Article pubs.acs.org/JAFC
Efficient Binding of Heavy Metals by Black Sesame Pigment: Toward Innovative Dietary Strategies To Prevent Bioaccumulation Paola Manini,† Lucia Panzella,*,† Thomas Eidenberger,‡ Antonella Giarra,† Pierfrancesco Cerruti,§ Marco Trifuoggi,† and Alessandra Napolitano† †
Department of Chemical Sciences, University of Naples Federico II, Via Cintia 4, I-80126 Naples, Italy School of Engineering and Environmental Sciences, Upper Austria University of Applied Sciences, Stelzhamerstraße 23, 4600 Wels, Austria § Institute for Polymers, Composites and Biomaterials (IPCB), National Research Council (CNR), Via Campi Flegrei 34, I-80078 Pozzuoli, Italy ‡
ABSTRACT: Black sesame pigment (BSP) was shown to bind lead, cadmium, and mercury at pH 7.0 and to a lower extent at pH 2.0. BSP at 0.05 mg/mL removed the metals at 15 μM to a significant extent (>65% for cadmium and >90% for mercury and lead), with no changes following simulated digestion. The maximum binding capacities at pH 7.0 were 626.0 mg/g (lead), 42.2 mg/g (cadmium), and 69.3 mg/g (mercury). In the presence of essential metals, such as iron, calcium, and zinc, BSP retained high selectivity toward heavy metals. Model pigments from caffeic acid, ferulic acid, and coniferyl alcohol showed lower or comparable binding ability, suggesting that the marked properties of BSP may result from cooperativity of different sites likely carboxy groups and o-diphenol and guaiacyl functionalities. Direct evidence for the presence of such units was obtained by structural analysis of BSP by solid-state Fourier transform infrared spectroscopy and 13C nuclear magnetic resonance spectroscopy. KEYWORDS: Sesamum indicum L., black sesame pigment, heavy metal, chelation, phenolic polymers, simulated digestion
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acid, an edible and biodegradable polymer.20,21 Several other biopolymers of plant or animal origin have been described for the binding capacities toward metals, including heavy metals.22 Beside essential metal ions,23 natural melanins from squid24 and fungi25 and melanin-based synthetic pigments26 are regarded as efficient chelators of heavy metals. Up to now, most of these materials, including also rough agricultural wastes, have been investigated only for applications in water and soil remediation27−30 but, in no case, have been evaluated as food supplements to prevent bioaccumulation of dietary heavy metals. Black sesame seeds (Sesamum indicum L.), traditionally used in Chinese folk medicine and as food for humans in China and other east Asian countries, have attracted interest because of its potent antioxidant activity, superior to that of white sesame seeds.31,32 We recently developed an improved purification procedure to obtain black sesame pigment (BSP), involving fat removal, followed by an optimized hydrolytic protocol.33 This pigment displayed good antioxidant efficiency in the 2,2diphenyl-1-picrylhydrazyl (DPPH) radical assay, high ferric-ionreducing capacity, and potent antinitrosating properties. Chemical and spectral analyses provided evidence for the presence of vanilloid structural units inside the pigment. In the frame of a research project aimed at assessing the potential of black sesame pigment as a food supplement, the
INTRODUCTION The adverse effects on human health associated with exposure to heavy metals are generally recognized and regularly reviewed by international bodies. Lead, cadmium, and mercury are highly toxic, even at low concentrations, because of their long biological half-lives (e.g., 30 days for lead in blood)1 and accumulation in several organs, particularly kidney and liver, causing impairing of the immune and central nervous systems as apparent from the onset of pathological states, including cognitive and behavioral abnormalities, chronic renal failure, and gastrointestinal toxicity.2,3 In recent years, metal intoxication raising from the diet has become an issue of increasing concern as a result of air and water pollution by wastes rich in heavy metals (mining and smelting, battery manufacturing, and pesticides) that are eventually taken up by certain crops and aquatic organisms and accumulate in the food chain. Cadmium and lead are primarily found in cereals, including rice,4 wheat, and products thereof, such as bread and pasta, at average levels of 24 and 14 μg/kg for bread5 and 50 and 19 μg/kg for pasta,6 respectively, and bioaccumulate in marine organisms and in cattle fed with contaminated products.7−9 Likewise, mercury is a typical contaminant of seafood.10 A number of synthetic chelating agents have been prepared for use in the treatment of metal intoxication.11−15 Alternative protective strategies have been evaluated against dietary chronic exposure based on consumption of food rich in ingredients that may play a chelating action, avoiding accumulation of the toxic metals in blood and tissues, among these, dietary fibers, such as wheat, rice, and oat bran cereal fibers,16−19 and polyglutamic © XXXX American Chemical Society
Received: October 28, 2015 Revised: January 3, 2016 Accepted: January 11, 2016
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DOI: 10.1021/acs.jafc.5b05191 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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mg in the case of coniferyl alcohol and 39 mg in the case of caffeic acid and ferulic acid). Binding Experiments: General Procedure. Stock solutions of the heavy metals at the appropriate concentration were prepared by dissolving the respective metal salts into a solution of 0.1 M HCl. Prior to the binding experiments, a suspension of the suitable pigment (BSP, digested BSP, or the acid-treated synthetic pigments) in 0.01 M phosphate buffer (pH 7.0 or 2.0) was obtained by homogenization in a Tenbroeck glass to glass homogenizer for 4 min. The in vitro binding experiments were carried out by adding proper aliquots of the metal solutions and the pigment suspensions to 0.01 M phosphate buffer at pH 7.0 or 2.0 and taking the mixture under stirring at 37 °C. After 2 h, the mixture was filtered through a nylon 0.45 μm membrane, acidified by the addition of 69% nitric acid (1:100, v/v), properly diluted with 1% nitric acid on the basis of the starting metal concentration, and analyzed by inductively coupled plasma mass spectrometry (ICP−MS). For comparative purposes, for each binding experiment, a blank experiment was planned, in which the heavy metal ion at the selected concentration was added in the phosphate buffer and incubated at 37 °C for 2 h without the addition of the pigment. Metal Analysis. Metal analysis was carried out on an ICP−MS instrument Aurora M90 model by Bruker. After initial instrumental optimization, the metal concentration was determined against a calibration curve built with five concentrations each of lead, cadmium, and mercury prepared from their respective stock solutions. The solutions obtained after BSP removal by filtration, properly diluted in the concentration range of the calibration curve, were analyzed, and the concentration of lead, cadmium, or mercury in the unknown sample aliquots was automatically determined. BSP Dose−Activity Relationship. To establish the BSP dose− activity relationship, a total of seven BSP doses, namely, 0.005, 0.01, 0.025, 0.05, 0.1, 0.15, and 0.25 mg/mL, were added separately to the Hg2+, Cd2+, and Pb2+ solutions at 15 μM in 0.01 M phosphate buffer at pH 7.0 or 2.0 (final volume of 10 mL). The suspensions were incubated for 2 h at 37 °C and then filtered through a nylon 0.45 μm membrane, acidified by the addition of 69% nitric acid (1:100, v/v), diluted with 1% nitric acid (1:10, v/v), and analyzed for metal concentration, as described above. Binding Isotherm Study. To determine the binding ability of the BSP with varying metal concentrations, 0.5 mg of the pigment was incubated separately in 0.01 M phosphate buffer at pH 7.0 (final volume of 10 mL) with Cd2+ and Pb2+ solutions at concentrations of 10, 15, 50, 100, 150, and 200 μM or with Hg2+ solutions at concentrations of 1, 2, 5, 10, and 15 μM. Suspensions were incubated for 2 h at 37 °C and then filtered through a nylon 0.45 μm membrane, acidified by the addition of 69% nitric acid (1:100, v/v), suitably diluted with 1% nitric acid on the basis of the starting metal concentration, and analyzed for metal concentration, as described above. Selectivity Study: Effect of Essential Metals. To determine the selectivity of Hg2+, Cd2+, and Pb2+ chelation by the BSP in the presence of selected essential metal ions, such as Zn2+, Ca2+, and Fe3+, suspensions of the BSP (0.5 mg) in 0.01 M phosphate buffer at pH 7.0 (final volume of 10 mL) were added with a metal binary mixture containing Zn2+, Ca2+, or Fe3+ (15 or 150 μM) and Hg2+, Cd2+, or Pb2+ (15 μM). The suspensions were incubated for 2 h at 37 °C and then filtered through a nylon 0.45 μm membrane, acidified by the addition of 69% nitric acid (1:100 v/v), diluted with 1% nitric acid (1:10, v/v), and analyzed for metal concentration, as described above. Binding of Digested BSP and Acid-Treated Synthetic Pigments. Digested BSP or the acid-treated pigments obtained from ferulic acid, caffeic acid, or coniferyl alcohol (0.5 mg) were suspended in 0.01 M phosphate buffer at pH 7.0 and treated separately with Hg2+, Cd2+, or Pb2+ at 15 μM (final volume of 10 mL). After incubation at 37 °C for 2 h, the suspensions were filtered through a nylon 0.45 μm membrane, acidified by the addition of 69% nitric acid (1:100, v/v), diluted with 1% nitric acid (1:10, v/v), and analyzed for metal concentration, as described above.
binding capacity of the purified pigment toward three representative heavy metals, namely, lead, cadmium, and mercury, was systematically investigated as a function of pH and other biologically relevant factors, including the presence of essential metal ions. The chelating properties of the pigment subjected to simulated gastrointestinal digestion were also evaluated. To provide a structural interpretation for the observed marked chelating ability, model pigments from different phenol precursors were as well prepared and comparatively investigated.
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MATERIALS AND METHODS
Materials. Black sesame seeds (S. indicum L.) were provided by Belan Ziviltechniker GmbH (Wels, Austria). Horseradish peroxidase (EC 1.11.1.7), hydrogen peroxide (30%, v/v, water solution), caffeic acid, ferulic acid, HgCl2 (Hg2+), CdCO3 (Cd2+), Pb(NO3)2 (Pb2+), CaSO4·2H2O (Ca2+), FeSO4 (Fe2+), ZnSO4 (Zn2+), HNO3 (≥69%, v/ v, TraceSELECT water solution), pepsin from porcine gastric mucosa, pancreatin from porcine pancreas, and porcine bile extract were from Sigma-Aldrich (Milan, Italy). Coniferyl alcohol was from Acros Organics (Geel, Belgium). HNO3 (1 and 3%) and HCl (0.1 M) solutions and 0.01 M phosphate buffers (pH 2, 6.8, and 7) were prepared using ultrapure deionized water with a conductivity of 65%) at lower doses (0.05 mg/mL). The binding ability decreases at acidic pH with R around 50% only at the highest doses. Binding of lead at pH 7.0 is highly efficient with R > 90% at doses as low as 0.025 mg/mL, while at pH 2.0, R > 60% is reached only at the highest doses. Similarly, binding of mercury is high (R > 90%) with BSP at 0.05 mg/mL at neutral pH but around 60% at the same dose at pH 2.0. On this basis, it seems that the activity of BSP is expected to be maximal in the intestinal environment. This is an important issue if we consider that food and toxic materials remain in the stomach for a shorter time (1−2 h) with respect to the small intestine, which is the main organ where absorption occurs. To evaluate whether the metal-chelating properties of BSP could be modified during the digestion processes, binding experiments were repeated using the pigment subjected to simulated gastrointestinal digestion according to a commonly used protocol.34,35 The method consists of two sequential steps: an initial pepsin/HCl digestion for 2 h at 37 °C to simulate gastric conditions, followed by a digestion with bile salts/pancreatin for 2 h at 37 °C at pH 7.5 to simulate small intestine conditions. The final pigment was tested for the binding activity at pH 7.0 at a dose of 0.05 mg/mL and the metal at 15 μM against untreated BSP. Figure 2 shows that the
Binding Ability of BSP at Different pH Values. The binding ability of BSP obtained from sesame seeds according to the previously developed purification protocol33 was assayed at pH 2.0 and 7.0 relevant to the gastrointestinal conditions. The concentration of Hg2+, Pb2+, and Cd2+ was initially set at 15 μM in 0.01 M phosphate buffer. The in vitro binding experiments were carried out by the addition of the pigment finely suspended and of a suitable amount of the metal to the phosphate buffer at the desired pH followed by incubation at 37 °C for 2 h. After filtration to remove the pigment, the incubation mixtures were acidified and analyzed for metal content by ICP−MS. Figure 1 reports the relationship between the amount of metal binding at different doses of BSP, expressed as R (%), with R = 100 × (C0 − Ceq)/C0, where C0 is the actual starting metal concentration as analytically determined from blank experiments and Ceq is the metal concentration remaining in solution after incubation with BSP. In all cases, binding is
Figure 2. Effect of simulated digestion treatment on the binding ability of BSP. Metal concentration, 15 μM; BSP doses, 0.05 mg/mL; temperature, 37 °C; contact time, 2 h. Shown are mean values of three separate experiments ± SD.
binding ability of digested BSP is decreased with respect to BSP to an extent similar (from 15 to 30%) for all of the metals investigated. This would suggest that the active components, likely those at higher molecular weight, are not lost in the simulated digestion, particularly at the slightly alkaline pH of the intestine, where the binding efficiency was shown to be higher. Binding Isotherms. The binding ability of BSP was further evaluated over a wide range of metal concentrations, using the pigment at the 0.05 mg/mL dose and fixing the pH of the incubation medium at 7.0 on the basis of the higher binding activity shown by the previous series of experiments under these conditions with respect to acid pH values. Figure 3 reports the metal binding capacity qe (milligrams of metal per gram of pigment) of the pigment calculated using the equation qe = (C0 − Ceq)(V/W), where C0 (mg/L) is the actual starting metal concentration as analytically determined from blank experiments, Ceq (mg/L) is the metal concentration remaining in solution after incubation with the pigment, V (L)
Figure 1. Binding of (A) mercury, (B) cadmium, and (C) lead by BSP at variable amounts at pH (■) 2.0 and (◆) 7.0. Metal concentration, 15 μM; BSP doses, 0.005−0.25 mg/mL; temperature, 37 °C; contact time, 2 h. Shown are mean values of three separate experiments ± standard deviation (SD). C
DOI: 10.1021/acs.jafc.5b05191 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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binding capacity and binding constant according to the Langmuir model, respectively. As shown in Table 1, a better fitting of the mercury and cadmium isotherms at pH 7.0 was obtained with the Langmuir Table 1. Isotherm Parameters for Mercury and Cadmium Binding by BSP at pH 7.0 modeled isotherm parameter Freundlich KF (Ln mg1 − n g−1) n r2 Langmuir qm (mg/g) b (L/mg) r2
mercury
cadmium
77.86 1.26 0.908
15.95 2.60 0.928
133.33 0.742 0.974
43.10 0.983 0.991
model with respect to the Freundlich model, suggesting a homogeneous adsorption. Quite different was the case of lead for which the sigmoid trend of the isotherm gave a poor correlation with either models, indicating that the interaction of BSP with this heavy metal is more complex, probably dictated by the presence in BSP of more than one type of binding site with different affinities for lead. To further confirm this interpretation, the isotherm data were subjected to Scatchard plot analysis according to the following equation:
qe /Ce = qmKb − Kbqe where qm (mg/g) is the maximum binding capacity and Kb (L/ mg) is the binding constant relative to the affinity of BSP toward the metal ion.37 As shown in Figure 4, in good agreement with what was previously observed with the modeling approach, for both mercury and cadmium, the Scatchard plot showed a linear trend confirming the homogeneous binding mode, whereas in the case of lead, the concave-down curve indicated a positive cooperativity between the binding sites during chelation. Effect of Essential Metal Ions on the Heavy Metal Binding by BSP. To assess whether essential metal ions may affect the chelating ability of BSP toward target heavy metals, the binding of BSP to the heavy metals under evaluation was measured in the presence of Zn2+, Ca2+, and Fe2+, taken as representative essential metal ions. Each of the essential metals at 15 or 150 μM was incubated separately with Hg2+, Cd2+, and Pb2+ at 15 μM in the presence of BSP (0.05 mg/mL) in 0.01 M phosphate buffer at pH 7.0. The results in Figure 5 are presented as the ratio REM/R, where REM is the removal of the heavy metal in the incubation run in the presence of the essential metal ion and R is the removal obtained in the absence of the essential metal ion. Neither calcium nor zinc affects the binding ability of any of the three heavy metals significantly at the concentrations tested, while iron ions at the lower concentration enhance BSP binding but only in the case of cadmium and lead. At the highest doses, calcium ions decrease to some extent the binding of lead, which can be reasonably accounted for considering the affinity of either cations for the same binding site, i.e., the carboxy groups.38 Thus, different from many chelating agents used for heavy metal detoxification that are reported in some cases to induce
Figure 3. Binding isotherm for (A) mercury, (B) cadmium, and (C) lead binding by BSP at pH 7.0. Initial concentration range, 10−200 μM for cadmium and lead and 1−15 μM for mercury; BSP dose, 0.05 mg/mL; temperature, 37 °C; contact time, 2 h. Shown are mean values of three separate experiments ± SD.
is the volume of the incubation mixture, and W (g) is the pigment dose. In all cases, the curves are characterized by an increased uptake following a rise in the metal concentration. With an increase of the starting metal concentration from 10 to 200 μM, the amount of Pb2+ binding onto BSP rises by 626.0 mg/g and the amount of Cd2+ binding onto BSP rises by 42.2 mg/g. Noteworthy is also the affinity of BSP for Hg2+, reaching a qe value of 69.3 mg/g versus an equilibrium concentration of 1.3 mg/L. Modeling of Isotherms. Modeling of data from isotherms of BSP with mercury, cadmium, and lead is a valuable approach to obtain information on binding characteristics, such as binding constants and maximum binding capacity. To this aim, two different models have been selected: the Freundlich model applicable to heterogeneous binding mode and the Langmuir model based on a monolayer adsorption mode.20,36 The nonlinear forms of these models can be represented according to the following equations: qe = KFCe1/ n
qe = (qmbCe)/(1 + bCe)
where KF (Ln mg1 − n g−1) and n are the Freundlich constants relative to the binding capacity and the binding intensity, respectively, and qm (mg/g) and b (L/mg) are the maximum D
DOI: 10.1021/acs.jafc.5b05191 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 5. Effect of essential metal ions at (A) 15 and (B) 150 μM on the binding of heavy metals (at 15 μM) by BSP at pH 7.0. BSP dose, 0.05 mg/mL; temperature, 37 °C; contact time, 2 h. Shown are mean values of three separate experiments ± SD.
Heavy Metal Binding Capacity of Model Pigments. A series of model synthetic phenolic polymers was tested for the binding activity toward Cd2+, Pb2+, and Hg2+ in comparison to BSP. The most representative precursors of biopolymers like lignins, namely, coniferyl alcohol, caffeic acid, and ferulic acid, were selected.
Figure 4. Scatchard plot isotherm analysis of (A) mercury, (B) cadmium, and (C) lead binding by BSP.
The choice of the model pigments to comparatively evaluate BSP binding properties was guided by the results of our previous studies,33 showing that it contains structural units featuring the o-methoxyphenol moiety as apparent from the formation of substantial amounts of vanillic acid by chemical degradation, but the possibility that other units either present in the native pigment or originating from the acid treatment that may as well contribute to the outstanding antioxidant and chelating properties should be also considered. Thus, the pigment precursors were chosen to allow for discrimination of the role played by the o-diphenol versus o-methoxyphenol functionalities (caffeic acid versus ferulic acid) as well as the role of the carboxy group in systems sharing the omethoxyphenol unit (ferulic acid versus coniferyl alcohol). Preparation of model polymer pigments was carried out according to a procedure previously developed33 involving horseradish peroxidase/H2O2 oxidation of the phenol at pH
essential metal deficiency,39 BSP showed a high selectivity toward heavy metals with respect to the essential metal cations selected in a 1:1 and 1:10 bisolute system. This may be ascribed to the presence of different types of binding sites in BSP compared to such compounds either natural or synthetic,20 including notably dietary fibers,18,19 that feature typically OH groups of carbohydrates or carboxy groups. Intriguing is also the synergistic effect observed in the case of iron, resulting in an increase of cadmium and lead binding. Such effects have been reported for polyglutamic acid20 with respect to lead binding at a very high concentration (100 mg/L) of both the heavy metal and the essential cation and interpreted as as a result of structural or conformational changes of the binding agent or possibly an extra mobility acquired by lead ions during competition that could facilitate binding of additional lead ions onto remote sites of the chelating polymer. E
DOI: 10.1021/acs.jafc.5b05191 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry 6.8, leading to separation of the polymer as a precipitate in good yields. The product was then subjected to the acid treatment under the same conditions developed in the case of BSP. The pigments were tested at 0.05 mg/mL at pH 7.0 with the metals Hg2+, Cd2+, and Pb2+ at 15 μM under the incubation conditions described for BSP. As shown in Figure 6, the
Although less efficient with respect to the other model pigments examined, the binding ability of the pigment from coniferyl alcohol toward the metals under study follows a trend closely similar to that exhibited by BSP. This would indicate that the o-methoxyphenol functionalities are essential for the binding properties, although products of the pinoresinol or dihydrodiconiferyl alcohol type arising from the peroxidase/ H2O2 oxidation that may feature alternative and peculiar metal chelating sites as described for these and other lignans43 may contribute to some extent. To support the hypotheses emerging from the binding behavior of the model pigments, in further experiments the structure of BSP was directly investigated using methodologies typically adopted for materials unsoluble in common solvents. The FTIR spectrum in ATR mode of BSP (Figure 7A) shows a broad band at about 3340 cm−1 attributed to −OH groups of
Figure 6. Binding activity of heavy metals by model synthetic pigments at pH 7.0 in comparison to BSP. Metals, 15 μM; synthetic pigments and BSP dose, 0.05 mg/mL; temperature, 37 °C; contact time, 2 h. Shown are mean values of three separate experiments ± SD.
polymer from caffeic acid exhibits a very high binding ability toward mercury, comparable to that observed with BSP, whereas in the case of cadmium, the pigment from caffeic acid shows an activity even higher (>70% removal) than BSP. In the case of lead, BSP appears as the strongest chelating agent with respect to all of the synthetic pigments. Among critical factors that may affect the relative binding ability observed is the ionization state of the pigments under the pH conditions of the incubation. Although data are available only for the monomers, it can be safely predicted that, at pH 7, the carboxyl group is in the anion form also in the polymers, while the ionization of both the o-methoxyphenol and o-diphenol functionalities is negligible.40 The difference behavior of the model pigments in chelation of lead compared to cadmium and mercury may be interpreted in terms of the different binding sites involved. Support to this view is offered by studies on caffeic acid, showing that the preferential binding site for lead is the carboxyl group rather than the catecholic functionalities.41 This is also supported by analysis of binding isotherms for BSP, indicating that lead chelation occurs at sites different from those involved in cadmium and mercury binding. The chemical pathways described for the peroxidasecatalyzed oxidation of caffeic and ferulic acids involve coupling of the phenoxy radical with the carbon-centered radical α to the carboxyl group, leading to oligomers of the type shown below featuring a variety of potential binding sites, including conjugated and non-conjugated carboxyl groups, together with hydroxyl and methoxyl functionalities.42
Figure 7. (A) FTIR−ATR and (B) solid-state BSP.
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C NMR spectra of
cellulose and hemicellulose, which were not completely removed following the acid hydrolysis, and of phenolic compounds. The presence of methyl and methylene groups is confirmed by the two sharp peaks at 2920 and 2850 cm−1. The band at 1701 cm−1 can be associated with the carbonyl (CO) stretching in the lipid components and cinnamic-acid-derived units. The low-intensity bands at 1600 and 1510 cm−1 are mainly due to CC vibration of aromatic rings of lignan-type molecules. The band at about 1450 cm−1 corresponds to C−H bending, including that of methoxyl groups. The region from 1000 to 1250 cm−1 exhibits several types of vibrations, F
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essential metals, a drawback exhibited by several dietary supplements, such as cereal fibers. In combination with our previous observations indicating the marked antioxidant properties of BSP, these results highlight the potential of this low-cost, easily accessible material from plant sources as an ingredient of functional food or as a food supplement.
including C−O−H and C−O−C, characteristic of lignans and carbohydrates. In particular, the absorptions at 1210 and 1110 cm−1 can be attributed to phenols and alkyl aromatic ethers (Ar−O) of lignans,44 while peaks at 1155, 1063, and 1030 cm−1 originate from the vibrational modes of the glycosyl units of cellulose and hemicellulose.45 NMR spectroscopy provided further information about the composition of BSP. The solid-state 13C cross-polarization magic angle spinning (CP-MAS) NMR spectrum of BSP (Figure 7B) shows main distinct signals at 147, 105, 75, 72, 56, 33, and 30 ppm. This complex pattern is suggestive of the presence of different aromatic, polysaccharide, and aliphatic compounds. More specifically, among the aromatic carbons, the sharp peak at around 147 ppm is due to aromatic carbons linked to oxygen atoms, such as those (C3 and C4) of guaiacyl or o-diphenol units.46 The broad signals at about 133, 123, and 114 ppm can be attributed to the other aromatic carbons of the same moieties, whereas the signal at 56 ppm is due to methoxy groups. Characteristic signals of the polysaccharide components range from 60 to 105 ppm and arise likely from cellulose, which was not completely removed following the acid hydrolysis. The sharp signal at 105 ppm can be attributed to the anomeric carbon (C1) of the glucosidic ring, while the weak resonances at 89 and 84 ppm are due to C4 of crystalline and amorphous domains in cellulose.47 At lower frequencies, C2, C3, and C5 carbons of cellulose resonate at 75 and 72 ppm, while C6 is responsible for the weak signals at about 65 and 62 ppm. The resonances in the range between 30 and 32 ppm are ascribed to alkyl carbon in long-chain aliphatic chains typical of fatty acids and not completely removed by the treatment of the crude ground sesame seeds with dichloromethane. A broad signal between 180 and 170 ppm assigned to conjugated and nonconjugated carboxyl groups would indicate the presence, beside the lipid components, of cinnamic acid units. Low resonances are also observed in the 40−50 ppm range, which would be compatible to those arising from the oxidation reactivity of cinnamic moieties, as shown in the structure above. In light of all of these results, the observation that, in most cases, BSP outcompetes the synthetic pigments for the binding activity toward the toxic metals tested may lead to the conclusion that, although o-methoxyhydroxy units are represented to a significant extent in the backbone of BSP, such units are not the main determinants of the marked binding activity but o-diphenols and to a lower extent carboxy groups provide a more critical contribution. In conclusion, in the present study, we have shown the high binding capacity of the pigment from black sesame seeds toward heavy metals, such as lead, cadmium, and mercury, that are commonly found in food. The highest activity is associated with neutral pH values simulating the intestinal environment compared to the acidic pH values peculiar of the gastric compartments. Even at low concentrations (0.05 mg/mL), the pigment proved able to remove the metals to a significant extent (>65% for cadmium and >90% for mercury and lead). At such doses, the pigment is able to remove the metals completely at the highest levels that have been reported in contaminated food. A good chelating ability against lead and to a lower extent cadmium is retained by BSP, even with the metal at concentrations up to 200 μM. The binding activity is not lost following digestion as simulated using a model of the gastrointestinal transit, and on the basis of competition experiments, it should not interfere with the absorption of
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AUTHOR INFORMATION
Corresponding Author
*Telephone: +39081674131. Fax: +39081674393. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED BSP, black sesame pigment; DPPH, 2,2-diphenyl-1-picrylhydrazyl; ICP−MS, inductively coupled plasma mass spectrometry; ATR, attenuated total reflectance; CP-MAS, crosspolarization magic angle spinning
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REFERENCES
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DOI: 10.1021/acs.jafc.5b05191 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.jafc.5b05191 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
