Utilizing Food Matrix Effects To Enhance Nutraceutical Bioavailability

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Utilizing food matrix effects to enhance nutraceutical bioavailability: Increase of curcumin bioaccessibility using excipient emulsions Liqiang Zou, Wei Liu, Chengmei Liu, Hang Xiao, and David Julian McClements J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf506149f • Publication Date (Web): 01 Feb 2015 Downloaded from http://pubs.acs.org on February 7, 2015

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Journal of Agricultural and Food Chemistry

220x184mm (150 x 150 DPI)

ACS Paragon Plus Environment


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Utilizing food matrix effects to enhance nutraceutical

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bioavailability: Increase of curcumin bioaccessibility

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using excipient emulsions

4 Liqiang Zouab, Wei Liua*, Chengmei Liua, Hang Xiaob, David Julian

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McClementsb,c*

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a

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Nanchang, No. 235 Nanjing East Road, Nanchang 330047, Jiangxi, China

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b

Department of Food Science, University of Massachusetts, Amherst, MA 01003

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c

Department of Biochemistry, Faculty of Science, King Abdulaziz University, P. O.

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Box 80203 Jeddah 21589 Saudi Arabia

State Key Laboratory of Food Science and Technology, Nanchang University,

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* These authors contributed equally to this manuscript. Contact information:

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Wei Liu, State Key Laboratory of Food Science and Technology, Nanchang

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University, Nanchang, 330047, Jiangxi, China Tel: + 86 791 88305872x8106. Fax:

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+86 791 88334509. E-mail:

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.Journal: Journal of Agricultural and Food Chemistry

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Submitted: December 18, 2014

E-mail:

[email protected].

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David Julian McClements, Department of Food Science, University of Massachusetts,

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Amherst, MA 01003, USA Tel: (413) 545-1019. Fax: (413) 545-1262. E-mail:

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[email protected].


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ABSTRACT

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Excipient foods have compositions and structures specifically designed to

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improve the bioaccessibility of bioactive agents present in other foods co-ingested

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with them.

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and bioaccessibility of curcumin from powdered rhizome turmeric (Curcuma longa).

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Corn oil-in-water emulsions were mixed with curcumin powder and the resulting

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mixtures were incubated at either 30 ºC (to simulate a salad dressing) or 100 ºC (to

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simulate a cooking sauce). There was an appreciable transfer of curcumin into the

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excipient emulsions at both incubation temperatures, but this effect was much more

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pronounced at 100 ºC. The bioaccessibility of curcumin measured using a simulated

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gastrointestinal tract model was greatly improved in the presence of the excipient

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emulsion, particularly in the system held at 100 ºC. This effect was attributed to the

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higher initial amount of curcumin solubilized within the oil droplets, as well as that

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solubilized in the mixed micelles formed by lipid digestion. This study highlights the

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potential of designing excipient food emulsions that increase the oral bioavailability

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of lipophilic nutraceuticals, such as curcumin.

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KEYWORDS: curcumin; excipient food; nanoemulsion; bioaccessibility;

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nutraceutical

In this study, an excipient emulsion was shown to improve the solubility

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INTRODUCTION Rhizome turmeric (Curcuma longa) is commonly used in foods as both a spice and

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a pigment because of its characteristic taste and yellow color (1, 2).

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also been used in traditional Asian medicine for thousands of years because of its

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perceived health benefits, and is now being actively investigated for its potential as a

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bioactive agent in pharmaceutical, food, and cosmetic products (1, 2).

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most highly biologically active constituents within turmeric is curcumin, i.e., 1,7-

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bis(4-hydroxy-3-methoxyphenyl)-1,6-hepadiene-3,5-dione. Curcumin has been used

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for the treatment of a variety of ailments, including coughs, fevers, jaundice, wounds,

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eczema, inflammatory joints, parasitic skin diseases, colds, liver diseases, urinary

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diseases, anemia, bacterial infections, and viral infections (1).

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basis for the claimed health benefits of curcumin can be attributed to various

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biological activities, including anti-inflammatory, antioxidant, antiviral, antibacterial,

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antifungal, and antitumor activities (3).

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Turmeric has

One of the

The physiological

The potential health benefits of curcumin have led to considerable interest in

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incorporating it into commercial food products as a nutraceutical agent.

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the high melting point, poor water-solubility, and low oral bioavailability of curcumin

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make it difficult to incorporate into many functional food products (4).

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curcumin is highly susceptible to chemical degradation when it is present within

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aqueous environments, particularly around neutral pH, which will also limit its

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bioavailability (5). One approach to increase the oral bioavailability of curcumin is to

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encapsulate it within food-grade biopolymers or colloidal delivery systems (6, 7).


However,

In addition,

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Numerous delivery systems have been investigated for their potential to improve the

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dispersibility, stability, and bioavailability of curcumin, including casein micelles (8,

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9), modified starch (10), curcumin nanoparticles (11), soy protein complexes (12),

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phospholipid complexes (13), colloidosomes (14), liposomes (15), nanoemulsions (4,

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16), and emulsions (17).

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An alternative approach for enhancing the oral bioavailability of curcumin is to

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develop an excipient food that is consumed with curcumin-rich foods.

In general, an

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excipient food has a composition and structure that is specially designed to increase

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the bioavailability of nutraceuticals that are present in other foods co-ingested with it

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(18).

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improved by consuming it with a specially designed sauce that increases its solubility

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within the intestinal fluids.

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effects when consumed in isolation, but it may boost the bioactivity of nutraceuticals

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in other foods and therefore enhance their health benefits. An excipient food may

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work by a number of different mechanisms, including reducing chemical degradation,

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modulating metabolism, increasing solubility, enhancing absorption, or inhibiting

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efflux (18).

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its ability to increase bioavailability, including lipids, carbohydrates, proteins,

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minerals, chelating agents and phytochemicals. For example, the solubilization and

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cellular uptake of carotenoids (β-carotene and lycopene) from fruits and vegetables

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can be increased within the small intestine phase by co-ingesting them with lipids (19-

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21).

For example, the bioavailability of curcumin within a natural spice may be

Thus, an excipient food may have no beneficial health

Many different components within an excipient food may contribute to

The bioaccessibility of curcumin can be increased in a similar manner using



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this approach (4).

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products to form mixed micelles that solubilize and transport highly lipophilic

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components to the epithelium cells.

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bioavailability of certain food components would be to incorporate efflux inhibitors,

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e.g., piperine (a constituent from black pepper) has been shown to act as an efflux

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inhibitor for curcumin (22).

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This effect has been attributed to the ability of the lipid digestion

An alternative strategy to improve the

In the present study, we examined the possibility of developing excipient

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emulsions to increase the bioaccessibility of powdered curcumin.

Crystalline forms

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of lipophilic bioactive agents usually have a much lower bioavailability than solubilized

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forms (23).

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excipient emulsion would increase its bioaccessibility due to its ability to increase the

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solubility of curcumin in the intestinal fluids.

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and time on the transfer of curcumin into the excipient emulsion prior to ingestion was

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measured, as well as the bioaccessibility of curcumin after exposure to a simulated

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gastrointestinal tract (GIT). Two incubation temperatures (30 and 100 ºC) were used to

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mimic conditions that curcumin might experience in food applications: (i) within

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ambient foods (such as salads); (ii) within cooked foods (such as curry sauces).

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information obtained from this study may be useful for the design of excipient

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emulsions to increase the bioavailability and bioactivity of lipophilic nutraceuticals.

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MATERIALS AND METHODS

We therefore hypothesized that mixing powdered curcumin with an

The effect of incubation temperature

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Materials. Corn oil purchased from a local supermarket was used as an

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example of a digestible long chain triglyceride. The following chemicals were

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purchased from the Sigma Chemical Company (St. Louis, MO): curcumin


The

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(SLBH2403V), mucin from porcine stomach (SLBH9969V), pepsin from porcine

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gastric mucosa (SLBL1993V), lipase from porcine pancreas pancreatin

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(SLBH6427V), porcine bile extract (SLBK9078), Tween 80 (BCBG4438V), and Nile

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Red (063K3730V). The supplier reported that the activity of the pepsin was around

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250 units/mg, and the activity of the lipase was around 100-400 units/mg protein

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(using olive oil). All other chemicals were of analytical grade. Double distilled

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water was used to prepare all solutions and emulsions.

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Excipient emulsion preparation. Initially, an aqueous phase was prepared by

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mixing 1% (w/w) Tween 80 with an aqueous buffer solution (10.0 mM phosphate

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buffer saline (PBS), pH 6.5). Coarse oil-in-water emulsions were prepared by

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homogenizing 10% (w/w) corn oil with 90% (w/w) aqueous phase using a high-speed

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blender for 2 min (M133/1281-0, Biospec Products, Inc., ESGC, Switzerland). Fine

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emulsions were then obtained by passing the coarse emulsions through a

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microfluidizer (M110Y, Microfluidics, Newton, MA) with a 75 μm interaction

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chamber (F20Y) at an operational pressure of 9,000 psi for 5 passes. The resulting

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excipient emulsions were stored in a refrigerator at 4 ºC before use.

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Preparation of curcumin-emulsion, curcumin-oil, and curcumin-buffer

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mixtures. Curcumin (3 mg) was weighed into a beaker and then excipient emulsion

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(10 mL) was added. The resulting mixtures were then incubated at either 30 or 100 ºC

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for different times (ranging from 10 min to 120 min for 30 ºC and from 10 min to 60

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min for 100 ºC). After incubation, selected samples were immediately placed in an ice

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water and then used for the following experiments. In some experiments, the



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curcumin was mixed with bulk corn oil or with buffer (PBS) solution rather than an

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emulsion.

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Curcumin solubility in mixtures. The solubility of curcumin in each mixture

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was measured spectrophotometrically based on the method of Ahmed, Li,

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McClements and Xiao (4) with some modifications. 10 mL of mixture was collected,

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and then centrifuged at 1750 rpm for 10 min at ambient temperature (CL10

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centrifuge, Thermo, Scientific, Pittsburgh, PA, USA) to remove non-dissolved

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(crystalline) curcumin. 1 mL of supernatant was then mixed with 5 mL choloroform,

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vortexed, and centrifuged at 1750 rpm for 10 min at ambient temperature. The bottom

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layer containing the solubilized curcumin was collected, while the top layer was

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mixed with an additional 5 mL of chloroform and the same procedure was repeated.

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The two bottom chloroform layers were combined, and diluted to an appreciate

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concentration to be analyzed by a UV–VIS spectrophotometer at 419 nm (Ultraspec

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3000 pro, GE Health Sciences, USA). A cuvette containing pure chloroform was used

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as a reference cell. The concentration of curcumin extracted from each mixture was

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calculated from a calibration curve of absorbance versus curcumin concentration in

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chloroform. The solubility of curcumin in each mixture was then calculated as the

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concentration of curcumin extracted from each mixture multiplied by the dilution

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factor.

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Particle characterization. The mean particle diameters (Z-average) and particle

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size distributions of the curcumin-emulsion mixtures after incubation at different

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temperatures were monitored using a dynamic light scattering instrument (Nano-ZS,


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Malvern Instruments, Worcestershire, UK). The electrical charge (-potential) of the

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particles in these samples was measured using a micro-electrophoresis instrument

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(Nano-ZS, Malvern Instruments, Worcestershire, UK). Samples were diluted with

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buffer solution (10.0 mM PBS, pH 6.5) prior to measurements to avoid multiple

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scattering effects.

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The mean particle diameter and particle size distribution of samples exposed to

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simulated gastrointestinal conditions was measured using static light scattering

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(Mastersizer 2000, Malvern Instruments Ltd., Worcestershire, UK). Samples were

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diluted with appropriate buffer solutions (same pH as GIT phase) and stirred in the

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dispersion unit at a speed of 1200 rpm. The particle size is reported as the surface-

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weighted mean diameter (d32).

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Microstructural analysis.

The microstructure of samples was characterized

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using confocal scanning fluorescence microscopy (Nikon D-Eclipse C1 80i, Nikon,

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Melville, NY). Samples analyzed by confocal microscopy were dyed with Nile Red to

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highlight the location of the lipid phase.

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ethyl alcohol at a concentration (1 mg/mL). Then, before analysis 2 mL emulsion

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samples were mixed with 0.1 mL Nile Red solution (1 mg/mL ethanol) to dye the oil

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phase. All images were captured with a 10× eyepiece and a 60× objective lens (oil

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immersion). Changes in the properties of curcumin crystals in the mixtures were

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observed using a cross-polarized lens (C1 Digital Eclipse, Nikon, Tokyo, Japan).

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The Nile red was dissolved in absolute

Curcumin oil-solubility characteristics.

The temperature-dependence of the

dissolution of crystalline curcumin into bulk corn oil was characterized by measuring



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the change in turbidity (600 nm) with temperature using a UV–visible

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spectrophotometer equipped with a temperature controller (Agilent Cary 200, Agilent,

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Santa Clara, CA). This method can be used to detect the presence of curcumin

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crystals in the oil phase and indirectly determine the solubility of curcumin in bulk

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corn oil.

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at ambient temperature, and then the mixture was heated from 25 to 100 ºC, and then

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cooled from 100 to 25 ºC at a rate 1 ºC/min with continuous stirring. In some

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experiments, the change in turbidity with time was measured at a fixed incubation

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temperature (30 or 100 ºC) to determine the kinetics of isothermal dissolution.

A weighed amount of curcumin (3 or 4 mg/mL) was dispersed in corn oil

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Simulated gastrointestinal digestion: Powdered curcumin was mixed with

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excipient emulsion, corn oil, or buffer solution and then held at 30 ºC for 30 min or

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100 ºC for 10 min. Each sample was passed through a three-step GIT model that

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consisted of mouth, gastric, and small intestine phases, which was slightly modified

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from the our previous study (24).

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Initial system: The initial systems were placed into a glass beaker in an incubator

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shaker at a rotation speed of 100 rpm for 15 min at 37 °C for preheating (Innova

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Incubator Shaker, Model 4080, New Brunswick Scientific, New Jersey, USA).

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Three different initial systems were tested: (i) curcumin and excipient emulsion; (ii)

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curcumin, corn oil, and buffer solution; or, (iii) curcumin and buffer solution. The

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initial concentration of curcumin was the same in all systems, while the initial

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concentration of corn oil in systems (i) and (ii) were the same.


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Mouth phase: A simulated saliva fluid (SSF) containing 3 mg/mL mucin and

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various salts was prepared as described previously (25). SSF was preheated to 37 °C

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and then mixed with the preheated curcumin mixture at a 1:1 mass ratio. The mixture

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was then adjusted to pH 6.8 and placed in an incubator shake at 100 rpm and 37ºC for

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10 min. This incubation time is longer than a food would normally spend in the

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mouth, but was used to minimize sample-to-sample variations that might occur if very

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short times were used.

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Stomach phase: Simulated gastric fluid (SGF) was prepared by placing 2 g NaCl

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and 7 mL HCl into a container, and then adding double distilled water to 1 L. The

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bolus sample from the mouth phase was then mixed with SGF containing 0.0032

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g/mL pepsin preheated to 37 °C at a 1:1 mass ratio. The mixture was then adjusted to

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pH 2.5 and placed in a shaker at 100 rpm and 37 ºC for 2 hours to mimic stomach

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digestion.

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because of the difficulty in obtaining a reliable and economically viable source of this

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digestive enzyme (26).

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digestion within the stomach phase, and should therefore its omission should only

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have a fairly modest impact on the gastrointestinal fate of the excipient emulsions.

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It should be noted that we did not include gastric lipase in the SGF

Gastric lipase typically promotes a limited amount of lipid

Small Intestine phase: 30 mL chyme samples from the stomach phase were

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diluted with buffer solution (10 mM PBS, pH 6.5) to obtain a final corn oil

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concentration of 1.25%. The diluted chyme was then incubated in a water bath (37 ºC)

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for 10 min and then the solution was adjusted back to pH 7.0. Next, 3 mL of

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simulated intestinal fluid (containing 0.5 M CaCl2 and 7.5 M NaCl) was added to 60



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mL digesta. Then, 7 mL bile extract, containing 375.0 mg bile extract (pH 7.0, PBS),

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was added with stirring and the pH was adjusted to 7.0. Finally, 5 mL of pancreatic

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suspension, containing 120 mg of lipase (pH 7.0, PBS), was added to the sample and

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an automatic titration unit (Metrohm, USA Inc.) was used to monitor the pH and

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control it to a fixed value (pH 7.0) by titrating 0.25 M NaOH solution into the reaction

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vessel for 2 h at 37 °C. The percentage of free fatty acids released in the sample was

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calculated from the number of moles of NaOH required to maintain neutral pH as

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described previously (27).

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not be fully ionized at pH 7, and therefore the FFAs determined by the pH stat method

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are only the titrable ones (28).

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It should be noted that some of the free fatty acids may

Curcumin bioaccessibility.

After in vitro digestion, 30 mL raw digesta of each

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mixture was centrifuged (18000 rpm, Thermo Scientific, USA) at 25 ºC for 30 min.

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The clear supernatant was collected and assumed to be the ‘‘micelle’’ fraction in

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which the curcumin was solubilized. In some samples, a layer of non-digested oil was

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observed at the top of the samples and it was removed from the micelle fraction.

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Aliquots of 5 mL of micelle fraction were mixed with 5 mL of chloroform, vortexed

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and centrifuged at 1750 rpm for 10 min at ambient temperature. The bottom layer

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containing the solubilized curcumin was collected, while the top layer was mixed with

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an additional 5 mL of chloroform and the same procedure was repeated. The two

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collected chloroform layers were mixed together, and then diluted to an appreciate

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concentration to be analyzed by a UV–VIS spectrophotometer at 419 nm. The

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concentration of curcumin was calculated from the absorbance using a standard curve


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using a suitable dilution factor.

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according to the following expression: BA% = 100 × cM / cD, where cM and cD are the

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curcumin concentrations in the mixed micelle phase and in the total digesta collected

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after the small intestine phase, respectively.

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Statistical analysis.

The bioaccessibility of curcumin was calculated

All experiments were carried out on at least two freshly

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prepared samples. The results are expressed as means ± standard deviation (SD). Data

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was subjected to statistical analysis using SPSS software version 18.0. Differences

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were considered significant at p < 0.05.

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RESULTS AND DISCUSSION

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Effect of incubation temperature on particle characteristics.

In this study,

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curcumin-containing samples were exposed to two different incubation temperatures

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to simulate different conditions that they might experience in food applications: (i) 30

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ºC for salads at ambient temperature; (ii) 100 ºC for sauces at cooking temperatures.

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The mixture of curcumin and excipient emulsion was stirred to ensure homogeneity,

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and then incubated at either 30 or 100 ºC for different times.

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diameter, particle size distribution, particle charge, and visual appearance of the

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different samples was then measured (Table 1, Figures 1a-c).

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The mean particle

There were no appreciable changes in the characteristics of the droplets in the

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curcumin-emulsion mixtures during incubation at 30 ºC for up to 120 min (Table 1

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and Figure 1a).

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diameter, polydispersity index (PDI), or ζ-potential of the curcumin-emulsion

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mixtures when compared to the initial emulsions. The curcumin-emulsion systems

Indeed, there were no significant changes in the mean particle



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were also stable to incubation at 100 ºC from 10 to 30 min, with no significant

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changes in mean particle diameter, PDI, or ζ-potential. However, there was evidence

265

of some droplet creaming in the curcumin-emulsion mixture after heating at 100 ºC

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for 60 min (indicated by an arrow in Figure 1c). Moreover, there was a significant

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increase in the mean particle diameter and PDI for these emulsions (Figure 1b and

268

Table 1). This effect may be due to droplet coalescence resulting from dehydration of

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the non-ionic surfactant head groups at elevated temperatures (29). There was no

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significant change in the magnitude of the electrical charge on the emulsion droplets

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when they were incubated at either 30 or 100 ºC, which suggests that the interfacial

272

composition remained relatively constant.

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The microstructures of the curcumin-emulsion mixtures after heating at 30 and

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100 ºC for different times were recorded using confocal fluorescence microscopy

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(Figure 2a). There was little change in the microstructure of the mixed systems

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incubated at 30 ºC for different times, confirming their high stability under these

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conditions. However, there was evidence of some larger oil droplets in the mixed

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systems after incubation at 100 ºC for prolonged times, suggesting that some

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coalescence had occurred.

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We also measured the amount of curcumin transferred into the excipient

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emulsions over time at different incubation temperatures.

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solubilized in the excipient emulsions depended on incubation temperature and time.

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There was a gradual increase in the amount of curcumin solubilized within the

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excipient emulsion for mixtures incubated at 30 ºC (Table 1).


The amount of curcumin

On the other hand,

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there was a rapid increase in the amount of curcumin solubilized in the excipient

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emulsions during the first 10 minutes of incubation at 100 ºC, followed by an

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appreciable decrease at longer incubation times.

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fact that droplet coalescence and oiling off occurred in the emulsions held at the

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higher temperature, which meant that some of the curcumin remained in the upper oil

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phase and was not therefore measured. Overall, the amount of curcumin solubilized

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within the excipient emulsions was considerable higher for the samples incubated at

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the higher holding temperatures.

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in the emulsions was around 54 and 218 g/mL after incubation for 60 min at 30 ºC

294

and 90 ºC, respectively.

This decrease was attributed to the

For example, the amount of curcumin solubilized

295

The presence of curcumin crystals within the different mixtures after exposure to

296

different incubation temperatures was observed using a crossed polarizer lens (Figure

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2b). Some crystalline material was clearly observed in the curcumin-emulsion

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mixtures incubated at 30 ºC from 10 to 120 min, which suggested that the curcumin

299

crystals did not completely dissolve when held at this temperature in the presence of

300

the excipient emulsion. Conversely, no crystals were observed in the curcumin-

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emulsion mixtures held at 100 ºC at any incubation time, which indicated that

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curcumin crystals rapidly and completely dissolved within the emulsions at this

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elevated temperature. However, there was evidence of some large droplets in these

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samples after heating for prolonged times at 100 ºC, again suggesting that droplet

305

coalescence occurred.

306

Temperature dependence of curcumin oil-solubility.


The dependence of the


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transfer of curcumin into the excipient emulsions on incubation temperature and time

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may be due to changes in its oil solubility with temperature (30). We therefore used

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turbidity measurements to monitor the solubility of curcumin crystals in bulk corn oil

310

at different temperatures.

311

curcumin-oil mixture is relatively high when curcumin crystals are present because

312

they scatter light strongly, but it is relatively low when the crystals have melted or

313

dissolved due to the decrease in light scattering.

314

The principle of this method is that the turbidity of the

The turbidity of curcumin in corn oil mixtures (3 mg/mL) decreased appreciably

315

upon heating from 25 to 100 ºC (Figure 3a) until it reached a value close to zero at

316

100 ºC indicating that the crystals had fully dissolved at this temperature. Upon

317

cooling, the turbidity stayed low suggesting that the curcumin remained dissolved

318

within the oil. This may have occurred because curcumin was below its saturation

319

temperature at the lower temperatures, or because of supersaturation/supercooling

320

effects (30).

321

curcumin/oil mixture containing a higher curcumin concentration (4 mg/mL).

322

case, the turbidity still decreased appreciably with increasing temperature, but the

323

final turbidity reached at high temperatures was considerably greater than that

324

observed at the lower curcumin level.

325

temperatures still appeared turbid, suggesting that not all of the curcumin crystals had

326

dissolved. When this sample was cooled down the turbidity remained relatively high

327

and even increased slightly, which can be attributed to the fact that the solubility of

328

curcumin decreases with decreasing temperature and the amount of curcumin present

We therefore measured the change in turbidity with temperature for a In this

Indeed, the samples at the higher


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was above the saturation level. These results suggest that the solubility of curcumin

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in corn oil at ambient temperature was somewhere between 3 and 4 mg/mL, which is

331

in agreement with the value of ≈ 3.2 ± 0.1 mg/mL reported previously (4).

332

Information about the kinetics of curcumin dissolution at the two incubation

333

temperatures was obtained by measuring changes in the turbidity of curcumin/oil

334

mixtures (3 mg/mL) over time (Figure 3b). There was little change in the turbidity of

335

the mixture held at 30 ºC suggesting that curcumin crystals only dissolved slowly at

336

this temperature.

337

held at 100 ºC, which indicated that the curcumin crystals rapidly dissolved in the

338

corn oil at this elevated temperature. This knowledge may be important for

339

developing effective processing operations for incorporating curcumin into food

340

products.

341

On the other hand, the turbidity decreased rapidly in the mixture

Influence of simulated digestion on particle properties.

In this section, we

342

examined the influence of the excipient emulsions on the potential biological fate of

343

curcumin using an in vitro gastrointestinal tract (GIT) model that simulates the mouth,

344

stomach, and small intestine phases. Curcumin-emulsion mixtures incubated at 30 ºC

345

for 30 min or at 100 ºC for 10 min were selected for the GIT study to simulate

346

ambient food applications (such as salad dressings) and cooking applications (such as

347

curry sauces).

348

emulsions that were physically stable, and that might simulate usage conditions.

349

should be noted that the amount of curcumin solubilized in the excipient emulsions at

350

the higher temperature was not strongly dependent on incubation temperature.

These incubation times were selected because they led to excipient


It

The


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results for the excipient emulsions were compared to curcumin-oil and curcumin-

352

buffer mixtures that initially contained the same amount of curcumin.

353

size, microstructure, particle charge, and overall appearance of the different samples

354

were then measured (Figures 4 to 6).

355

Particle size and system microstructure.

The particle

The properties of the curcumin-

356

emulsion mixture were evaluated at all stages of the GIT model, but the curcumin-oil

357

mixture was only evaluated after incubation in the small intestine phase since reliable

358

samples could not be obtained from the mouth or stomach phases. This was because

359

the bulk oil tended to form a separate layer at the top of the mixtures, and therefore it

360

was difficult to collect a representative sample.

361

curcumin-buffer mixture were not determined in this series of experiments because it

362

was difficult to collect reliable samples when there were only a few curcumin crystals

363

present in a large volume of buffer solution.

364

Similarly, the characteristics of the

In general, fairly similar trends were observed in the gastrointestinal behavior of

365

curcumin-emulsion mixtures that had previously been incubated at either 30 or 100

366

ºC, and so the results are discussed together. The mean particle diameter (d32)

367

determined by static light scattering remained relatively constant after exposure to the

368

mouth and stomach phases, but increased appreciably after exposure to the small

369

intestine phase (Figure 4a). Examination of the full particle size distributions of these

370

emulsions indicated that a large fraction of the droplets had fairly similar sizes to the

371

initial emulsions after exposure to the mouth and stomach phases, which suggested

372

that they were relatively stable to coalescence, presumably because they had a non-


Page 19 of 44


373

ionic surfactant coating that was resistant to changes in pH, salt, and protease activity.

374

Nevertheless, confocal microscopy images of the same samples indicated that

375

extensive droplet flocculation occurred within the mouth and stomach phases (Figure

376

6), which may have been due to depletion or bridging flocculation promoted by mucin

377

originating from the artificial saliva (31). There appeared to be some dissociation of

378

the flocs formed in the emulsions when they moved from the mouth to the stomach

379

stage (Figure 6). This behavior is in agreement with previous studies (31), and may

380

be caused by a number of factors including sample dilution, changes in solution

381

composition, and/or mechanical agitation (32, 33).

382

The fact that the large particles observed in many of the samples by confocal

383

microscopy were not observed by static light scattering suggests that the flocs

384

dissociated upon dilution and stirring during sample preparation for the particle size

385

analysis.

386

measurements with microscopy measurements for this type of complex colloidal

387

system.

388

This result highlights the importance of confirming light scattering

Light scattering measurements indicated that a population of relatively large

389

particles was present in the curcumin-emulsion mixtures after exposure to the small

390

intestine phase (Figures 5a and b), which was confirmed by confocal microcopy

391

(Figure 6).

392

intestinal digesta may contain various types of colloidal species, including non-

393

digested lipids, micelles, vesicles, liquid crystals, and insoluble matter (such as

394

calcium soaps).

It is difficult to identify the precise nature of these particles because the



395

Page 20 of 44

Light scattering (Figure 5) and confocal microscopy (Figure 6) indicated that

396

there were much larger particles present in the curcumin-oil mixtures after exposure

397

to the small intestine phase than in the curcumin-emulsion mixtures.

398

explanation for this effect is that the bulk corn oil was digested more slowly than the

399

pre-emulsified corn oil (see later), and therefore there were some large non-digested

400

lipid droplets present.

401

Electrical characteristics.

A possible

The electrical characteristics (ζ-potential) of the

402

particles in emulsion-curcumin mixtures exposed to different incubation temperatures

403

followed similar trends after passage through each stage of the simulated GIT (Figure

404

7), and so they will again be considered together.

405

curcumin-emulsion mixtures had relatively low negative charges (≈ −4 mV), which

406

was due to the fact that a non-ionic surfactant was used to coat the droplets. The

407

particles became appreciably more negative after exposure to the mouth phase (≈ −9

408

mV), which may have been due to association of anionic species (such as mucin) with

409

the lipid droplet surfaces (31). The particle charge became much less negative (≈ −2

410

mV) after exposure to the stomach phase, which can be attributed to the relatively low

411

pH and high ionic strength of the gastric fluids. Finally, the particle charge became

412

highly negative (≈ −47 mV) after exposure to the small intestinal fluids, which is

413

probably due to the presence of various anionic constituents associated with this phase

414

such as bile salts, phospholipids, and free fatty acids (31). There were no significant

415

differences between the electrical characteristics of the particles in the small intestine

416

phase for the curcumin-emulsion and curcumin-oil mixtures at either incubation

The particles in the initial


Page 21 of 44


417

temperature. This effect can be attributed to the fact that the electrical characteristics

418

were dominated by the presence of bile salts, phospholipids, and free fatty acids, and

419

were not strongly affected by sample microstructure.

420

Lipid digestion.

In this section, we used the pH stat method to determine the

421

rate and extent of lipid digestion of curcumin-emulsion and curcumin-oil mixtures

422

that had previously been incubated at either 30 or 100 ºC. The volume of NaOH that

423

had to be titrated into the samples to maintain a constant pH (7.0) was measured as a

424

function of digestion time, and then the fraction of free fatty released from the

425

mixture was calculated (Figure 8).

426

The initial incubation temperature (30 or 100 ºC) had no effect on the rate and

427

extent of lipid digestion for the curcumin-emulsion mixtures, which was probably due

428

to the fact that the interfacial areas and compositions of the lipid droplets entering the

429

small intestine phase were fairly similar (Figure 8). In these systems, there was a

430

rapid increase in FFAs during the first 10 minutes, followed by a more gradual

431

increase at longer times.

432

FFAs released over time in the curcumin-oil mixtures for both incubation times

433

(Figure 8).

434

oil exposed to lipase for the emulsified corn oil than for the bulk corn oil.

435

the confocal microscopy images clearly highlighted that the fat droplets in the small

436

intestine phase were much larger for the bulk oil than the emulsified oil (Figure 6).

437

Additionally, the rate and extent of lipid digestion in the curcumin-oil mixture that

438

had been incubated at 100 ºC (56% FFAs released after 2 hours) was higher than the

Conversely, there was a much less steep increase in the

The origin of this effect can be attributed to the higher surface area of


Indeed,


Page 22 of 44

439

one that had been incubated at 30 ºC (49% FFAs released after 2 hours).

440

of this effect is unknown, but it may have been because the corn oil that had been

441

incubated at the higher temperature was initially dispersed better in the water phase,

442

i.e. formed smaller fat droplets (higher surface area).

443

Curcumin solubilization and mixed micelle properties.

The origin

The characteristics of

444

the particles in the mixed micelle phase formed after exposure of the samples to

445

simulated small intestine conditions were measured, as well as the amount of

446

curcumin solubilized within the mixed micelle phase (Table 2).

447

that the mixed micelle phase was collected by centrifugation, so that any large

448

particles observed in the small intestine digesta should have been removed.

449

the mixed micelle samples contained highly negatively charged particles, which can

450

be attributed to the fact that they consisted primarily of bile salts, phospholipids, and

451

free fatty acids. The mean particle diameters in the micelle phase were around 100 to

452

200 nm, which suggests that they were probably vesicles since true micelles are much

453

small than this (< 10 nm). Surprisingly, the particles in the mixed micelle phase

454

collected from digestion of the bulk oils were appreciably smaller than those collected

455

from digestion of the emulsified oils (Table 2). There are a number of potential

456

mechanisms that could account for this observation, such as differences in the rate and

457

extent of lipid digestion, and the presence of non-ionic surfactant in the emulsions.

458

For both the curcumin-emulsion and curcumin-oil systems, there was no major

459

difference between the size of the particles in samples that had been incubated at 30 or

460

100 ºC.


It should be noted

All of

Page 23 of 44

461


The amount of curcumin present in the mixed micelle phase is a good indication

462

of its bioaccessibility, i.e., the amount available for absorption.

463

concentration of curcumin measured in the mixed micelle phase was higher for the

464

digested curcumin-emulsion mixtures that for the digested curcumin-oil mixtures

465

(Table 2). This suggests that there was more efficient transfer of the curcumin into

466

the mixed micelles for the emulsion than for the bulk oil.

467

reasons for this phenomenon: (i) some of the bulk oil was not digested, and so some

468

of the curcumin may have remained dissolved within this oil phase; (ii) more of the

469

emulsified oil was digested, and so there will have been more mixed micelles

470

available to solubilize the curcumin.

471

micelles obtained from digestion of the curcumin-emulsion mixture incubated at 100

472

ºC was appreciably higher than that for the mixture incubated at 30 ºC.

473

was probably due to the fact that a higher fraction of the crystalline curcumin was

474

solubilized within the emulsion held at the higher incubation temperature.

475

amount of curcumin present within the mixed micelle phase resulting from digestion

476

of the curcumin-oil mixture was also higher for the sample incubated at 100 ºC than

477

for the one incubated at 30 ºC. This effect may again be due to the fact that a higher

478

amount of curcumin was solubilized in the oil phase (rather than present as crystals)

479

prior to digestion.

480

bulk oil incubated at 100 ºC, which may have resulted in greater curcumin release and

481

solubilization.

482

Overall, the

There are two major

The curcumin concentration in the mixed

This effect

The

In addition, there was a greater extent of lipid digestion in the

Potential Mechanisms. The potential mechanisms responsible for the increase in



Page 24 of 44

483

bioaccessibility of curcumin by excipient emulsions are highlighted in Figure 9.

484

Prior to ingestion, curcumin may be solubilized within the oil droplets when

485

powdered curcumin is incubated with the emulsions. This may occur due to

486

diffusion of curcumin molecules through the aqueous phase, and occurs more rapidly

487

for emulsifier oil than for bulk oil due to the higher surface area and shorter diffusion

488

pathway. In addition, this process occurs more rapidly at higher temperatures due to

489

the higher solubility of curcumin in the oil and water phases. After ingestion,

490

curcumin may be solubilized within the mixed micelles resulting from digestion of the

491

oil droplets. The transfer of curcumin into the mixed micelles may be more efficient

492

for emulsified oil than for bulk oil due to the faster rate and greater extent of lipid

493

digestion. Consequently, there are more mixed micelles available for the curcumin

494

to the solubilized within.

495

In summary, the present work has shown that excipient emulsions can be used

496

to increase the bioaccessibility of powdered curcumin. We have shown that a greater

497

amount of curcumin is transferred from the powder into the lipid droplets for

498

curcumin-emulsion mixtures incubated at 100 ºC than for those incubated at 30 ºC.

499

This effect was attributed to the fact that solubility of curcumin in the water and oil

500

phases increases with increasing temperature, as well as the mass transport rate.

501

was shown that the curcumin concentration in the mixed micelle phase formed after

502

exposure to a simulated gastrointestinal tract depended on the nature of the food

503

matrix, decreasing in the following order: emulsified oil > bulk oil > buffer solution.

504

This effect was attributed to the increased solubilization capacity of the small


It

Page 25 of 44


505

intestinal fluids when a triglyceride oil is broken down into free fatty acids and

506

monoglycerides that are incorporated into mixed micelles.

507

concentration in the mixed micelle phase was higher for curcumin-emulsion or

508

curcumin-oil mixtures that had been incubated at 100 ºC than for those that had been

509

incubated at 30 ºC, which was attributed to a greater solubilization of the curcumin

510

into the oil phase prior to digestion.

511

designing food matrices to increase the oral bioavailability of lipophilic nutraceuticals

512

and vitamins.

513

ACKNOWLEDGMENTS

514

This material is based upon work supported by the Cooperative State Research,

515

Extension, Education Service, United State Department of Agriculture, Massachusetts

516

Agricultural Experiment Station (Project No. 831) and by the United States

517

Department of Agriculture, NRI Grants (2011-03539, 2013-03795, 2011-67021, and

518

2014-67021).

519

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612 613



Page 28 of 44

70

Relative Intensity (%)

30 °C 120 min

60 50

30 °C 60 min

40 30 °C 30 min

30 20

30 °C 20 min

10 30 °C 10 min

0 10

100

1000

10000

Droplet Diameter (nm) Figure 1 (a). Particle size distributions of mixtures of curcumin and excipient emulsion after incubation at 30 ºC for different times;


Page 29 of 44


90

Relative Intensity (%)

80 100 °C 60 min

70 60

100 °C 30 min

50 40

100 °C 20 min

30 100 °C 10 min

20 10

Unheated

0 10

100

1000

10000

Droplet Diameter (nm) Figure 1 (b). Particle size distributions of mixtures of curcumin and excipient emulsion after incubation at 100 ºC for different times.



Figure 1 (c). Photographs of mixtures of curcumin and excipient emulsion after incubation at 30 and 100 ºC.

Note: yellow sediment (curcumin crystals) was

observed at the bottom of the test tubes held at 30 ºC, whereas a yellow oil layer was observed at the top of the test tubes after heating at 100 ºC for 60 minutes (red arrows).


Page 30 of 44

Page 31 of 44


Figure 2. (a) Effect of temperature on microstructure of mixtures of curcumin and excipient emulsion; (b). Effect of temperature on polarized light microscopy of curcumin and excipient emulsion.



4

3 mg/mL curcumin (Heating) 3 mg/mL curcumin (Cooling)

3.5

4 mg/mL curcumin (Heating) 4 mg/mL curcumin (Cooling)

3

Turbidity (cm-1)

Page 32 of 44

2.5 2 1.5 1 0.5 0

20

30

40

50

60

70

80

90 100

Temperature (ºC ) Figure 3 (a). Absorbance versus temperature profile of curcumin-corn oil mixtures (3 and 4 mg/mL),


Page 33 of 44


3

Turbiditiy (cm-1)

2.5 2

30 C

1.5

1 0.5 100 C

0

0

20

40

60

80

100

120

Time (min)

Figure 3b. Absorbance versus time profile of a curcumin-corn oil mixture (3 mg/mL) at different isothermal storage temperatures.



30 ℃ 30 min (Emulsion)

Mean Particle Diameter (µm)

0.8

100 ℃ 10 min (Emulsion)

Page 34 of 44

cd

cd

30 ℃ 30 min (Oil) 100 ℃ 10 min (Oil)

0.6

bcd abc

0.4

ab a

abc ab

abc a

0.2

0

Initial

Mouth Stomach Intestine

Figure 4. (a). Influence of simulated gastrointestinal conditions on the mean droplet diameter (d32) of curcumin-emulsion and curcumin-oil mixtures after incubation at 30 ºC for 30 min or at 100 ºC for 10 min. Samples designated with different letters (a, b, c) were significantly different (Duncan, p < 0.05);


Page 35 of 44


Figure 4 (b). Image of micelle phase collected from curcumin-emulsion and curcumin-oil mixtures.



Page 36 of 44

60 Initial

Mouth

Stomach

Intestine

50

Volume (%)

40

30

20

10

0 0.01

1

100

10000

Diameter (μm) Figure 5a. Influence of simulated gastrointestinal conditions on the particle size distributions of curcumin-emulsion mixture after 30 min incubation at 30 ºC.


Page 37 of 44


60 Initial

Mouth

Stomach

Intestine

50

Volume (%)

40 30 20 10 0 0.01

1

100

10000

Diameter (μm) Figure 5b. Influence of simulated gastrointestinal conditions on the particle size distributions of curcumin-emulsion mixture after 10 min incubation at 100 ºC.



Page 38 of 44

8

7

Volume (%)

6 5 100 ℃ 10 min Intestine

4 30 ℃ 30 min Intestine

3 2 1 0 0.01

1

100

10000

Diameter (μm) Figure 5c. Influence of simulated gastrointestinal conditions on the particle size distributions of curcumin-oil mixture in the small intestine (measurements could not be made in the initial, mouth, or stomach phases for this sample).


Page 39 of 44


Figure 6. Influence of simulated gastrointestinal conditions on microstructure of curcumin-emulsion and curcumin-oil mixtures exposed to different incubation conditions (30 ºC for 30 min or 100 ºC for 10 min) determined by confocal fluorescence microscopy. The scale bars represent a length of 20 μm, and the red regions represent lipids.



Page 40 of 44

Figure 7. Influence of simulated gastrointestinal conditions on the particle charge of curcumin-emulsion and curcumin-oil mixtures exposed to different incubation conditions (30 ºC for 30 min or 100 ºC for 10 min). Samples designated with different letters (a, b, c) were significantly different (Duncan, p < 0.05).

-5

d d

cd bc

ζ-Potential (mV)

bc b

-15

-25

30 ℃ 30 min (Emulsion) 100 ℃ 10 min (Emulsion)

-35

30 ℃ 30 min (Oil) 100 ℃ 10 min (Oil)

a

-45 a

a a

-55 Initial

Mouth

Stomach


Intestine

Page 41 of 44


Figure 8. Influence of incubation temperature on the free fatty acids (FFA %) release profile for curcumin-emulsion and curcumin-oil mixtures exposed to different incubation conditions (30 ºC for 30 min or 100 ºC for 10 min).

80

FFA Released (%)

70 60 50 40 30

30 ℃ 30 min (Emulsion) 30 ℃ 30 min (Oil)

20

100 ℃ 10 min (Emulsion) 100 ℃ 10 min (Oil)

10 0 0

20

40

60

80

100

Digestion Time (min)


120


Figure 9.

Page 42 of 44

Schematic diagram of the pre-ingestion and post-ingestion solubilization

of curcumin in excipient emulsions.

Prior to ingestion, curcumin may be solubilized

in oil droplets when powdered curcumin is incubated with the emulsions. After ingestion, curcumin may be solubilized within the mixed micelles resulting from digestion of the oil droplets.

Curcumin

Pre-Ingestion

Incubation

Solubilization

Excipient Emulsion

Curcumin Powder

Post-Ingestion Digestion

Solubilization Mixed micelles


Page 43 of 44


Table 1. Effect of incubation time and temperature on the mean particle diameter (Z-average), polydispersity index (PDI), ζ-potential, and curcumin solubility of mixtures of curcumin and excipient emulsion.

The “initial” sample was the oil-in-water emulsion before adding

curcumin and before incubation. ND = not determined. 30 ºC

Mean diameter

100 ºC

Initial

10 min

20 min

30 min

60 min

120 min

10 min

20 min

30 min

60 min

2089 a

2057 a

22521 a

21316 a

21819 a

20812 a

25925 a

26725 a

25818 a

984551 b

(nm) PDI

ζ-potential (mV)

Solubility

0.200.0

0.200.0

0.260.0

0.230.0

0.230.0

0.200.0

0.270.0

0.340.0

0.280.0

0.620.3

a

1a

5a

6a

4a

3a

9a

8a

6a

b

-6.81.2

-7.03.7

-5.71.8

-4.60.9

-6.94.2

-5.51.0

-3.6 1.0

-3.70.7

-3.90.6

-7.62.7

a

a

a

a

a

a

a

a

a

a

ND

3110 a

3610 ab

495 bc

549 c

9910 d

27421 f

26430 f

26634 f

21866 e

(µg/mL)



Page 44 of 44

Table 2. Influence of incubation temperature on the curcumin concentration, particle size, polydispersity index (PDI), and ζ-potential of the micelle phase isolated from mixtures containing curcumin dispersed in emulsions, corn oil or buffer solution. Samples designated with different letters (a, b, c) were significantly different (Duncan, p < 0.05). ND = not determined.

30 ºC Emulsion Curcumin

Oil

83.519.5 bc 68.1  6.8 b

(µg/mL) Mean diameter

100 ºC Buffer

Emulsion

Oil

Buffer

12.76.5

119.5 

95.2  5.1

14.6 1.0

a

23.8 d

c

a

194 9 b

134  7 a

ND

202 9 b

146  19 a

ND

0.47  0.06

0.20  0.06

ND

0.38  0.01

0.21 

ND

c

a

b

0.01 a

-56.6  1.4

-44.4  4.7

a

b

(nm) PDI

ζ-potential (mV)

ND

-58.0  0.2 a -55.6  1.0


a

ND

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