Article Cite This: J. Agric. Food Chem. 2018, 66, 1488−1497
pubs.acs.org/JAFC
Enhancement of Curcumin Bioavailability by Encapsulation in Sophorolipid-Coated Nanoparticles: An in Vitro and in Vivo Study Shengfeng Peng,† Ziling Li,†,‡ Liqiang Zou,† Wei Liu,*,† Chengmei Liu,† and David Julian McClements*,§ †
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 Jiangxi, P.R. China School of Life Science, Jiangxi Science and Technology Normal University, Nanchang, 330013 Jiangxi, P.R. China § Biopolymers and Colloids Laboratory, Department of Food Science, University of Massachusetts, Amherst, Massachusetts 01003, United States ‡
ABSTRACT: There is great interest in developing colloidal delivery systems to enhance the water solubility and oral bioavailability of curcumin, which is a hydrophobic nutraceutical claimed to have several health benefits. In this study, a natural emulsifier was used to form sophorolipid-coated curcumin nanoparticles. The curcumin was loaded into sophorolipid micelles using a pH-driven mechanism based on the decrease in curcumin solubility at lower pH values. The sophorolipid-coated curcumin nanoparticles formed using this mechanism were relatively small (61 nm) and negatively charged (−41 mV). The nanoparticles also had a relatively high encapsulation efficiency (82%) and loading capacity (14%) for curcumin, which was present in an amorphous state. Both in vitro and in vivo studies showed that the curcumin nanoparticles had an appreciably higher bioavailability than that of free curcumin crystals (2.7−3.6-fold), which was mainly attributed to their higher bioaccessibility. These results may facilitate the development of natural colloidal systems that enhance the oral bioavailability and bioactivity of curcumin in food, dietary supplements, and pharmaceutical products. KEYWORDS: curcumin, bioavailability, nanoparticles, bioaccessibility, stability, biosurfactant, sophorolipid
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INTRODUCTION Nutraceuticals are bioactive components found in foods that are claimed to have certain health benefits when ingested orally.1 Curcumin is a polyphenolic nutraceutical typically isolated from the herb turmeric, which is a member of the ginger family.2 It has been reported to exhibit a broad range of biological activities that may be beneficial to human health, including anticancer, anti-inflammatory, antimicrobial, and antioxidant activities.2 However, the utilization of curcumin as a nutraceutical ingredient in functional food and beverage products is currently limited by its poor water solubility, chemical stability, and oral bioavailability characteristics.3 These challenges can often be overcome by encapsulating curcumin within colloidal delivery systems that contain small particles dispersed within an aqueous medium, such as emulsions,4,5 nanoemulsions,5 micelles,6,7 liposomes,8−10 biopolymer microgels,4 and polymer nanoparticles.11−14 The functional performance of each colloidal delivery system depends on the composition and structure of the particles it contains. Consequently, they must be carefully designed to exhibit the functional attributes required for a specific application. These functional attributes include physical and chemical stability under different environmental conditions, rheological characteristics, optical properties, flavor profile, and gastrointestinal fate (including bioavailability).15 The food industry is increasingly moving toward formulating its products from natural ingredients rather than synthetic ones.16,17 Consequently, it would be beneficial to create curcumin delivery systems from natural components. Synthetic surfactants are often used to facilitate the formation and/or © 2018 American Chemical Society
increase the stability of the particles in colloidal delivery systems. They do this by adsorbing to particle surfaces and generating strong repulsive forces between the particles, such as steric or electrostatic repulsion.18 Synthetic surfactants can often be replaced with natural alternatives (“biosurfactants”), which can be isolated from animal, plant, or microbial sources.16,17 In this research, we focus on the potential application of sophorolipids as biosurfactants for forming curcumin-loaded colloidal delivery systems. Sophorolipids are surface-active glycolipids composed of a polar sophorose group and a nonpolar fatty acid group, which can be produced by microbial fermentation.19 In addition, a recently developed method of forming curcumin-loaded nanoparticles is utilized, which is based on changes in the solubility of curcumin with pH.9,20,21 At relatively low pH, the curcumin has no charge and is a highly hydrophobic molecule with a low water solubility.15 However, at a sufficiently high pH, some of the hydroxyl groups on curcumin become deprotonated, leading to a negative charge that increases the water solubility. Consequently, curcumin can be loaded into the hydrophobic core of surfactant micelles by mixing an alkaline solution of curcumin with an acidic solution of surfactant micelles. When the two solutions are mixed, the pH is reduced, which causes the curcumin to become more hydrophobic and move into the surfactant micelles (Figure 1). Previous studies have shown that Received: Revised: Accepted: Published: 1488
November 22, 2017 January 22, 2018 January 29, 2018 January 29, 2018 DOI: 10.1021/acs.jafc.7b05478 J. Agric. Food Chem. 2018, 66, 1488−1497
Article
Journal of Agricultural and Food Chemistry
Figure 1. Schematic of sophorolipid-coated curcumin nanoparticle fabrication using the pH-driven loading mechanism. An acidic sophorolipid micelle solution is mixed with a basic curcumin solution, which causes the curcumin molecules to become more hydrophobic and move into the hydrophobic core of the surfactant micelles. curcumin concentration of 2.0 mg/mL. This curcumin level was used because it was fully soluble within the initial alkaline solution as well as within the sophorolipid micelles formed after the following step. The alkaline curcumin solutions were then added to similar volumes of acidic sophorolipid solutions while stirring continuously at 500 rpm on a magnetic stir-plate. The resulting mixtures were incubated for 30 min at room temperature and then centrifuged at 10,000g for 10 min to remove any insoluble curcumin particles. The curcumin samples used for the X-ray diffraction and storage stability studies were in a powdered form, which was produced by lyophilization using a freezedrier (Alpha 2-4 LD plus, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). Nanoparticle Characterization. The particle size distribution and electrical characteristics (ζ-potential) of the curcumin nanoparticles were measured at 25 °C using a combined dynamic light scattering (DLS)−electrophoresis instrument (Nicomp 380 ZLS, Santa Barbara, CA, USA). The time-dependent fluctuations in light intensity were measured at an angle of 90° after the nanoparticle suspensions had been diluted 4-fold with water to avoid multiple scattering effects. All data were calculated as the mean and standard deviation based on at least three samples with each sample being measured in triplicate. Microstructure images of the nanoparticle suspensions were obtained using an atomic force microscope (AFM). An aliquot of nanoparticle suspension was placed on a freshly cleaved mica substrate, and then images of the samples were acquired using an AFM (Agilent 5500, Agilent Technologies, Santa Clara, CA, USA) with a silicon cantilever of force constant of 0.58 N m−1 operated in tapping mode at room temperature. The X-ray diffraction (XRD) patterns of pure curcumin powder and powdered sophorolipid-coated curcumin nanoparticles were recorded using a D8 Advance X-ray diffractometer (Bruker, Germany). The divergence slit was set at 1°, and the receiving slit was set at 0.1 mm
polyphenols such as curcumin and rutin can be loaded into synthetic and natural surfactant micelles using the pH-driven method.9,20,21 Some of the advantages of this loading method are that it is simple to perform and does not require heating or organic solvents. To the best of our knowledge, this is the first study of the preparation of sophorolipid-coated curcumin nanoparticles using the pH-driven method. The main objectives of the current study are to determine whether curcumin nanoparticles could be successfully produced from sophorolipids using the pH-driven method, characterize the physicochemical properties of the nanoparticles formed, and assess their potential for increasing curcumin bioavailability using both in vitro and in vivo methods. The results of this study may lead to the development of novel all natural curcumin delivery systems that can be incorporated into functional foods, dietary supplements, and pharmaceutical preparations.
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MATERIALS AND METHODS
Materials. Powdered curcumin (composed of 76.4% curcumin, 17.3% demethoxycurcumin, and 3.8% bisdemethoxycurcumin) was purchased from the Aladdin Industrial Corporation (Shanghai, China). Sophorolipid was bought from the Boliante Chemical Company (Xian, China). Ethanol, phosphoric acid, sodium hydroxide, and other reagent chemicals were all of analytical grade. Nanoparticle Preparation. Curcumin nanoparticles were prepared using the pH-driven method described in our previous study with some slight modifications.9 Briefly, a series of solutions were prepared by dissolving different amounts of the biosurfactant in 20 mM phosphoric acid to obtain sophorolipid concentrations of 2, 4, 8, 12, and 16 mg/mL. Powdered curcumin was then weighed into a 30 mM sodium hydroxide solution and stirred for 5 min to reach a final 1489
DOI: 10.1021/acs.jafc.7b05478 J. Agric. Food Chem. 2018, 66, 1488−1497
Article
Journal of Agricultural and Food Chemistry
sample was then maintained at a constant pH of 7.0 by addition of 50 mM NaOH solution using an automatic titration unit (pH stat). In Vitro Bioavailability Determination. The in vitro bioavailability of curcumin was estimated by measuring changes in its concentration and location after passing through the simulated GIT. It was assumed that the overall bioavailability is mainly limited by its chemical degradation (transformation) and solubility in the mixed micelle phase (bioaccessibility) rather than by its uptake by the epithelium cells (absorption). Thus, the in vitro bioavailability (BA) = bioaccessibility (B*) × transformation (T*). The transformation is defined as the fraction of curcumin that remains in an active state after passage through the GIT. The raw digesta of each sample was centrifuged at 40,000g for 30 min at 4 °C to remove any insoluble matter. The supernatants were collected and assumed to be the mixed micelle fraction in which the bioactive agent is solubilized in a form suitable for absorption. The solubilized curcumin was diluted with methanol and assayed using high-performance liquid chromatography (1260 HPLC, Agilent Technologies, Santa Clara, CA, USA) equipped with a UV−visible detector. Curcumin was separated on a Sunfire C18 column (250 mm × 4.6 mm, 5 μm; Waters Corporation, Milford, MA, USA) using a mobile phase consisting of 0.1% (v/v) acetic acid and acetonitrile (45:55 v/v) at a flow rate of 1.0 mL min−1 with detection by UV absorption at 420 nm. The transformation and bioaccessibility of curcumin were then calculated using the equations
for the incident beam. The scan rate was 2° per min over a 2θ angle range of 5−40°. The encapsulation efficiency (EE) and loading capacity (LC) of the curcumin nanoparticles were determined according to the procedures described in our previous study.9 Briefly, nanoparticle suspensions were centrifuged at 10,000g for 10 min to remove any nonencapsulated curcumin. The supernatant was then removed and diluted with anhydrous ethanol. The absorbance at 420 nm was determined using a UV−vis spectrophotometer (Pgeneral T6, China), and the concentration of loaded curcumin was calculated from a calibration curve. The EE and LC were calculated using the expressions
EE (%) = mL,C /mIC × 100
(1)
LC (%) = mL,C /mP × 100
(2)
where mL,C is the mass of curcumin trapped within the nanoparticles, mI,C is the initial mass of curcumin present in the system, and mP is the total mass of the nanoparticles (curcumin + sophorolipids). The total mass of the nanoparticles was determined by freeze-drying the centrifuged nanoparticle suspension and then weighing the resulting powder. The mass of curcumin loaded in the nanoparticles was determined by rehydrating the nanoparticles in an aqueous solution and then measuring the absorbance using a UV−vis spectrophotometer. The initial mass of curcumin present in the system was calculated from the known quantities of the nutraceutical added initially taking into account the various dilution steps. Physical Stability of Nanoparticles. Influence of pH and Ionic Strength. A series of nanoparticle suspensions with pH values ranging from 2.0 to 8.0 were prepared by adding different amounts of either HCl or NaOH solution. A series of nanoparticle suspensions with different ionic strengths was prepared by incorporating different levels of salt into them: 10, 20, 50, 100, 200, or 1000 mM NaCl. The resulting solutions were then stirred for 1 h at ambient temperature, and any changes in particle size and charge were measured using the methods described earlier. Storage Stability. The influence of storage temperature and time on the stability of the nanoparticle suspensions was measured to provide some insights into their potential long-term stability. Powdered (lyophilized) curcumin nanoparticles were stored at 25 °C, and aqueous suspensions containing curcumin nanoparticles were stored at 4 or 25 °C for 30 days. The particle size, charge, and encapsulation efficiency were then recorded at different time intervals during storage. In Vitro Bioavailability. The in vitro bioavailability of curcumin was determining by passing the samples through a static simulated gastrointestinal tract (GIT) and then measuring the amounts of curcumin that had degraded and that had been solubilized in the mixed micelle phase. Simulated Gastrointestinal Tract (GIT). The static GIT method used in this study consisted of simulated mouth, stomach, and small intestine phases and has been described previously.9 This method is fairly similar to the international consensus INFOGEST method that has recently been developed.22 Oral Phase. A test sample (7.5 mL) was mixed with 7.5 mL of simulated saliva fluid containing mucin (30 mg/mL) and various salts prepared as described elsewhere.23 The resulting mixtures were then adjusted to pH 6.8 and shaken at 90 rpm for 10 min at 37 °C to mimic oral conditions. Gastric Stage. A simulated gastric fluid was prepared that contained NaCl (2 mg/mL), HCl (7 mg/mL), and pepsin (3.2 mg/mL), which was then heated to 37 °C. Fifteen milliliters of the simulated gastric fluid was then added to 15 mL of the bolus solution resulting from the oral phase. The resulting mixtures were then adjusted to pH 2.5 and shaken at 100 rpm for 2 h to mimic gastric conditions. Small Intestine Phase. Samples from the simulated gastric phase were adjusted to pH 7.0 with 2 M NaOH. Simulated small intestinal fluid containing pancreatin (24 mg/mL, 2.5 mL), bile extract solution (50 mg/mL, 3.5 mL), and saline solution (0.5 M CaCl2 and 7.5 M NaCl, 1.5 mL) were then added to the reaction vessel. The pH of the
transformation (%) = C D,C/C I,C × 100
(3)
bioaccessibility (%) = CM,C/C D,C × 100
(4)
where CM,C and CD,C are the concentrations of curcumin in the mixed micelle fraction and in the overall digesta at the end of the simulated GIT model and CI,C is the concentration of curcumin initially added (taking into account the various dilution steps). This latter value is therefore a measure of the total amount of curcumin that would be present in the small intestine phase if there were no losses due to chemical degradation. It should be noted that a simple in vitro GIT model cannot accurately simulate the complex processes occurring within a living gastrointestinal tract, but it is useful for rapidly screening different samples and for identifying important physicochemical mechanisms. In Vivo Bioavailability. The in vivo bioavailability of curcumin was evaluated by oral administration to 12 male Sprague−Dawley (SD) rats weighing between 260 and 300 g. The rats were randomly divided into two groups (n = 6). Group 1 was administrated 100 mg/ kg body weight crystalline curcumin, and group 2 was administrated 100 mg/kg body weight curcumin nanoparticles by oral gavage. Aqueous suspensions (10 mg/mL) of crystalline curcumin were prepared by dispersing powdered curcumin into 1.0% sodium carboxymethyl cellulose (as a stabilizer). Aqueous suspensions (10 mg/mL) of curcumin nanoparticles were prepared by dispersing lyophilized sophorolipid-coated curcumin nanoparticles in distilled water. A total of 0.5 mL of blood sample was collected from the retroorbital plexus of each rat at different times (0.5, 1, 2, 4, and 8 h) into heparinized microcentrifuge tubes (containing 20 μL of 1000 IU heparin/mL of blood). These samples were then immediately centrifuged at 4000g for 10 min at 4 °C to isolate the plasma, which was then stored at −80 °C until analysis by LC−MS/MS. According to previous studies,24−26 curcumin is mainly present in a conjugated form (curcumin glucuronide) when it is absorbed through the intestinal cells in rats. Thus, the concentration of curcumin and curcumin glucuronide in rat plasma were analyzed. However, only curcumin glucuronide could be detected in the rat plasma, and so this value was used to determine the in vivo oral bioavailability. Plasma (100 μL) was mixed with 200 μL of acetonitrile by vortexing and centrifuged at 10000g for 5 min at 4 °C. Aliquots of the extracts were injected onto a C18 column (Zorbax Eclipse Plus C18 column, 100 mm × 2.1 mm, I.D., 3.5 μm, Agilent, USA) kept at 40 °C. The mobile phase consisted of two components: A, acetonitrile and B, 0.1% formic acid. The gradient profile was as follows: 0−1 min, 80% B → 20% B; 1−3 min, 20%; 3−3.5 min, 20% B → 80% B. The flow rate was 1490
DOI: 10.1021/acs.jafc.7b05478 J. Agric. Food Chem. 2018, 66, 1488−1497
Article
Journal of Agricultural and Food Chemistry
Table 1. Impact of Sophorolipid Concentration on the Physicochemical Characteristics of (A) Freshly Prepared Curcumin Nanoparticles and (B) Rehydrated Powdered Curcumin Nanoparticles with a Curcumin Concentration of 1 mg/mLa sophorolipid concentration (mg/mL) A
B
a
1 2 4 6 8 1 2 4 6 8
mean diameter (nm) 114 101.8 60.8 59.5 60.9 400 290 58.8 57.9 59.8
± 13b ± 9.8b ± 3.7a ± 0.9a ± 0.8a ± 34d ± 15c ± 2.8a ± 0.4a ± 1.1a
polydispersity index 0.160 0.276 0.295 0.153 0.089 0.709 0.657 0.222 0.077 0.089
± 0.015ab ± 0.035c ± 0.045c ± 0.023ab ± 0.017a ± 0.029d ± 0.015d ± 0.006bc ± 0.024a ± 0.008a
ζ-potential (mV) −39.6 −40.9 -41.2 −26.4 −20.50 −40.6 −39.9 -41.1 −26.77 −19.00
± 1.9a ± 1.2a ± 2.0a ± 1.1b ± 0.56c ± 1.8a ± 2.4a ± 0.9a ± 1.63b ± 1.98c
encapsulation efficiency (%) 40.9 64.9 82.2 89.3 88.6 25.8 59.1 79.2 88.7 89.9
± 1.3b ± 2.4c ± 0.7de ± 1.6e ± 1.3e ± 3.9a ± 7.8c ± 6.1d ± 0.3e ± 1.1e
Different superscript letters indicate statistically significant differences.
0.3 mL/min. Curcumin and curcumin glucuronide were analyzed using a 6410 QQQ MS/MS system (Agilent Technologies, USA) equipped with an electrospray ionization source (ESI) operating in positive mode. The mass spectrometer ion source parameters were as follows: gas temperature, 350 °C; gas flow, 10 L/min; nebulizer gas, 40 psi; spray voltage, 4000 kV. Nitrogen served as a nebulizer and collision gas. Curcumin and curcumin glucuronide were determined using the multiple reaction monitor mode as follows: CUR, m/z 369 > 285, m/z 369 > 177. CUR-G, m/z 545 > 369, m/z 545 > 177. Statistical Analysis. All measurements were replicated at least three times. The results were then expressed as means ± standard deviations. Data were subjected to statistical analysis using SPSS software, version 18.0 (SPSS Inc., Chicago, IL, USA). The Student− Newman−Keuls test was performed to check significant comparisons, and P < 0.05 was considered statistically significant.
increased from 1 to 8 mg/mL. The encapsulation efficiency increased from approximately 41 to 89% as the sophorolipid concentration increased from 1 to 8 mg/L. An understanding of the physicochemical basis of the pHdriven loading mechanism is required to interpret the observed results. At a sufficiently high pH, curcumin has a negative charge and is soluble in water, but when the pH is reduced, it loses its charge and becomes more hydrophobic.15 In the presence of biosurfactant micelles, there are two possibilities for the hydrophobic curcumin molecules to reduce their thermodynamically unfavorable contact with water: (i) they can form crystals or (ii) they can be solubilized within the hydrophobic interior of the micelles (Figure 1). At relatively low biosurfactant levels, there may not be enough biosurfactant micelles present to completely solubilize all of the curcumin, and thus, some curcumin crystals are formed. Alternatively, the rate of crystal formation may exceed the rate of solubilization so that some curcumin crystals are generated before solubilization can occur. At relatively high biosurfactant levels, there may be enough sophorolipid micelles available to solubilize all of the curcumin molecules present and/or the solubilization rate may exceed the crystallization rate, thereby leading to fewer curcumin crystals being formed. The curcumin crystals are likely to be larger than the curcumin-swollen micelles and are likely to have different electrical characteristics, which would account for the observed decrease in mean particle diameter and change in ζ-potential with increasing biosurfactant concentration (Table 1). During the solubilization process, it is likely that some of the original sophorolipid micelles incorporated curcumin and swelled, whereas others dissociated so that they could release biosurfactant molecules that could then cover the increased surface area of the curcumin-swollen micelles. The PDI was relatively small at low sophorolipid levels because the system mainly contained only large curcumin aggregates and was relatively small at high sophorolipid levels because the system mainly contained curcumin-loaded micelles. On the other hand, the PDI was relatively large at intermediate sophorolipid levels because the system contained a mixture of small micelles and large curcumin crystals. The observed increase in encapsulation efficiency with increasing sophorolipid concentration may also be ascribed to a similar physicochemical mechanism. At relatively low biosurfactant levels, more curcumin crystals are formed in the aqueous phase because of the low micelle solubilization capacity or slow solubilization rate. Consequently, these relatively large crystals would be removed during the
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RESULTS AND DISCUSSION Potential Impact of Fabrication Method on Curcumin Stability. Curcumin has been reported to chemically break down upon prolonged storage in aqueous alkaline conditions,3,5 and so it was possible that some of it may have degraded when using the pH-driven mechanism used to fabricate the curcumin nanoparticles. Nevertheless, a previous study by our group indicated that only 0.39 ± 0.27% of curcumin was degraded when it was dissolved in an aqueous alkaline solution (pH 12.0) for 10 min.9 The pH-driven loading method used in the current study only involved holding curcumin at pH 12.0 for 5 min, and thus the degradation of curcumin during this procedure is expected to be relatively small. Effect of Biosurfactant Concentration. Initially, experiments were carried out to determine the optimum sophorolipid concentration required to form the curcumin nanoparticles. Consequently, the impact of sophorolipid concentration on the mean particle diameter, polydispersity index, ζ-potential, and encapsulation efficiency of the curcumin nanoparticles was determined (Table 1, A). For the fresh samples (A), the mean particle diameter decreased from approximately 114 to 61 nm when the sophorolipid concentration increased from 1 to 4 mg/mL, but then it remained relatively constant at higher sophorolipid levels. The polydispersity index (PDI) of most of the samples was relatively small (
