spectrophotometer.
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The initial mass of curcumin present in the system was calculated from the known
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quantities of the nutraceutical added initially, taking into account the various dilution steps.
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Physical stability of nanoparticles
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Influence of pH and ionic strength: A series of nanoparticle suspensions with pH
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values ranging from 2.0 to 8.0 were prepared by adding different amounts of either HCl or
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NaOH solution. A series of nanoparticle suspensions with different ionic strengths was
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prepared by incorporating different levels of salt into them: 10, 20, 50, 100, 200 or 1000
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mM NaCl. The resulting solutions were then stirred for 1 hour at ambient temperature and
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any changes in particle size and charge were measured using the methods described earlier.
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Storage stability: The influence of storage temperature and time on the stability of the
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nanoparticle suspensions was measured to provide some insights into their potential long-
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term stability. Powdered (lyophilized) curcumin nanoparticles were stored at 25 ºC, and
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aqueous suspensions containing curcumin nanoparticles were stored at 4 ºC or 25 ºC for
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30 days. The particle size, charge, and encapsulation efficiency were then record at 8
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different time intervals during storage.
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In vitro bioavailability. The in vitro bioavailability of curcumin was determining by
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passing the samples through a static simulated gastrointestinal tract (GIT), and then
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measuring the amounts of curcumin that had degraded and that had been solubilized in the
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mixed micelle phase.
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Simulated gastrointestinal tract (GIT). The static GIT method used in this study
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consisted of simulated mouth, stomach, and small intestine phases, and has been described
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previously .
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method that has recently been developed .
9
This method is fairly similar to the international consensus INFOGEST 22
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Oral phase: 7.5 mL of a test sample were mixed with 7.5 mL of simulated saliva fluid
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containing mucin (30 mg/mL) and various salts, prepared as described elsewhere . The
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resulting mixtures was then adjusted to pH 6.8 and shaken at 90 rpm for 10 min at 37 °C
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to mimic oral conditions.
23
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Gastric stage: A simulated gastric fluid was prepared that contained NaCl (2 mg/mL),
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HCl (7 mg/mL), and pepsin (3.2 mg/mL), which was then heated to 37 °C. 15 mL of the
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simulated gastric fluid was then added to 15 mL of the bolus solution resulting from the
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oral phase. The resulting mixtures were then adjusted to pH 2.5 and shaken at 100 rpm for
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2 h to mimic gastric conditions.
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Small intestine phase: Samples from the simulated gastric phase were adjusted to pH
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7.0 with 2 M NaOH. Simulated small intestinal fluid containing pancreatin (24 mg/mL, 2.5 9
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mL), bile extract solution (50 mg/mL, 3.5 mL) and saline solution (0.5 M CaCl and 7.5 M
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NaCl, 1.5 mL) were then added into the reaction vessel. The pH of the sample was then
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maintained constant at pH 7.0 by addition of 50 mM NaOH solution using an automatic
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titration unit (pH stat).
2
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In vitro bioavailability determination. The in vitro bioavailability of curcumin was
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estimated by measuring changes in its concentration and location after passing through the
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simulated GIT. It was assumed that the overall bioavailability is mainly limited by its
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chemical degradation (transformation) and solubility in the mixed micelle phase
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(bioaccessibility), rather than by its uptake by the epithelium cells (absorption). Thus, the
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in vitro bioavailability (BA) = bioaccessibility (B*) ´ transformation (T*).
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transformation is defined as the fraction of curcumin that remains in an active state after
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passage through the GIT. The raw digesta of each sample was centrifuged at 40,000 g for
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30 min at 4°C to remove any insoluble matter. The supernatants were collected and
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assumed to be the mixed micelle fraction, in which the bioactive agent is solubilized in a
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form suitable for absorption. The solubilized curcumin was diluted with methanol and
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assayed using high performance liquid chromatography (1260 HPLC, Agilent
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Technologies, Santa Clara, CA, USA) equipped with a UV-visible detector. Curcumin was
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separated on a Sunfire C18 column (250 mm × 4.6 mm, 5 μm; Waters Corporation, Milford,
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MA, USA), using a mobile phase consisting of 0.1% (v/v) acetic acid and acetonitrile
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(45:55 v/v) at a flow rate of 1.0 mL min , with detection by UV absorption at 420 nm.
The
−1
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The transformation and bioaccessibility of curcumin were then calculated using the following equations:
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Transformation (%) = C / C × 100 (3)
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Bioaccessibility (%) = C /C × 100 (4)
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Here, C and C are the concentrations of curcumin in the mixed micelle fraction and
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in the overall digesta at the end of the simulated GIT model, and C is the concentration of
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curcumin initially added (taking into account the various dilution steps). This latter value
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is therefore a measure of the total amount of curcumin that would be present in the small
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intestine phase if there were no losses due to chemical degradation. It should be noted that
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a simple in vitro GIT model cannot accurately simulate the complex processes occurring
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within a living gastrointestinal tract, but it is useful for rapidly screening different samples
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and for identifying important physicochemical mechanisms.
D,C
M,C
M,C
I,C
D,C
D,C
I,C
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In vivo bioavailability. The in vivo bioavailability of curcumin was evaluated by oral
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administration to 12 male Sprague Dawley (SD) rats weighing between 260 and 300 g. The
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rats were randomly divided into two groups (n=6). Group 1 was administrated 100 mg/kg
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body weight crystalline curcumin and group 2 was administrated 100 mg/kg body weight
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curcumin nanoparticles by oral gavage. Aqueous suspensions (10 mg/mL) of crystalline
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curcumin were prepared by dispersing powdered curcumin into 1.0% sodium
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carboxymethyl cellulose (as a stabilizer). Aqueous suspensions (10 mg/mL) of curcumin
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nanoparticles were prepared by dispersing lyophilized sophorolipid-coated curcumin 11
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nanoparticles in distilled water. A total of 0.5 mL blood sample was collected from the
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retro-orbital plexus of each rat at different times (0.5, 1, 2, 4 and 8 h) into heparinized
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microcentrifuge tubes (containing 20 μL of 1000 IU heparin/mL of blood). These samples
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were then immediately centrifuged at 4000 g for 10 min at 4 °C to isolate the plasma, which
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was then stored at -80 °C until analysis by LC–MS/MS. According to previous studies
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curcumin is mainly present in a conjugated form (curcumin glucuronide) when it is
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absorbed through the intestinal cells in rats. So, the concentration of curcumin and
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curcumin glucuronide in rat plasma were analyzed for.
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glucuronide could be detected in the rat plasma, and so this value was used to determine
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the in vivo oral bioavailability.
24-26
,
However, only curcumin
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Plasma (100 µL) was mixed with 200 µL acetonitrile by vortexing and centrifuged at
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10000 g for 5 min, at 4 °C. Aliquots of the extracts were injected onto a C18 column
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(Zorbax Eclipse Plus C18 column, 100mm×2.1mm, I.D., 3.5 μm, Agilent, USA) kept at
230
40 °C. The mobile phase consisted of two components: A, acetonitrile and B, 0.1% formic
231
acid. The gradient profile was as follows: 0-1min, 80%B→20%B; 1-3min, 20%; 3-3.5min,
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20%B→80%B. The flow rate was 0.3 ml/min. Curcumin and curcumin glucuronide were
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analyzed using a 6410 QQQ MS/MS system (Agilent Technologies, USA) equipped with
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an electrospray ionization source (ESI), operating in positive mode. The mass spectrometer
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ion source parameters were as follow: gas temperature, 350℃; gas flow, 10 L/min;
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nebulizer gas, 40 psi; spray voltage 4000 kV. Nitrogen served as a nebulizer and collision 12
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gas. Curcumin and curcumin glucuronide were determined using the multiple reaction
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monitor mode as follows: CUR, m/z 369 > 285, m/z 369 >177. CUR-G, m/z 545 > 369,
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m/z 545 >177.
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Statistical analysis:
All measurements were replicated at least three times. The
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results were then expressed as means ± standard deviations. Data were subjected to
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statistical analysis using SPSS software, version 18.0 (SPSS Inc., Chicago, IL, USA). The
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Student-Newman-Keuls test was performed to check significant comparisons and P < 0.05
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was considered statistically significant.
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RESULTS AND DISCUSSION
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Potential impact of fabrication method on curcumin stability: Curcumin has been
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reported to chemically breakdown upon prolonged storage in aqueous alkaline conditions
248
3, 5
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mechanism used to fabricate the curcumin nanoparticles. Nevertheless, a previous study
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by our group indicated that only 0.39 ± 0.27% of curcumin was degraded when it was
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dissolved in an aqueous alkaline solution (pH 12.0) for 10 min . The pH-driven loading
252
method used in the current study only involved holding curcumin at pH 12.0 for 5 min, and
253
so the degradation of curcumin during this procedure is expected to be relatively small.
, and so it was possible that some of it may have degraded when using the pH-driven
9
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Effect of biosurfactant concentration: Initially, experiments were carried out to
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determine the optimum sophorolipid concentration required to form the curcumin 13
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nanoparticles. Consequently, the impact of sophorolipid concentration on the mean particle
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diameter, polydispersity index, z-potential, and encapsulation efficiency of the curcumin
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nanoparticles was determined (Table 1, A).
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For the fresh samples (A), the mean particle diameter decreased from around 114 to
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61 nm when the sophorolipid concentration increased from 1 to 4 mg/mL, but then it
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remained relatively constant at higher sophorolipid levels. The polydispersity index (PDI)
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of most of the samples was relatively small (< 0.2), indicating that they had fairly narrow
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particle size distributions.
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sophorolipid levels had higher PDI values, indicating that they had broad distributions.
265
The electrical characteristics of the nanoparticles also depended on biosurfactant
266
concentration, with the z-potential changing from around -41 to -21 mV when the
267
sophorolipid level increased from 1 to 8 mg/mL. The encapsulation efficiency increased
268
from around 41 to 89% as the sophorolipid concentration increased from 1 to 8 mg/L.
However, some of the samples containing intermediate
269
An understanding of the physicochemical basis of the pH-driven loading mechanism
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is required to interpret the observed results. At a sufficiently high pH, curcumin has a
271
negative charge and is soluble in water, but when the pH is reduced it loses its charge and
272
becomes more hydrophobic . In the presence of biosurfactant micelles, there are two
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possibilities for the hydrophobic curcumin molecules to reduce their thermodynamically
274
unfavorable contact with water: (i) they can form crystals; or, (ii) they can be solubilized
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within the hydrophobic interior of the micelles (Fig. 1). At relatively low biosurfactant
15
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levels, there may not be enough biosurfactant micelles present to completely solubilize all
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of the curcumin, and so some curcumin crystals are formed. Alternatively, the rate of
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crystal formation may exceed the rate of solubilization, so that some curcumin crystals are
279
generated before solubilization can occur. At relatively high biosurfactant levels, there may
280
be enough sophorolipid micelles available to solubilize all of the curcumin molecules
281
present, and/or the solubilization rate may exceed the crystallization rate, thereby leading
282
to fewer curcumin crystals being formed. The curcumin crystals are likely to be larger than
283
the curcumin-swollen micelles, and are likely to have different electrical characteristics,
284
which would account for the observed decrease in mean particle diameter and change in z-
285
potential with increasing biosurfactant concentration (Table 1). During the solubilization
286
process, it is likely that some of the original sophorolipid micelles incorporated curcumin
287
and swelled, whereas others dissociated so that they could release biosurfactant molecules
288
that could then cover the increased surface area of the curcumin-swollen micelles. The
289
PDI was relatively small at low sophorolipid levels because the system mainly contained
290
only large curcumin aggregates, and was relatively small at high sophorolipid levels
291
because the system mainly contained curcumin-loaded micelles. On the other hand, the
292
PDI was relatively large at intermediate sophorolipid levels because the system contained
293
a mixture of small micelles and large curcumin crystals.
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The observed increase in encapsulation efficiency with increasing sophorolipid
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concentration may also be ascribed to a similar physicochemical mechanism. At relatively 15
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low biosurfactant levels, more curcumin crystals are formed in the aqueous phase because
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of the low micelle solubilization capacity or slow solubilization rate. Consequently, these
298
relatively large crystals would be removed during the centrifugation step, thereby leading
299
to a low measured encapsulation efficiency. Conversely, at high biosurfactant levels, most
300
of the curcumin will be solubilized within the biosurfactant micelles leading to the
301
formation of sophorolipid-coated curcumin nanoparticles. These nanoparticles are
302
relatively small and are not removed by centrifugation, thereby leading to a higher
303
measured encapsulation efficiency.
304
Effect of rehydration on nanoparticle stability: For commercial applications, it is
305
often advantageous to have a powdered form of a nutraceutical ingredient, as this improves
306
handling, transport and storage, and allows it to be incorporated into a broader range of
307
products. For this reason, the impact of converting the aqueous suspension of curcumin
308
nanoparticles into a powder using freeze drying (lyophilization) was assessed. The
309
physiochemical properties of the curcumin nanoparticles were then measured after the
310
powder had been rehydrated to establish the influence of lyophilization on their
311
performance (Table 1, B).
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As with the freshly prepared systems, the mean particle diameter of the curcumin
313
nanoparticles arising from the rehydrated powders decreased steeply as the sophorolipid
314
concentration increased from 1 to 4 mg/mL, but then remained fairly constant at higher
315
biosurfactant levels. At sophorolipid concentrations of 4 mg/mL or higher, the rehydrated 16
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curcumin nanoparticles rapidly dispersed in water and had physicochemical characteristics
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similar to those of the original systems (Table 1). At lower sophorolipid concentrations,
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the rehydrated curcumin nanoparticles had much larger particle sizes and polydispersity
319
indices than the original systems, suggesting that appreciable particle aggregation occurred
320
during the drying and/or rehydration stages. Indeed, large precipitates could even be
321
observed by eye in these samples. At low biosurfactant concentrations, there may have
322
been more large curcumin crystals present, which were more prone to aggregation than the
323
smaller curcumin nanoparticles. In addition, the encapsulation efficiency of the curcumin
324
was much lower in the rehydrated samples than in the original systems at low biosurfactant
325
levels. This result can be attributed to the fact that any large curcumin crystals would have
326
been removed during the centrifugation step, and so there would be a smaller amount of
327
water-dispersible curcumin remaining in the final nanoparticle suspensions.
328
From a practical point of view, it is important to create delivery systems using the
329
lowest amount of surfactant possible, so as to reduce costs, potential toxicity, and any
330
undesirable off-flavors. For this reason, a biosurfactant concentration of 4 mg/mL was used
331
in the remainder of the studies, since it led to relatively small highly-charged nanoparticles
332
with a high encapsulation efficiency.
333
Characterization of curcumin nanoparticles: A variety of analytical techniques
334
were employed to provide additional information about the nature of the sophorolipid-
335
coated curcumin nanoparticles formed using the pH-driven method. As discussed in the 17
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336
previous section, the curcumin nanoparticles were relatively small (61 nm) and highly
337
negative charged (-41 mV) when formed using a sufficiently high sophorolipid level (4
338
mg/mL). In addition, they had a relatively high encapsulation efficiency (82.2 ± 0.7%) and
339
loading capacity (13.7 ± 0.2%). In other words, over 82% of the curcumin added to the
340
system was trapped inside the nanoparticles, and each nanoparticle consisted of around 14%
341
curcumin and 86% sophorolipid. The encapsulation efficiency and loading capacity values
342
for our nanoparticles were in good agreement with those reported for other systems in
343
previous studies. For instance, the encapsulation of curcumin in Pluronic P123 micelles
344
loaded using a heating method resulted in an EE of 46% and LC of 4.4% . Curcumin
345
encapsulation in copolymer mPEG-PCL micelles using a nanoprecipitation method yielded
346
an EE of 89% and LC of 21% . Curcumin encapsulation in casein micelles using the pH-
347
driven method led to an EE of 81% and LC of 4%. The biosurfactant used in our study
348
therefore had similar or better encapsulation characteristics as other synthetic and natural
349
surfactants used previously. At the same time, the pH-driven method is relatively fast,
350
simple, inexpensive, and does not require the use of organic solvents or sophisticated
351
processing equipment, which would be advantageous for commercial applications.
27
28
352
Additional information about the microstructure of the curcumin nanoparticles was
353
obtained using AFM. The curcumin nanoparticles appeared as smooth spheres that were
354
evenly distributed throughout the images (Fig. 2), with dimensions consistent with those
355
determined by dynamic light scattering. The physical state of the curcumin within the 18
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356
sophorolipid-coated nanoparticles was investigated using X-ray diffraction. For the sake
357
of comparison, the X-ray diffraction patterns of pure curcumin, pure sophorolipid, and
358
sophorolipid-coated curcumin nanoparticles were determined (Fig. 3). For pure curcumin
359
powder, diffraction peaks were detected at 2θ values ranging from 5° to 30°, indicating a
360
highly crystalline structure . Conversely, no peaks were observed for the pure sophorolipid,
361
indicating that the pure biosurfactant was not in a crystalline form. In addition, no peaks
362
were observed for the powdered sophorolipid-coated curcumin nanoparticles, which
363
suggested that the curcumin was in an amorphous form in these systems. This result
364
suggests that encapsulation of curcumin within the hydrophobic interior of nanoparticles
365
inhibited its tendency to crystallize. Studies in the pharmaceutical industry suggest that
366
delivering solid bioactive agents in an amorphous (rather than crystalline) form increases
367
their oral bioavailability . Consequently, the nanoparticle delivery systems developed in
368
our study may be particularly suitable for the oral delivery of curcumin in a highly
369
bioavailable form.
29
30
370
Stability of curcumin nanoparticles: Knowledge of the impact of pH and ionic
371
strength on the physicochemical properties and stability of curcumin nanoparticles is
372
important because they may experience different solution conditions when added to food
373
products or when they travel through the human GIT. The nanoparticle suspensions were
374
therefore incubated at different pH values (1.5 to 8) for 30 minutes, and then their visual
375
appearance and particle size were determined. From pH 3 to 8, there was no obvious change 19
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376
in the visual appearance or mean particle diameter of the nanoparticle suspensions (Fig.
377
4A), suggesting that they were relatively stable to aggregation in this pH range. However,
378
the nanoparticle suspensions became turbid and the mean particle diameter increased
379
steeply when the pH was reduced to 2.0 and 1.5. The aggregation of curcumin nanoparticles
380
at low pH values can be explained by the pK values of the hydrophilic sugar residues on
381
the sophorolipid molecules. The curcumin nanoparticles had a relatively high negative
382
charge (-30 mV) at neutral pH, which is mainly due to charged carboxylic acid groups (-
383
COO ) on the sugar residues of the sophorolipid. Typically, carboxylic groups have pK
384
values around pH 3.5, which means that lose their charge (-COOH) when the pH is around
385
or below this value . Consequently, at pH 2 and below, the negative charge on the curcumin
386
nanoparticles was insufficient to generate an electrostatic repulsion strong enough to
387
overcome any attractive interactions (such as van der Waals), and so nanoparticle
388
aggregation occurred.
a
-
a
31
389
The influence of ionic strength on the stability of the curcumin nanoparticles was
390
determined by incubating them in solutions containing different NaCl levels (Fig. 4B). At
391
< 500 mM NaCl, the curcumin nanoparticles were relatively stable to aggregation without
392
any changes in their visual appearance and mean particle diameter, which suggested that
393
the electrostatic repulsion between the nanoparticles was still strong enough to overcome
394
any attractive interactions. Conversely, at 500 mM NaCl and above, the nanoparticles were
395
highly unstable to aggregation, as seen by changes in visual appearance and the increase in 20
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396
mean particle size. This phenomenon can be attributed to the ability of the cationic counter-
397
ions in salt (Na ) to accumulate around the anionic curcumin nanoparticles and screen the
398
electrostatic repulsion between them . As described earlier, the reduced electrostatic
399
repulsion would then not be strong enough to overcome the attractive interactions between
400
the nanoparticles, thereby leading to aggregation.
+
32
401
The ability of a delivery system to remain stable during long-term storage under
402
different conditions is important for many commercial applications. Consequently, the
403
stability of the curcumin nanoparticles was determined by measuring changes in their
404
appearance, particle size, surface potential, and encapsulation efficiency during storage.
405
Aqueous suspensions of curcumin nanoparticles were stored at 4 and 25 °C, whereas
406
powdered curcumin nanoparticles were stored at 25 °C and then rehydrated prior to
407
analysis. There was little change in the appearance of the nanoparticle suspensions when
408
they were stored at 4 °C in aqueous form or at 25 °C in powdered form for one month (Fig.
409
5A). Moreover, the mean particle diameter, z-potential, and encapsulation efficiency of the
410
curcumin nanoparticles changed little when stored under the same conditions (Fig. 5B-D).
411
However, the nanoparticle suspensions turned form yellow to brown, and large flocs
412
became visible to the eye, when they were stored at 25 °C in aqueous form for one month.
413
In addition, the mean particle diameter increased from around 54 to 114 nm, the z-potential
414
changed from around -41 to -19 mV, and the encapsulation efficiency deceased from
415
around 82 to 59%. Taken together, these results indicate that storage conditions have a 21
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416
major impact on the stability of the curcumin nanoparticles. The systems rapidly degrade
417
when stored in aqueous form at ambient temperature, but their shelf-life can be extended
418
by reducing the storage temperature or converting them into a powder. The origin of the
419
instability mechanism is currently unknown, but it could be due to chemical degradation
420
of curcumin in aqueous solutions, which is known to lead to color changes and precipitation.
421
3
422
In Vitro Bioavailability of Curcumin: The in vitro bioavailability of curcumin in the
423
sophorolipid-coated nanoparticles was evaluated by passing them through a simulated
424
gastrointestinal tract (GIT), and then measuring the total amount of curcumin remaining,
425
as well as the fraction that was present in the mixed micelle phase. These results were then
426
used to calculate the transformation (T*), bioaccessibility (B*), and in vitro bioavailability
427
(BA) of curcumin. The transformation was defined as the percentage of curcumin
428
remaining in the overall system after passage through the simulated GIT, whereas the
429
bioaccessibility was defined as the fraction of curcumin in the small intestine phase that
430
was solubilized in the mixed micelles and therefore available for absorption. As shown in
431
Fig. 6, the fraction of curcumin remaining was appreciably higher when it was in the free
432
crystal form (88.3%) than when it was in the nanoparticle form (48.0%). Curcumin
433
degradation under simulated GIT conditions occurs mainly due to exposure to aqueous
434
neutral or alkaline environments, but may also be partly due to digestive enzymes (such as
435
pepsin) . The surface area of curcumin exposed to the aqueous phase would be much larger 33
22
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436
for the nanoparticles than for the free crystals due to the much smaller dimensions of the
437
nanoparticles. Consequently, the curcumin in the nanoparticles may have been more
438
susceptible to degradation than the curcumin in the larger crystals. Conversely, the
439
bioaccessibility of curcumin in the free crystals (9.1%) was significantly lower than that in
440
the nanoparticles (61.3%). A number of physicochemical phenomena may account for this
441
large difference in bioaccessibility . First, the water-solubility of hydrophobic materials is
442
known to increase as their particle size decreases, thereby leading to a higher local
443
concentration of curcumin around the nanoparticles than around the crystals. Consequently,
444
the driving force for solubilization into the mixed micelles would be greater for the
445
nanoparticles. Second, the surface area of particles increases as their dimensions decrease,
446
which would have led to a faster dissolution rate of the curcumin from the nanoparticles
447
than the larger crystals. Third, the curcumin within the nanoparticles was in an amorphous
448
form, which is known to be more soluble than the crystalline form
449
of curcumin solubilized by the mixed micelle phase after passage through the simulated
450
GIT is a measure of the amount available for absorption, and can therefore be used as a
451
measure of in vitro bioavailability . The amount of bioavailable curcumin was much higher
452
(2.7-fold) in the nanoparticles (294 ± 20 µg/mL) than in the crystals (80.1 ± 2.1 µg/mL).
453
This effect can mainly be attributed to the much higher bioaccessibility of the curcumin in
454
the nanoparticles (since the chemical stability in the nanoparticles was actually lower).
455
Overall, these results suggest that the in vitro bioavailability of curcumin can be increased
34
30, 34
. The absolute amount
33
23
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456
appreciably by delivering it in the form of sophorolipid-coated nanoparticles, which is
457
mainly due to a large increase in bioaccessibility.
458
In Vivo Bioavailability of Curcumin: In vitro measurements provide valuable
459
information about the physicochemical mechanism that impact the bioavailability of
460
nutraceuticals, but they cannot accurately model the complexity of a living GIT. For this
461
reason, the in vivo bioavailability of the different forms of curcumin was determined by
462
oral administration to rats at a dose of 100 mg/Kg. The impact of delivery system type on
463
the pharmacokinetics was determined by measuring the serum curcumin concentration
464
over time after administration (Fig. 7). Numerous studies have reported that curcumin
465
undergoes metabolism after oral administration. For instance, in vivo studies with rats
466
and mice have shown that curcumin undergoes metabolic O-conjugation to curcumin
467
glucuronide and curcumin sulfate, as well as reduction to tetrahydrocurcumin,
468
hexahydrocurcumin, octahydrocurcumin, and hexahydrocurcuminol
469
metabolites formed, curcumin glucuronide is the major one found in the plasma after oral
470
administration of curcumin in rats . In our study, the actual curcumin concentration in the
471
blood was too low to determine and so the concentration of curcumin glucuronide was used
472
to evaluate the in vivo bioavailability of the ingested curcumin delivery systems. A number
473
of important pharmacokinetic parameters were then calculated from these curves, including
474
the maximum plasma concentration (C ), the time to reach this maximum (T ), and the
475
area under the curve from 0 to 8 hours (AUC ). Oral administration of the curcumin in the
35-36
. Among the
24
max
max
0-8 h
24
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476
form of nanoparticles led to an appreciably higher and more prolonged level of curcumin
477
in the blood of the rats (C = 2.74 µg/mL; T = 1 h; AUC = 6.51 µg h/mL), than in the
478
form of free crystals (C
479
differences were statistically significant (p < 0.01). The AUC
480
approximately 3.6-fold greater when it was delivered as nanoparticles than as free crystals.
481
This enhancement in bioavailability is in good agreement with the results from the in vitro
482
model, and highlights the potential of the curcumin nanoparticles as nutraceutical delivery
483
systems. In this study, we used sophorolipid-coated curcumin nanoparticles to improve the
484
in vivo bioavailability characteristics of curcumin.
485
have also been reported to improve the bioavailability of curcumin
486
and co-workers utilized different kinds of emulsifier to prepare curcumin nanosuspensions
487
and reported that the oral bioavailability of curcumin increased between 1.2- and 3.7-times
488
compared to free curcumin depending on the type of emulsifier used . Rice bran protein
489
nanoparticles have also been utilized to encapsulate curcumin and the oral bioavailability
490
of the curcumin was shown to increase around 9.2-times compared to free curcumin .
491
Compared to these delivery systems, the main advantages of sophorolipid-coated curcumin
492
nanoparticles are that they are simple to prepare, do not require organic solvents, and can
493
be fabricated from natural ingredients.
max
max
max
= 0.47 µg/mL; T
max
0-8 h
= 1 h; AUC
= 1.43 µg h/mL). These
0-8 h
0-8 h
value of curcumin was
Numerous other delivery systems 37-39
. For instance, Wang
39
40
494
In summary, it was shown that curcumin could be loaded into sophorolipid micelles
495
using a simple pH-driven method based on the decrease in its water-solubility at lower pH 25
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496
values. Under basic conditions, curcumin has a negative charge that makes it water-soluble
497
and so it can be mixed with an aqueous suspension of sophorolipid micelles. When the pH
498
is reduced, the curcumin loses its charge and so its water-solubility decreases, which causes
499
it to move into the hydrophobic core of the surfactant micelles. As a result, the micelles
500
swell in size leading to the formation of sophorolipid-coated curcumin nanoparticles.
501
Experiments showed that these nanoparticles were relatively small and negatively charged,
502
had a relatively high encapsulation efficiency, and that the curcumin was in amorphous
503
form. Both in vitro and in vivo studies showed that encapsulating curcumin in sophorolipid-
504
coated nanoparticles greatly increased its oral bioavailability, which was mainly attributed
505
to their ability to increase the solubilization of the curcumin in the mixed micelle phase. In
506
summary, a simple method of producing curcumin nanoparticles has been developed that
507
may be useful for the development of nutraceutical delivery systems suitable for
508
application in functional foods, supplements, and pharmaceuticals.
509
510
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FUNDING STATEMENT
624
We appreciate the financial support by the National Science Foundation of China (No.
625
21766018, 31601468). This material was also partly based upon work supported by the
626
National Institute of Food and Agriculture, USDA, Massachusetts Agricultural Experiment
627
Station (MAS00491) and USDA, AFRI Grants (2014-67021 and 2016-25147). Shengfeng
628
Peng thanks the Chinese Scholarship Council for funding to support his work (No.
629
201706820021).
630 631
30
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632
Figure captions
633
Fig. 1 Schematic of sophorolipid-coated curcumin nanoparticle fabrication using the pH-driven
634
loading mechanism. An acidic sophorolipid micelle solution is mixed with a basic curcumin
635
solution, which causes the curcumin molecules to become more hydrophobic and move into the
636
hydrophobic core of the surfactant micelles.
637
Fig. 2 Atomic forces microscopy image of sophorolipid-coated curcumin nanoparticles.
638
Fig. 3 XRD spectra of pure curcumin, pure sophorolipid, and powdered sophorolipid-coated
639
curcumin nanoparticles.
640
Fig. 4 Effect of (A) pH and (B) NaCl concentration on the mean particle size and appearance of
641
sophorolipid-coated curcumin nanoparticles.
642
Fig. 5 Physicochemical stability of sophorolipid-coated curcumin nanoparticles stored in the form
643
of aqueous suspensions at 4 and 25 °C, or powders at 25 °C for one month: (A) change of
644
appearance, (B) change of encapsulation efficiency, (C) change of the average diameter, (D) change
645
of the zeta potential.
646
Fig. 6 The transformation and bioaccessibility of free curcumin and sophorolipid-coated curcumin
647
nanoparticles after pass through gastrointestinal tract.
648
Fig. 7 Bioavailability of free curcumin and sophorolipid-coated curcumin nanoparticles. (Inset)
649
Pharmacokinetics parameters of two curcumin samples. AUC, area under the plasma
650
concentration−time curve from 0 h to 8 h; Cmax, peak concentration; Tmax, time to reach peak
651
concentration. 31
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652
Table 1. Impact of sophorolipid concentration on the physicochemical characteristics of (A) freshly
653
prepared curcumin nanoparticles and (B) rehydrated powdered curcumin nanoparticles. The
654
curcumin concentration was 1 mg/mL. Sophorolipid Mean
Polydispersity
z-potential
Encapsulation
diameter (nm)
index
(mV)
efficiency (%)
1
114 ± 13
0.160 ± 0.015
ab
-39.6 ± 1.9
a
40.9 ± 1.3
b
2
101.8 ± 9.8
0.276 ± 0.035
c
-40.9 ± 1.2
a
64.9 ± 2.4
c
4
60.8 ± 3.7
a
0.295 ± 0.045
c
-41.2 ± 2.0
a
82.2 ± 0.7
de
6
59.5 ± 0.9
a
0.153 ± 0.023
ab
-26.4 ± 1.1
b
89.3 ± 1.6
e
8
60.9 ± 0.8
a
0.089 ± 0.017
a
-20.50 ± 0.56
88.6 ± 1.3
e
1
400 ± 34
d
0.709 ± 0.029
d
-40.6 ± 1.8
a
25.8 ± 3.9
a
2
290 ± 15
c
0.657 ± 0.015
d
-39.9 ± 2.4
a
59.1 ± 7.8
c
4
58.8 ± 2.8
a
0.222 ± 0.006
bc
-41.1 ± 0.9
a
79.2 ± 6.1
d
6
57.9 ± 0.4
a
0.077 ± 0.024
a
-26.77 ± 1.63
b
88.7 ± 0.3
e
8
59.8 ± 1.1
a
0.089 ± 0.008
a
-19.00 ± 1.98
c
89.9 ± 1.1
e
concentration (mg/mL)
A
B
b
b
c
655
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Fig. 1
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Fig. 2
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Fig. 3
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Fig. 4
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Fig. 5
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Fig. 6
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Fig. 7
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GRAPHIC FOR TABLE OF CONTENTS
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