Magnetic Purification of Curcumin from Curcuma longa Rhizome by

Article pubs.acs.org/JAFC

Magnetic Purification of Curcumin from Curcuma longa Rhizome by Novel Naked Maghemite Nanoparticles Massimiliano Magro,†,‡ Rene Campos,§ Davide Baratella,† Maria Izabela Ferreira,§ Emanuela Bonaiuto,† Vittorino Corraducci,† Maíra Rodrigues Uliana,§ Giuseppina Pace Pereira Lima,§ Silvia Santagata,⊥ Paolo Sambo,⊥ and Fabio Vianello*,†,‡ †

Department of Comparative Biomedicine and Food Science, University of Padua, Legnaro, 35020 PD, Italy Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Palacky University in Olomouc, 771 47 Olomouc, Czech Republic § Department of Chemistry and Biochemistry, Universidade Estadual Paulista (UNESP), 18.610-307 Botucatu, São Paulo Brazil ⊥ Department of Agronomy, Food, Natural Resources, Animals and the Environment, University of Padua, Legnaro, 35020 PD, Italy ‡

S Supporting Information *

ABSTRACT: Naked maghemite nanoparticles, namely, surface active maghemite nanoparticles (SAMNs), characterized by a diameter of about 10 nm, possessing peculiar colloidal stability, surface chemistry, and superparamagnetism, present fundamental requisites for the development of effective magnetic purification processes for biomolecules in complex matrices. Polyphenolic molecules presenting functionalities with different proclivities toward iron chelation were studied as probes for testing SAMN suitability for magnetic purification. Thus, the binding efficiency and reversibility on SAMNs of phenolic compounds of interest in the pharmaceutical and food industries, namely, catechin, tyrosine, hydroxytyrosine, ferulic acid, coumaric acid, rosmarinic acid, naringenin, curcumin, and cyanidin-3-glucoside, were evaluated. Curcumin emerged as an elective compound, suitable for magnetic purification by SAMNs from complex matrices. A combination of curcumin, demethoxycurcumin, and bisdemethoxycurcumin was recovered by a single magnetic purification step from extracts of Curcuma longa rhizomes, with a purity >98% and a purification yield of 45%, curcumin being >80% of the total purified curcuminoids. KEYWORDS: magnetic purification, curcumin, Curcuma longa, curcuminoids, magnetic nanoparticles, polyphenols

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INTRODUCTION The isolation of biomolecules, for the pharmaceutical and food industries, is usually performed using various chromatographic, electrophoretic, ultrafiltration, or precipitation and solvent extraction methods.1 All of these processes show important drawbacks when applied at the industrial scale, such as expensive instrumentation, time-consuming procedures, or large quantities of organic solvent waste. As an example, affinity ligand based systems represent one of the most important available technologies for downstream processing, in terms of both selectivity and recovery. The power of column affinity chromatography has been shown in many successful applications, especially at the laboratory scale.2 However, the disadvantage of all standard column liquid chromatography procedures is the impossibility to cope with biological samples containing particulate material, so these techniques are not suitable for working in the early stages of the isolation/ purification process, when suspended solids and fouling components are present in the sample.3 Among the existing purification protocols, separation and purification by magnetic techniques represent promising alternatives for selective and reliable capture of specific molecules,4 in particular, for the isolation and purification of proteins and peptides.5 Magnetic separation techniques present some advantages in comparison with standard separation procedures, as they are often characterized by the extreme easiness of the process: only a few handling steps take place in a © XXXX American Chemical Society

single test tube or vessel, even for very large volumes. Furthermore, expensive liquid chromatography systems, centrifuges, filters, or other instrumentations are not required. Moreover, the separation process can be performed directly on crude samples containing suspended solid material. In addition, as a further advantage with respect to standard chromatography techniques, magnetic separations do not require large volumes of solvents; thus, an excessive dilution of the target molecule in solution is avoided. In recent years, an increasing number of examples of aqueous colloidal suspensions constituted of magnetic nanoparticles were reported. In all of the studies, magnetic nanoparticles were coated by low or high molecular weight organic polymers or inorganic shells, used as steric stabilizers to prevent particle aggregation and to maintain long-term stability, pH and electrolyte tolerance, and, of course, proper surface chemistry. Several alternatives have been developed to modify the surface of magnetic nanoparticles, generally constituted of iron oxides (magnetite or maghemite), such as coating with metallic gold, organic polymers, and silica.6−9 The applications of coated magnetic nanoparticles for magnetic separations still have drawbacks. As an example, polymeric coating stabilizers often Received: September 30, 2014 Revised: December 23, 2014 Accepted: January 1, 2015

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DOI: 10.1021/jf504624u J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Molecular structures of phenolic compounds tested in the present experimental work.

37-7) is a homodimer of feruloyl-methane, containing a methoxy group and a hydroxyl group, a heptadiene with two Michael acceptors, and an α,β-diketone (see Figure 1). Curcumin has shown significant efficacy as a chemopreventive in cell culture studies, as it elicits efficacy in various clinical studies.17,18 Numerous experimental studies on in vitro and in vivo activities of this compound have been reported.19 A special attribute of curcumin resides in its antioxidant potency,20 namely, the ability to scavenge free radicals in blood and body tissues, helping the prevention of various cardiovascular, viral, and other chronic diseases.21 In particular, curcumin’s ability to affect gene transcription and induce apoptosis in preclinical models advocates its potential utility in cancer chemoprevention and chemotherapy.22 In Curcuma longa rhizome, a combination of substances called curcuminoids, namely, curcumin, demethoxycurcumin, and bis-demethoxycurcumin, can be found, curcumin being the major compound. In the present work naked SAMNs were successfully applied for the purification of curcuminoids, and curcumin, from extracts of C. longa rhizome.

bind weakly to nanoparticle surfaces and eventually desorb or exchange with bulk solution, affecting the stability of colloidal suspensions. In addition, processes to coat nanoparticles are often cumbersome, time-consuming, expensive, and characterized by low yield, limiting massive productions. Finally, nanoparticle coating reduces the average magnetic moment of the material by introducing a nonmagnetic component in the final nanoparticle.4,10 Therefore, magnetic nanoparticles, to be a real competitor for standard technologies, need to respond to specific requirements, that is, colloidal stability, proper surface reactivity, and superparamagnetism. Recently, we developed a novel synthetic procedure for nanostructured superparamagnetic material in the size range around 10 nm, constituted of stoichiometric maghemite (γFe2O3) and showing peculiar surface chemical behavior, called surface active maghemite nanoparticles (SAMNs).11,12 SAMNs present a high average magnetic moment and high water stability as colloidal suspensions without any superficial modification or coating derivatization. Because of their unique physical and chemical properties, these naked iron oxide nanoparticles are currently used to immobilize various biomolecules.13−16 Biomolecule immobilization is carried out in water, without any chemical reactant, and the total derivatization process is mild, minimizing alteration/oxidation of ligands, low cost, ecologically green, and reversible. These peculiarities make SAMNs an elective competitor to standard methods for biomolecule purification. In the present work, the binding properties of SAMNs were tested with different ligands of high interest in the pharmaceutical and food industries. Interestingly, among tested molecules, SAMNs displayed high specificity for curcumin binding. Curcumin (IUPAC name, (1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione; CAS Registry No. 458-

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MATERIALS AND METHODS

Materials. Chemicals were purchased at the highest commercially available purity, and they were used without further treatment. Iron(III) chloride hexahydrate (97%), sodium borohydride (NaBH4), and tetramethylammonium hydroxide (N(CH3)4OH) were obtained from Aldrich (Sigma-Aldrich, Italy), as well as catechin, tyrosine, hydroxytyrosine, ferulic acid, coumaric acid, rosmarinic acid, naringenin, curcumin, and cyanidin-3-glucoside. A mixture of curcuminoids (curcumin, demethoxycurcumin, and bis-demethoxycurcumin) (purity ≥ 94.0%), being curcumin ≥80.0% (by TLC), was obtained from Aldrich (cod. C7727, Sigma-Aldrich, Italy). B


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Journal of Agricultural and Food Chemistry The cultivations of Eryngium fetidum and Curcuma longa were carried out in a greenhouse in the experimental farm of the State University of São Paulo ‘Júlio de Mesquita Filho’ in Botucatu, São Paulo, Brazil (22°53′09″ S latitude, 48°26′42″ W longitude, and at 804 m altitude), with harvest at the optimal period. The soil used in the experiment showed the following characteristics: pH 6.3; M.O = 23 g dm−3; P = 60 mg dm−3; H + Al = 19 mmol dm−3; K = 7 mmol dm−3; Ca = 44 mmol dm−3; Mg = 25 mmol dm−3; SB = 77 mmol dm−3; CTC = 95, V% = 80; B = 0.31 mg dm−3; Cu = 2.2 mg dm−3; Fe = 26 mg dm−3; Mn = 2.4 mg dm−3; Zn = 5.3 mg dm−3. Instrumentation. Optical spectroscopy measurements were performed in 1 cm quartz cuvettes using a Cary 50 spectrophotometer (Varian Inc., Palo Alto, CA, USA). Transmission electron microscope (TEM) micrographs were taken on a JEOL 2010 operating at 160 kV with a point-to-point resolution of 1.9 Å. A series of Nd−Fe−B magnets (N35, 263−287 kJ/m3 BH, 1170−1210 mT flux density by Powermagnet, Germany) was used for the magnetic driving of nanoparticles. Synthesis of Surface Active Magnetic Nanoparticles. A typical synthesis of nanoparticles was already described11,12 and can be summarized as follows: FeCl3·6H2O (10.0 g, 37 mmol) was dissolved in Milli-Q grade water (800 mL) under vigorous stirring at room temperature. NaBH4 solution (2 g, 53 mmol) in ammonia (3.5%, 100 mL, 4.86 mol mol−1 Fe) was quickly added to the mixture. Soon after the reduction reaction occurrence, the temperature of the system was increased to 100 °C and kept constant for 2 h. Then, the material was cooled at room temperature and aged in water, as prepared, for another 24 h. This product was separated by imposition of an external magnet and washed several times with water. This material can be transformed into a red-brown powder (final synthesis product) by drying and curing at 400 °C for 2 h. The resulting nanopowder showed a magnetic response upon exposure to a magnetic field. The final mass of product was 2.0 g (12.5 mmol) of Fe2O3, and a yield of 68% was calculated. The nanoparticulated resulting material was characterized by Mössbauer spectroscopy, FT-IR spectroscopy, high-resolution transmission electron microscopy, XRD, and magnetization measurements12 and was constituted of stoichiometric maghemite (γ-Fe2O3) with a mean diameter (dav) of 11 ± 2 nm, which can lead to the formation, upon ultrasound application in water (Bransonic, model 221, 48 kHz, 50 W), of a stable colloidal suspension, without any organic or inorganic coating or derivatization. The surface of these bare maghemite nanoparticles shows peculiar binding properties and can be reversibly derivatized with selected organic molecules. We called these bare nanoparticles surface active maghemite nanoparticles (SAMNs). Quantitative Determination of Free Phenolic Acids and Curcuminoids by HPLC. Curcuminoids, chlorogenic acid, ferulic acid, p-coumaric acid, caffeic acid, gallic acid, cyanidin-3-glucoside, and synaptic acid were separated and quantified using an HPLC (model XLC, Jasco, Japan). The liquid chromatography apparatus consisted of a PU-2080 pump, an MD-2015 diode array detector, an AS-2055 autosampler, and a CO-2060 column oven. ChromNAV chromatography software was used for chromatographic data analysis. The separation of phenolic acids was obtained on a Tracer Extrasil ODS2 (5 mm, 250 mm, Teknokroma, Spain) operating at room temperature. The mobile phase consisted of formic acid, pH 2.8 (A), and acetonitrile (B). Elution gradient was carried out in 50 min, and the flow rate was 0.8 mL min−1 (10−80% B). All standards were dissolved in 50% ethanol, and the calibration curves were built with standard concentrations ranging from 0.06 to 60 mg L−1. Two wavelengths (325 and 310 nm) were used to detect phenolic acids. Measurements at 325 nm were used for the identification of chlorogenic acid, caffeic acid, and ferulic acid. The identification of p-coumaric acid was performed at 310 nm. HPLC determination of curcuminoids (bis-demethoxy-curcumin, demethoxy-curcumin, and curcumin) was carried out according to a procedure derived from that of He with some modification.23 A UHPLC system (Ultimate 3000 BioRS, Dionex-Thermo Fisher Scientific Inc., USA) equipped with a diode array detector was used.

An Ace 5 C18 (Advanced Chromatography Technologies, UK) column (5 μm, 25 cm × 4.6 mm) was used for curcuminoid analysis, at 48 °C. The gradient profile of the mobile phase, (A) water (0.25% acetic acid, Mallinckrodt) and (B) acetonitrile (HPLC grade, Mallinckrodt), was as follows: 0−17 min, 40−60% B; 17−32 min, 60−85% B; 32−38 min, 85% B; 38−40 min, 85−40% B; 40−45 min, 40% B; the flow rate was 0.5 mL min−1. The injection volume was 20 μL (full loop). All samples were filtered through 0.22 μm membrane filters before the injections. Curcuminoid peaks were identified by retention times and injections tests with standards (curcumin, demethoxy-curcumin, and bisdemethoxy-curcumin).

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RESULTS A list of biomolecules of interest for the pharmaceutical and food industries, namely, catechin, tyrosine, hydroxytyrosine, ferulic acid, coumaric acid, rosmarinic acid, naringenin, curcumin, and cyanidin-3-glucoside, was screened to test the recognition properties of SAMN surface toward different compounds. Thus, the binding efficiency and reversibility on SAMNs were evaluated by UV−vis spectroscopy. Moreover, with the aim of broadening the applicability spectrum of SAMNs in the field of biomolecule purification, a general approach for predicting SAMN suitability for selective magnetic separation of compounds of interest from complex matrices was studied. Selected molecules present chemical features potentially able to anchor, by metal chelation, on iron(III) available sites on the SAMN surface, the presence of which was recently demonstrated.16 The comparison of the interactions of different molecular functionalities with SAMNs, from binding to desorption, allowed the individuation of chemical features required for stable and, at the same time, reversible binding on the SAMN surface. Structural and magnetic properties of as-prepared iron oxide nanoparticles were investigated by zero-field and in-field Mössbauer spectroscopy and SQUID magnetization measurements.12 In-field Mössbauer spectroscopy was used due to its unique ability to distinguish between structurally isomorphous maghemite and magnetite and also to quantify the distribution of cations in tetrahedral (T) an octahedral (O) sites of these spinel structures. The in-field Mössbauer spectrum, collected at 5 K in an external magnetic field of 5 T, corresponded with that reported for maghemite. Taking into account the maghemite stoichiometric formula, given by (Fe3+)T(Fe3+5/3 o1/3)OO4, where “o” stands for vacancies, the prepared maghemite was perfectly stoichiometric. To monitor the magnetic response of SAMNs, hysteresis loops were measured at 2 and 300 K. At 2 K, the isothermal magnetization curve showed hysteresis with the values of coercivity and remanent magnetization comparable with those reported for bulk maghemite. As prepared SAMNs showed a maximum magnetization of 71.4 Am2 kg−1, which was similar to that of bulk maghemite12 and higher than values reported for other magnetic nanoparticles in the literature.24 The value of the maximum magnetization at 7 T was slightly lower than that reported for bulk maghemite, which was caused by a nanometer size of the particles. The hysteresis loop at 2 K was symmetric (i.e., equal values of positive and negative coercivity and positive and negative remanent magnetization), and no exchange bias phenomenon was present. Thus, the degree of magnetic disorder occurring at the surface layers of nanoparticles was not significant. Some misalignment of the surface atomic magnetic moments of each particle is present, in accordance with in-field Mössbauer data; however, it does not crucially govern the overall magnetic response of the system. C


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Journal of Agricultural and Food Chemistry From the application point of view, it is important that a very high magnetization is achievable already at very low applied fields at room temperature. Thus, we can conclude that the prepared magnetic particles would act as an excellent magnetic carrier due to their well-defined stoichiometric structure, singlephase character, suitable magnetic properties, and uniform size distribution. Moreover, as a unique aspect of the preparation procedure, the particles exhibit colloidal behavior with no indications of the sedimentation and/or aggregation process even within several months. Tentatively, phenolic compounds were incubated with aqueous suspensions of SAMNs, and the resulting nanoconjugates were extensively washed with water, by alternating magnetic separations, to eliminate loosely bound molecules. Finally, the release of the phenolic compounds from the nanoconjugate was tested under different conditions. All tested compounds present at least one phenol ring in their molecular structure (see Figure 1); thus, the whole process was easily followed by UV−vis spectroscopy. The characteristic wavelength of maximum absorbance and the molar extinction coefficients of tested compounds are reported in Table S1 in the Supporting Information. Because the focus of the present paper is providing a purification strategy for real applications, we aimed at obtaining the best compromise between yield and purity of phenolic compounds, minimizing the operations during the purification steps. For this last reason we avoided any other modification of the extracting and binding solutions, such as pH or ionic strength modifications. First, because the tested substances could possibly be easily oxidized at room temperature, their stability was, as well, assessed by UV−vis spectroscopy. Optical spectra were acquired within 24 h, and the residual concentration of phenolics, with respect to freshly prepared solutions, is reported in Table S1 (in the Supporting Information, last column). Experimental data indicated that the stability of tested substances was acceptable, and, anyway, the magnetic purification tests carried out with SAMNs was performed within 2 h. The tested phenolic compounds (10.0 μM) were incubated in the presence of SAMNs (0.5 g L−1), and experimental results evidenced different behaviors in terms of efficiency and reversibility of the binding. In all of the explored cases, phenolic−SAMN nanoconjugates showed remarkable stability and no oxidation of bound phenolics was observed. It is known that catechols and ene-diols, bearing multiple hydroxyl groups, as well as carboxylic groups, are able to form complexes with transition metals and metal oxide-based nanomaterials.25,26 Recently, we demonstrated the presence of available under-coordinated iron(III) atoms on the surface of SAMNs,27 and these phenolic ligands can lead to the formation of very stable complexes, as they convert under-coordinated iron atoms on the nanoparticle surface to a bulk-like lattice structure.28 Interestingly, most of the tested phenolic substances led to the formation of irreversible complexes with SAMNs, in agreement with Rajh, who reported, with bidentate ene-diol ligands, the formation of very stable complexes with metal oxide nanoparticles.29 In fact, the protocol used for releasing ligands from the nanoparticle surface, consisting of an incubation of the complex in 0.5 M NH4OH,27 was inefficient with the tested group of molecules (see Table 1). As an example, a remarkable binding efficiency on the SAMN surface was shown by catechin, and the subsequent incubation

Table 1. Binding and Release Efficiencies of Tested Compounds on the Surface of SAMNsa polyphenol catechin tyrosin 3-hydroxytyrosine ferulic acid coumaric acid rosmarinic acid naringenin curcumin cyanidin-3-glucoside

binding (%) 61.7 0.23 37.5 17.2 26.6 57.7 45.3 94.3 94.1

± ± ± ± ± ± ± ± ±

5.0 0.1 6.3 0.7 0.7 3.4 3.7 1.4 0.8

release (%) 4.7 nd 4.2 1.7 nd 1.0 7.7 97.2 3.0

± 1.1 ± 0.6 ± 0.2 ± ± ± ±

0.8 0.4 2.4 0.2

a Incubations were performed with 50.0 μM of the tested compound in the presence of 1.0 g L−1 SAMNs. The releasing process from the nanoparticles was performed with 99.6% ethanol. Values are the mean of three experiments.

of the complex of SAMNs with catechin (SAMN@catechin) in 99.6% ethanol led to the release of only 4.7 ± 1.1% of the bound molecule, indicating that catechin formed stable complexes on the iron oxide surface, as it completed the dangling boundaries of truncated crystalline structure of the nanoparticle.30 Moreover, the comparison between the behavior of tyrosine and 3-hydroxytyrosine highlighted the role of the catechol structure, with respect to simple phenolic groups. The catechol structure was responsible for a 160-fold increase of substance binding with respect to simple phenolic rings. As in the case of catechin, the catechol structure of 3hydroxytyrosine generated an irreversible binding, and the incubation of its SAMN complex in 99.6% ethanol led to a releasing yield of 4.2 ± 0.6% (n = 5). The strong intimate interaction between phenolics and the surface iron oxide nanoparticles was evidenced by UV−vis spectroscopy. The electronic absorption spectrum of bare SAMNs, acquired in water, showed a wide band with a maximum at about 400 nm (Figure 2, black line) characterized by an extinction coefficient of 1520 M−1cm−1, expressed as Fe2O3 molar concentration. Differently, the SAMN@catechin spectrum was characterized by a peak at 440 and a shoulder at 520 nm (see Figure 2, red line). The observed red shift and

Figure 2. UV−vis spectra of naked and catechin-coated SAMNs (SAMN@catechin): (black line) SAMN; (red line) SAMN@catechin. Spectra were acquired in water. The concentrations in suspension were 40 and 200 μg mL−1 for SAMN and SAMN@catechin, respectively. D


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create stable complexes with iron(III).31 In addition, its complex with SAMN (SAMN@curcumin) was completely reversible, and the molecule was completely released from the nanoparticle surface by incubation in 99.6% ethanol (see Table 1). It should be mentioned that different solvent mixtures, based on ethanol, acetic acid, and ammonia, were tested for phenolic release from the SAMN surface (data not shown). Some of them were already used for controlling SAMN complex formation.13 Ethanol was chosen for subsequent experiments aimed at the release of phenolic compounds from the SAMN surface. The use of ethanol for releasing oleic acid from iron oxide nanoparticles was already reported in the literature.32 Comparable binding efficiency on SAMNs with respect to curcumin was observed for cyanidin-3-glucoside, which presents a sugar ring as a distinctive chemical feature, a resorcinol, and a catechol moiety. As a general result, substances characterized by catechol and/ or keto−enol functionalities were able to efficiently bind on the SAMN surface, even if, at the same time, no easy general strategy for compound release from the nanoparticle surface was individuated. For comparison, as negative controls, two alkaloid molecules were tested, namely, caffeine and theophylline (Figure 1), both presenting no chemical features particularly prone to iron(III) chelation. Even at high concentration (1 mM), the incubation of these two substances with SAMNs did not lead to significant complex formation. In both cases, a negligible binding efficiency, with respect to previously tested phenolics, was observed (see Table 1). As a first test on a real sample, SAMNs were subsequently used for the magnetic separation of biomolecules in water− ethanol extracts of Eryingium fetidum L. (Apiaceae family). E. fetidum is an aromatic plant, used as a spice and medicinal plant in tropical regions. The leaves are used to treat several diseases, such as fever, pains, and hypertension.33−35 This species is also an important item in the perfumery and cosmetic industries due to the properties of its essential oil.36,37 Several studies have reported the presence of phenolic compounds in leaves of E. fetidum.38,39 However, researchers mainly focused their attention on the essential oil, and scarce information is available on other phytochemicals,40 such as phenolic compounds. The HPLC profiles of phenolic acids extracted from E. fetidum in 50% ethanol showed peaks attributable to chlorogenic, ferulic, p-coumaric, caffeic, gallic, and sinapic acid. All of these compounds may form complexes with undercoordinated iron(III) on the SAMN surface (see Figure 1). Ethanolic extracts of E. fetidum leaves (20 mg mL−1) were incubated in the presence of 0.5 g L−1 SAMNs to test binding specificities. After magnetic separation, SAMN-bound molecules were removed by incubation in 99.6% ethanol, and supernatants were analyzed by HPLC for the determination of phenolic acids. No enrichment of a specific phenolic acid was evidenced, and the chromatographic profile was unaltered with respect to that of the untreated sample, demonstrating that phenolic acids were purified by magnetic purification with SAMNs, but without any specificity (see Figure S1 in the Supporting Information). This behavior can be attributed to the concomitant presence of the catechol function on most of the phenolic acids in the extract. Molecules bearing chemical features with similar affinity toward iron(III) chelation compete for complex formation with the SAMN surface, without showing particular selectivity.

change in the absorption envelope (shape) of the SAMN spectrum upon catechin binding was not surprising. A similar phenomenon was observed with other biomolecules following their interaction with the nanoparticle surface.14 We suggest that the recorded red shift in the absorption spectrum of modified nanoparticles was due to the excitation of localized electrons from the surface modifier to the conduction band of iron oxide nanoparticles.29 In the present case, it was due to the presence of catechin on the SAMN surface. Moreover, the SAMN@catechin complex was studied by electron transmission microscopy (see Figure 3). TEM

Figure 3. TEM micrograph of SAMN@catechin complex.

microscopy images of the SAMN@catechin complex indicated the presence of an organic matrix, forming a shell of about 2.0 nm around iron oxide nanoparticles, characterized by a lower electron density, attributable to catechin molecules layering on the SAMN surface. The resulting SAMN@catechin complex was extremely stable and, if stored at 4 °C, catechin remained firmly bound to the iron oxide nanoparticle surface for at least 12 months. Considering the binding behavior of similar molecules (ferulic, coumaric, and rosmarinic acid), we observed the best binding efficiency on SAMN surface by rosmarinic acid, which presents two catechol moieties, with free hydroxyl groups, at the extremities of the organic chain (see Figure 1), therefore confirming that catechol-containing ligands showed a more efficient anchoring function on the SAMN surface with respect to simple phenol moieties and single-carboxylate ligands. Furthermore, the presence of a methoxy substituent (−OCH3) in the aromatic ring, such as in the molecule of ferulic acid, led to a considerably less efficient ligand binding. Narigenin and curcumin, presenting a keto−enol functionality, showed very good binding proclivity toward the SAMN surface. In particular, curcumin appeared as one of the best ligands for the SAMN surface (Table 1), confirming the literature reporting that curcumin has a remarkable ability to E


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Journal of Agricultural and Food Chemistry Curcumin Purification from Curcuma longa Rhizome. Experimental results showed that curcumin stands out, among all tested molecules, in terms of both binding efficiency on SAMNs and complex reversibility. Therefore, the magnetic separation of this molecule by SAMNs was tested on real samples of C. longa rhizome. HPLC analyses were performed to evaluate the efficiency of the magnetic purification and isolation yield of curcumin and curcuminoids as a function of experimental conditions and nanoparticle concentrations. As a control, commercial curcuminoid mixture, purified from C. longa rhizome at the highest commercial purity available, namely, a combination of curcumin, demethoxy-curcumin, and bis-demethoxy-curcumin, was subjected to chromatographic analysis. Rhizomes of C. longa collected after 65 days of cultivation were manually finely ground and then dried at 45 °C in a ventilated oven. Dried and powdered C. longa rhizome was extracted (20 mg mL−1) with different water−ethanol mixtures, where ethanol concentration was in the 0−100% range. Then, the extracts were incubated with SAMNs, in the 1.25− 10.0 g L−1 concentration range. After 1 h of incubation, SAMNs and the corresponding bound material were magnetically recovered. Then, SAMNs were resuspended in 99.6% ethanol for the release of bound curcuminoids. After 1 h of incubation and a further magnetic separation, SAMNs were finally removed, and the resulting supernatants were collected and characterized by HPLC. As reported in the literature,41 we found that curcumin represented the main curcuminoid compound in the C. longa rhizome (87.02% curcumin, 2.08% demethoxy-curcumin, and 10.90% bis-demethoxy-curcumin). As shown in Figure 4, the isolation yield of curcuminoids increased with ethanol concentration used during the extraction and depended, as well, on SAMN concentration. The best results were observed by extraction in 99.6% ethanol and using 10 g L−1 SAMNs for

the magnetic purification. Under these conditions it was possible to isolate >80% of curcuminoids from C. longa rhizome extracts, namely, 155.37 mg L−1. Notwithstanding the highest ethanol concentration (99.6%) represented the worst binding condition on nanoparticles, as it is used for the release of curcuminoids from SAMNs, it represented the best extraction condition in terms of yield. According to the le Chatellier principle, the high concentration of curcuminoids in the extract strongly favored the binding to the formation of the complex with nanoparticles; thus, under these conditions, curcuminoids successfully competed for SAMNs in the presence of extraction contaminants. Conversely, when curcuminoid-loaded nanoparticles were incubated in 99.6% pure ethanol, the equilibrium leads to the dissociation of the complexes. Moreover, the curcumin purification yield, with respect to the total amount of curcuminoids, obtained from the magnetic purification, was assessed by comparison of chromatographic peak areas by HPLC. In Figure S2 in the Supporting Information the fractions of purified curcumin are reported as a function of ethanol concentration, for all of the tested concentrations of SAMNs. The highest yield of curcumin, with respect to total curcuminoids, was observed using 25% ethanol for extraction. Interestingly, the combination of 25% ethanol for C. longa rhizome extraction and 1.25 g L−1 SAMNs for magnetic purification, which is the lowest nanoparticle concentration tested, led to the highest curcumin/curcuminoid ratio (>80.0%). For other tested ethanol and SAMN concentrations, the curcumin/curcuminoid ratio was about 65%. The proposed procedure for curcumin purification can be summarized as follows: dried powder of C. longa rhizome (10 g) was extracted with 99.6% ethanol (600 mL) with the aid of 5 min of sonication (Bransonic, model 221, 48 kHz, 50 W). Control experiments showed that the extraction of the same amount of powdered C. longa rhizome with ethanol led to the isolation, after drying in a rotary evaporator, of 50.7 mg g−1 of a residual oil. As reported in the literature, C. longa rhizome contains a considerable amount of essential oil; thus, the separation of curcuminoids from this hydrophobic matrix represents a problematic task.42 After decantation (10 min), the supernatant was incubated with SAMNs (10 g L−1) for 1 h, under stirring, at room temperature. The nanoparticle-containing suspension was subjected to magnetic separation, and the resulting supernatant was eliminated. The release of curcuminoids bound to nanoparticles was accomplished by subsequent incubation of SAMN@curcumin in 99.6% ethanol (600 mL) for 1 h, at room temperature. Released curcumin in ethanol was dried under low vacuum (100 torr) in a rotary evaporator at 40 °C, leading to 3.95 mg g−1 of a yellow powder, consisting of 64.0% curcumin, 17.0% demethoxy-curcumin, and 19.0% bis-demethoxy-curcumin (by HPLC), with a purity >98% with respect to the total dry mass of the extract. The total process can be accomplished within 80 min. The HPLC profile of curcuminoids after magnetic separation by SAMNs is shown in Figure 5. The amount of purified curcuminoids from C. longa extracts well agreed with literature values,43 and a yield of 45% with respect to its total content in the first ethanol extract was calculated. It should be mentioned that the total amount of curcuminoids, as well as the curcumin/curcuminoids ratio, will depend on the original composition of the C. longa rhizome sample.

Figure 4. Isolation yield of curcuminoids as a function of ethanol and SAMN concentrations. Powdered C. longa rhizome was extracted (20 mg mL−1) with different water−ethanol mixtures, and the resulting solutions were incubated with SAMNs. Recovered SAMNs and the corresponding bound material were resuspended in 99.6% ethanol for the release of bound molecules. SAMN concentrations used for the magnetic purification of curcuminoids were (black symbols) 1.25 g L−1, (green symbols) 2.5 g L−1, (blue symbols) 5.0 g L−1, and (red symbols) 10.0 g L−1. F


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Figure 5. HPLC profile of curcuminoids after magnetic purification by SAMN on Curcuma longa extracts. The separation of curcuminoids in the supernatant obtained from magnetic purification was carried out as described under Materials and Methods.

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DISCUSSION

ASSOCIATED CONTENT

S Supporting Information *

Novel maghemite nanoparticles (SAMNs) were studied for phenolic binding and were applied for the purification of curcumin from C. longa rhizome extracts. The peculiar properties of the SAMN surface, due to the presence of under-coordinated iron(III) sites, confer high specificity to select iron chelating molecules from complex matrices. These nanoparticles easily match with interests in biotechnology, revealing different surface characteristics from other reported magnetic nanoparticles, justifying the peculiar behavior of SAMN suspensions, their high colloidal stability in aqueous solutions, and their ability to reversibly adsorb biomolecules. These features led us to apply SAMNs as a novel tool to isolate and purify substances from natural matrices, which could be attractive for the pharmaceutical and food industries. SAMNs showed high specificity to reversibly bind curcumin and were used for isolating this biomolecule from C. longa rhizome extracts. The present paper envisages new possibilities offered by SAMNs for the isolation of curcumin and, moreover, proposes an easy procedure based on magnetic purification of curcuminoids, which could be easily modified for scaling-up, moving from a laboratory scale to an industrial level. The complete purification process, from SAMN synthesis to application, is fast and ecologically green, avoiding the use of large volumes of solvents, as typically required by elution in standard chromatographic methods, and the ethanol employed for extraction is easily recyclable. As well, SAMNs can be easily regenerated after the purification process by sonication in water and reutilized for further purification cycles. Therefore, the proposed process is cost-effective and does not involve production of wastes, being based on green chemistry processes, suitable for scaling-up. In the present case an estimated yield of 45% of pure curcuminoids, characterized by a purity >98%, was obtained from C. longa rhizomes.

HPLC profiles of E. fetidum extracts in 50% ethanol, before and after incubation with SAMNs (Figure S1), curcumin purification yield with respect to total curcuminoids after magnetic purification of Curcuma longa rhizome extracts as a function of ethanol concentration and SAMN concentration (Figure S2), and optical characteristics of catechin, tyrosine, 3hydroxytyrosine, ferulic acid, coumaric acid, rosmarinic acid, naringenin, curcumin and cyanidin-3-glucoside (Table S1). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(F.V.) Mail: Department of Comparative Biomedicine and Food Safety, University of Padua, Agripolis, Viale dell’Università 16, Legnaro, 35020 (PD), Italy. Phone: +39-0498276863. Fax: +39-049-8073310. E-mail: fabio.vianello@unipd. it. Funding

The present experimental work was partially funded by “Fondazione CARIPARO” and by Italian Institutional Ministry Grants cod. 60A06-7411 and 60A06-8055. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the support by the São Paulo Research Foundation, Process 2013/05644-3, and Brazilian National Counsel of Technological and Scientific Development, Process 478372/2013-2.

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ABBREVIATIONS USED SAMNs, surface active maghemite nanoparticles; TEM, transmission electron microscope; FT-IR, Fourier transform infrared G


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curcumin and its analogues (congeners) made by man and mother nature. Biochem. Pharmacol. 2008, 76, 1590−1611. (21) Maheshwari, R. K.; Singh, A. K.; Gaddipati, J.; Srimal, R. C. Multiple biological activities of curcumin: a short review. Life Sci. 2006, 78, 2081−2087. ́ (22) Grynkiewicz, G.; Slifirski, P. Curcumin and curcuminoids in quest for medicinal status. Acta Biochim. Polym. 2012, 59, 201−212. (23) He, X.-G.; Lin, L.-Z.; Lian, L.-Z.; Lindenmaier, M. Liquid chromatography−electrospray mass spectrometric analysis of curcuminoids and sesquiterpenoids in turmeric (Curcuma longa). J. Chromatogr., A 1998, 818, 127−132. (24) Gupta, A. K.; Gupta, M. Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomaterials 2005, 26, 3995−4021. (25) Thanh, N. T. K.; Green, L. A. W. Functionalization of nanoparticles for biomedical applications. Nano Today 2010, 5, 213− 230. (26) Laurent, S.; Forge, D.; Port, M.; Roch, A.; Robic, C.; Vander Elst, L.; Muller, R. N. Magnetic iron oxide nanoparticles: synthesis, stabilization, vectorization, physicochemical characterizations, and biological applications. Chem. Rev. 2008, 108, 2064−2110. (27) Magro, M.; Baratella, D.; Salviulo, G.; Polakova, K.; Zoppellaro, G.; Tucek, J.; Kaslik, J.; Zboril, R.; Vianello, F. Core−shell hybrid nanomaterial based on prussian blue and surface active maghemite nanoparticles as stable electrocatalyst. Biosens. Bioelectron. 2014, 52, 159−165. (28) Chen, L. X.; Liu, T.; Thurnauer, M. C.; Csencsits, R.; Rajh, T. Fe2O3 nanoparticle structures investigated by X-ray absorption nearedge structure, surface modifications, and model calculations. J. Phys. Chem. B 2002, 106, 8539−8546. (29) Rajh, T.; Chen, L. X.; Lukas, K.; Liu, T.; Thurnauer, M. C.; Tiede, D. M. Surface restructuring of nanoparticles: an efficient route for ligand−metal oxide crosstalk. J. Phys. Chem. B 2002, 106, 10543− 10552. (30) Rodriguez, J. A.; Fernández-García, M. Synthesis, Properties, and Applications of Oxide Nanomaterials; Wiley: Hoboken, NJ, USA, 2007. (31) Borsari, M.; Ferrari, E.; Grandi, R.; Saladini, M. Curcuminoids as potential new iron-chelating agents: spectroscopic, polarographic and potentiometric study on their Fe(III) complexing ability. Inorg. Chim. Acta 2002, 328, 61−68. (32) White, M. A.; Johnson, J. A.; Koberstein, J. T.; Turro, N. J. Toward the syntheses of universal ligands for metal oxide surfaces: controlling surface functionality through click chemistry. J. Am. Chem. Soc. 2006, 128, 11356−11357. (33) Singh, S.; Singh, D. R.; Banu, S.; Salim, K. M. Determination of bioactives and antioxidant activity in Eryngium foetidum L.: a traditional culinary and medicinal herb. Proc. Natl. Acad. Sci. India Sect. B: Biol. Sci. 2013, 83, 453−460. (34) Singh, B. K.; Ramakrishna, Y.; Ngachan, S. V. Spiny coriander (Eryngium foetidum L.): a commonly used, neglected spicing-culinary herb of Mizoram, India. Genet. Resour. Crop Evol. 2014, 61, 1085− 1090. (35) Duke, J. Duke’s Handbook of Medicinal Plants of Latin America; Taylor and Francis, CRC Press: Boca Raton, FL, USA, 2009. (36) Essia Ngang, J. J.; Nyegue, M. A.; Ndoye, F. C.; Tchuenchieu Kamgain, A. D.; Sado Kamdem, S. L.; Lanciotti, R.; Gardini, F.; Etoa, F.-X. Characterization of Mexican coriander (Eryngium foetidum) essential oil and its inactivation of Listeria monocytogenes in vitro and during mild thermal pasteurization of pineapple juice. J. Food Prot. 2014, 77, 435−443. (37) Garcia, M.; Sáenz, M.; Gómes, M.; Fernándes, M. Topical antiinflammatory activity of phytosterols isolated from Eryngium foetidum on chronic and acute inflammation models. Phytother. Res. 1999, 13, 78−80. (38) Craig, W. Health-promoting properties of common herbs. Am. J. Clin. Nutr. 1999, 70, 491s−499s. (39) Wong, S.; Leong, L.; Koh, J. Antioxidant activities of aqueous extract of selected plants. Food Chem. 2006, 99, 775−783.

spectroscopy; XRD, X-ray powder diffraction; SQUID, superconducting quantum interference device

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REFERENCES

(1) Kitts, D. D.; Weiler, K. Bioactive proteins and peptides from food sources. Applications of bioprocesses used in isolation and recovery. Curr. Pharm. Des. 2003, 9, 1309−1323. (2) Hage, D. S.; Cazes, J. Handbook of Affinity Chromatography, 2nd ed.; Taylor and Francis, CRC Press: Boca Raton, FL, USA, 2005. (3) Turkova, J. Affinity Chromatography, 1st ed.; Elsevier Scientific Publishing: Amsterdam, The Netherlands, 1978. (4) Horak, D.; Babic, M.; Mackova, H.; Benes, M. J. Preparation and properties of magnetic nano- and microsized particles for biological and environmental separations. J. Sep. Sci. 2007, 30, 1751−1772. (5) Safarik, I.; Safarikova, M. Magnetic techniques for the isolation and purification of proteins and peptides. Biomagn. Res. Technol. 2004, 2, 7−24. (6) Lyon, J. L.; Fleming, D. A.; Stone, M. B.; Schiffer, P.; Williams, M. E. Synthesis of Fe oxide core/Au shell nanoparticles by iterative hydroxylamine seeding. Nano Lett. 2004, 4, 719−723. (7) Zhang, Y.; Kohler, N.; Zhang, M. Surface modification of superparamagnetic magnetite nanoparticles and their intracellular uptake. Biomaterials 2002, 23, 1553−1561. (8) Lu, Y.; Yin, Y. D.; Mayers, B. T.; Xia, Y. N. Modifying the surface properties of superparamagnetic iron oxide nanoparticles through a sol−gel approach. Nano Lett. 2002, 2, 183−186. (9) Urbanova, V.; Magro, M.; Gedanken, A.; Baratella, D.; Vianello, F.; Zboril, R. Nanocrystalline iron oxides, composites and related materials as a platform for electrochemical, magnetic, and chemical biosensors. Chem. Mater. 2104, 26, 6653−6673. (10) Mackay, M. E.; Tuteja, A.; Duxbury, P. M.; Hawker, C. J.; Van Horn, B.; Guan, Z.; Chen, G.; Krishnan, R. S. General strategies for nanoparticle dispersion. Science 2006, 311, 1740−1743. (11) Magro, M.; Valle, G.; Russo, U.; Nodari, L.; Vianello, F. Maghemite nanoparticles and method for preparing thereof. World Patent WO/2012/010200, 2012. (12) Magro, M.; Sinigaglia, G.; Nodari, L.; Tucek, J.; Polakova, K.; Marusak, Z.; Cardillo, S.; Salviulo, G.; Russo, U.; Stevanato, R.; Zboril, R.; Vianello, F. Charge binding of rhodamine derivative to OHstabilized nanomaghemite: universal nanocarrier for construction of magnetofluorescent biosensors. Acta Biomater. 2012, 8, 2068−2076. (13) Sinigaglia, G.; Magro, M.; Miotto, G.; Cardillo, S.; Agostinelli, E.; Zboril, R.; Bidollari, E.; Vianello, F. Catalytically active bovine serum amine oxidase bound to fluorescent and magnetically drivable nanoparticles. Int. J. Nanomed. 2012, 7, 2249−2259. (14) Magro, M.; Faralli, A.; Baratella, D.; Bertipaglia, I.; Giannetti, S.; Salviulo, G.; Zboril, R.; Vianello, F. Avidin functionalized maghemite nanoparticles and their application for recombinant human biotinylSERCA purification. Langmuir 2012, 28, 15392−15401. (15) Venerando, R.; Miotto, G.; Magro, M.; Dallan, M.; Baratella, D.; Bonaiuto, E.; Zboril, R.; Vianello, F. Magnetic nanoparticles with covalently bound self-assembled protein corona for advanced biomedical applications. J. Phys. Chem. C 2013, 117, 20320−20331. (16) Magro, M.; Campos, R.; Baratella, D.; Lima, G.; Holà, K.; Divoky, C.; Stollberger, R.; Malina, O.; Aparicio, C.; Zoppellaro, G.; Zboril, R.; Vianello, F. A magnetically drivable nanovehicle for curcumin with antioxidant capacity and MRI relaxation properties. Chem.−Eur. J. 2014, 20, 11913−11920. (17) Nagpal, M.; Sood, S. Role of curcumin in systemic and oral health: an overview. J. Nat. Sci. Biol. Med. 2013, 4, 3−7. (18) Liu, D.; Chen, Z. The effect of curcumin on breast cancer cells. J. Breast Cancer 2013, 16, 133−137. (19) Yallapu, M. M.; Jaggi, M.; Chauhan, S. C. Cyclodextrincurcumin self-assembly enhances curcumin delivery in prostate cancer cells. Colloids Surf. B: Biointerfaces 2010, 79, 113−125. (20) Anand, P.; Thomas, S. G.; Kunnumakkara, A. B.; Sundaram, C.; Harikumar, K. B.; Sung, B.; Tharakan, S. T.; Misr, K.; Priyadarsini, I. K.; Rajasekharan, K. N.; Aggarwal, B. B. Biological activities of H


Article

Journal of Agricultural and Food Chemistry (40) Dawilai, S.; Muangnoi, C.; Praengamthanachoti, P.; Tuntipopipat, S. Anti-inflammatory activity of bioaccessible fraction from Eryngium foetidum leaves. BioMed. Res. Int. 2013, 2013, No. 958567. (41) Wichitnithad, W.; Jongaroonngamsang, N.; Pummangura, S.; Rojsitthisak, P. A simple isocratic HPLC method for the simultaneous determination of curcuminoids in commercial turmeric extracts. Phytochem. Anal. 2009, 20, 314−319. (42) Funk, J. L.; Frye, J. B.; Oyarzo, J. N.; Zhang, H.; Timmermann, B. N. Anti-arthritic effects and toxicity of the essential oils of turmeric. J. Agric. Food Chem. 2010, 58, 842−849. (43) Tayyem, R. F.; Heath, D. D.; Al-Delaimy, W. K.; Rock, C. L. Content of turmeric and curry powders. Nutr. Cancer 2006, 55, 126− 131.

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Magnetic Purification of Curcumin from Curcuma longa Rhizome by

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