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Operationally defined species characterization and bioaccessibility evaluation of cobalt, copper and selenium in Cape gooseberry (Physalis Peruviana L.) by SEC-ICP MS
Journal of Trace Elements in Medicine and Biology 34 (2016) 15–21
Contents lists available at ScienceDirect
Journal of Trace Elements in Medicine and Biology journal homepage: www.elsevier.com/locate/jtemb
Analytical methodology
Operationally defined species characterization and bioaccessibility evaluation of cobalt, copper and selenium in Cape gooseberry (Physalis Peruviana L.) by SEC-ICP MS Justyna Wojcieszek, Lena Ruzik ∗ Chair of Analytical Chemistry, Warsaw University of Technology, Poland
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Article history: Received 21 August 2015 Received in revised form 28 October 2015 Accepted 1 December 2015 Keywords: Bioaccessibility Physalis Peruviana SEC-ICP MS Selenomethionine Vitamin B12
a b s t r a c t Physalis peruviana could attract great interest because of its nutritional and industrial properties. It is an excellent source of vitamins, minerals, essential fatty acids and carotenoids. Physalis Peruviana is also known to have a positive impact on human health. Unfortunately, still little is known about trace elements present in Physalis Peruviana and their forms available for the human body. Thus, the aim of this study was to estimate bioaccessibility and characterization of species of cobalt, copper and selenium in Physalis Peruviana fruits. Total and extractable contents of elements were determined by mass spectrometer with inductively coupled plasma (ICP MS). In order to separate the different types of metal complexes Physalis peruviana fruits were treated with the following solvents: Tris–HCl (pH 7.4), sodium dodecyl sulfate (SDS) (pH 7.4) and ammonium acetate (pH 5.5). The best efficiency of extraction of: cobalt was obtained for ammonium acetate (56%) and Tris–HCl (60%); for copper was obtained for SDS (66%), for selenium the best extraction efficiency was obtained after extraction with SDS (48%). To obtain information about bioaccessibility of investigated elements, enzymatic extraction based on in vitro simulation of gastric (pepsin) and intestinal (pancreatin) digestion was performed. For copper and selenium the simulation of gastric digestion leads to the extraction yield above 90%, while both steps of digestion method were necessary to obtain satisfactory extraction yield in the case of cobalt. Size exclusion chromatography (SEC) coupled to on-line ICP MS detection was used to investigate collected metal species. The main fraction of metal compounds was found in the 17 kDa region. Cobalt and copper create complexes mostly with compounds extracted by means of ammonium acetate and SDS, respectively. Cobalt, copper and selenium were found to be highly bioaccessible from Physalis Peruviana. Investigation of available standards of cobalt and selenium allows confirming the presence of vitamin B12 and probably selenomethionine in the fraction bioaccessible by human body (obtained during enzymatic extraction). It should be noted that the presence of small seleno-compounds in Cape gooseberry was performed for the first time. The results show that the combination of SEC and ICP MS could provide a simple method for separating of soluble element species. © 2015 Elsevier GmbH. All rights reserved.
1. Introduction In recent years berries are becoming more popular due to their nutritional value and health benefits [1,2]. Cape gooseberry (Physalis peruviana L.), also known as goldenberry is a promising exotic fruit that could be a subject of many novel foods. It belongs to
∗ Corresponding author at: Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland. E-mail address: [email protected] (L. Ruzik). http://dx.doi.org/10.1016/j.jtemb.2015.12.001 0946-672X/© 2015 Elsevier GmbH. All rights reserved.
the Solanaceae family and originated in the South America. Nowadays Colombia is the largest Cape gooseberry exporter in the world [3–5]. Physalis Peruviana forms a dome-shaped shrub that can grow to 1 m and the fruits with an approximate weight of 4–5 g are covered by a shiny yellow peel. The flowers, produced in winter, are yellow with purple blotches [5,6]. The pulp is nutritious, containing particularly high levels of carotenoids, minerals and vitamin C [7]. The golden berry is also an excellent source of the vitamin B-complex and essential fatty acids. The protein content is exceptionally high for a fruit [8,9]. Physalis Peruviana is becoming more and more popular, it has increased interest worldwide
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because of its nutritional composition and the presence of biologically active compounds that provide health benefits and reduce the risk of various diseases such as cancer, malaria, asthma, hepatitis, dermatitis and rheumatism [10–13]. Although a lot of available publications focus on C. gooseberry and its properties there is a lack of information about speciation analysis of metals present and their bioavailability by human organism. Carrying out further studies, Physalis Peruviana could become a fruit of particular interest to the world’s up-scale food industry. The aim of the study was to separate complexes of selected elements with different bioligands and characterization of species of chosen metal. Determination of total concentration of metals in food does not provide information about their bioavailability in the human organism. Knowledge about content of element in the bioaccessible fraction is necessary to evaluate bioavailability. The information on the bioaccessibility of important nutrients in food and food supplements seems to be essential. For that reason the second objective of the study was to estimate elements bioaccessibility in C. gooseberry by in vitro simulation of gastrointestinal digestion using two-step model with pepsin as a gastric juice and pancreatin as an intestinal juice. To the best of our knowledge this is the first attempt to carry out characterization of species of chosen metal and bioaccessibility evaluation of metals in C. gooseberry by SEC-ICP MS.
2. Experimental 2.1. Reagents and chemicals Sodium chloride, ammonium acetate, sodium dodecyl sulphate, Tris(hydroxymethyl) aminomethane and hydrochloric acid were purchased from Sigma–Aldrich and were of analytical reagent grade. Selenomethionine and vitamin B12 standards (assay ≥ 98%) were also purchased from Sigma–Aldrich. Pepsin from porcine gastric mucosa and pancreatin were of biological grade (Sigma-Aldrich, Buchs, Switzerland). Deionized water (18 M cm) prepared with a Milli-Q system (Millipore Elix 3, Millipore, Saint–Quentin, France) was used throughout. The SEC column was calibrated using size exclusion standard (BIO-RAD, Warsaw, Poland).
2.2. Instrumentation The screening for the metal complexes was carried out by means of size exclusion chromatography coupled to ICP MS. Prepared samples were analysed on a Superdex200 10/300GL (GE Healthcare Life Sciences) exclusion column with bed volume of 24 mL. Before the analysis the column was calibrated with a mixture of thyroglobulin (670 kDa), ␥-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), vitamin B12 (1,35 kDa). Chromatographic separations were performed using Agilent 1100 gradient HPLC pump (Agilent Technologies, Waldbronn, Germany) as the sample delivery system. Agilent 7500a ICP MS (Tokyo, Japan) was used as on-line HPLC detector. All connections were made with PEEK tubing (0.17 mm i.d.). Operational parameters are summarized in Table 1. The determination of concentration of elements in C. gooseberry samples (after mineralization, buffer and enzymatic extraction) was carried out by Agilent 7500a ICP MS as an element-specific detector. Ni/Cu-skimmer was installed in the interface, the position of torch and nebulizer gas flow was adjusted daily with special emphasis to decrease the level of CsO+ below 0.2% with the aim to minimize the risk of polyatomic interferences caused by oxides. The working conditions were optimized daily using a 10 g L−1 solution of 7 Li+ , 89 Y+ and 209 Bi+ in 2% HNO3 , with a dwell time of 0.1 ms for each isotope.
Table 1 Operational parameters for SEC and ICP MS. Settings SEC separation Pump Column Mobile phase Elution program Flow Injection volume Column temperature ICP-MS RF Power Plasma, auxiliary, nebulizer gas flow Cones Monitored isotopes Dwell time
Agilent 1100 Superdex 200 (10 × 300 mm × 10 m)-GE Healthcare Life Sciences 10 mM ammonium acetate buffer (pH 7.4) Isocratic 0.7 mL min−1 100 L 24 ◦ C Agilent 7500a 1350 W 15.0, 1.0 and 1.05 L min−1 Sampler–Pt, Skimmer–Ni 55 Mn, 59 Co, 63 Cu, 65 Cu, 66 Zn, 67 Zn, 68 Zn, 82 Se, 95 Mo 0.1 ms
A Bandelin Sonorex Model 1210 ultrasonic bath (Germany) and MPW Model 350R centrifuge (MPW Warsaw, Poland) were used for extraction procedures. 2.3. Sample preparation 2.3.1. Determination of total content of elements and buffer extraction method The dried C. gooseberry was obtained from Kenay (Poland, imported from Peru) and stored at 4 ◦ C until analysis. For determination of total amount of elements in Physalis Peruviana, samples (0.3 g of dry mass) were digested by microwaveassisted mineralization with a mixture of 5 mL of HNO3 and 3 mL of H2 O2 . After cooling down the digests were diluted with MQ water to the volume of 25 mL and then diluted before ICP MS analysis. Samples and standard solutions were prepared with addition of 89 Y as the internal standard. The external calibration curves were linear in the investigated range from 2 g L−1 to 150 g L−1 with r2 above 0.997. Samples of C. gooseberry (0.07 g) were extracted during 1 h in ultrasonic bath, using 1 mL of the following buffers: (1) 10 mM ammonium acetate (pH 5.5), (2) 30 mM Tris–HCl (pH 7.4), (3) 2% sodium dodecyl sulphate (SDS) in water (pH 7.4). After extraction, the obtained solutions were centrifuged for 20 min at 15 000 rpm at 15 ◦ C. The final supernatants were filtered with 0.45 m syringe filter (Sigma–Aldrich, Bellefonte, PA, USA), two first drops were discarded and only the remaining part of the filtrates was injected on the size exclusion column. 2.3.2. In vitro simulation of gastrointestinal digestion The in vitro digestion method was based on Luten et al. [14] modified to the studied berries. 2.5 mL of gastric juice (6% w/v pepsin in 0.15 M NaCl, acidified to pH 1.8 by means of HCl) was added to 0.07 g of C. gooseberry and then shaken and sonicated for 10 min in ultrasonic bath. In the next step the mixture was incubated in the thermostatic water bath for 3.5 h at 37 ◦ C. After incubation the mixture was centrifuged at 4 ◦ C for 20 min at 15 000 rpm. The supernatants were filtered through 0.45 m syringe filters (Sigma–Aldrich, Bellefonte, PA, USA) and analyzed. For intestinal digestion, NaHCO3 solution was added to the remaining part of sample to obtain the neutral pH. After that 2.5 mL of intestinal juice (1.5% w/v pancreatin in 0.15 M NaCl) was added and the mixture was incubated for 2 h at 37 ◦ C. Following centrifugation and filtration of supernatant, the gastrointestinal extract was analyzed by SEC-ICP MS. To collect the information about the bioaccessibility of important nutrients from Physalis Peruviana by human organism, the
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Fig. 1. The scheme of analytical procedure for buffer extraction and enzymatic digestion of cobalt, copper and selenium analysis.
efficiency of enzymatic extraction was estimated by establishing the amounts of elements in digestion extracts against total content of elements in mineralized samples. The procedure of samples preparation is presented on Fig. 1. 2.3.3. Preservation of element species Preparation of the sample is a primary concern in speciation analysis of elements. It is important to preserving the original species or chemical form of element and obtaining a high extraction efficiency from the sample matrix. Some selenium species are particularly susceptible to oxidation, which could lead to false identification of selenium species [15]. During the investigation, non-volatile species have been collected by commonly practiced solid–liquid extraction (leaching). During this procedure sonication assisted forms had been used to remove copper, cobalt and selenium compounds from the sample matrix. During enzymatic
extractions (with pepsin and pancreatin) pH and temperature were controlled and maintained in the optimum range of activity of enzymes used during simulation of digestion. During speciation analysis non-oxidizing acid was used. The deep-freezing of samples was used in order to minimize any bacterial or enzymatic degradation or loss from volatility. 3. Results and discussion 3.1. Total content of elements in Physalis Peruviana C. gooseberry is known as an important source of trace elements. Cobalt, copper and selenium were selected for monitoring during samples analysis. Despite great progress made in the analytical instrumentation, the speciation analysis of cobalt and copper in the food is largely limited. Those elements were chosen due to the
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fact that to the best of our knowledge there was no previous studies of their speciation in analyzed fruits. Additionally, all described elements belong to the group “challenging” for analysts but the speciation process can provide information vital for human health. The total concentration of selected elements was determined after microwave-assisted digestion of berries by means of ICP MS. The results were obtained from three independent experiments. As the interest of this study the following total amount of elements -was identified: cobalt (0.27 ± 0.09 g g−1 ), copper (7.3 ± 0.3 g g−1 ) and selenium (0.09 ± 0.03 g g−1 ). Alongside of those elements, total content of other microelements was determined: manganese (16.0 ± 0.9 g g−1 ), zinc (15.4 ± 0.4 g g−1 ) and molybdenum (0.26 ± 0.13 g g−1 ). The results are not in good agreement with the earlier studies reported by other authors but it should be pointed that to our knowledge, this is the first study when the total content of trace elements was determined in dried fruits of Physalis Peruviana, not in the pulp or juice. Concentrations of the same elements in buffer and enzymatic extracts were also determined in C. gooseberry in order to evaluate their dietary intake. The efficiency of extraction of buffer and digestion methods was estimated by comparison the amounts of elements in extracts against total amount of elements obtained by mineralization. The yields of extraction of investigated elements obtained by different extraction methods and enzymatic digestion are presented in the Table 2. In cobalt case, the best efficiency of extraction was obtained for ammonium acetate (56%) and Tris–HCl (60%), what indicate that cobalt is present mostly in the form of water soluble compounds. For copper, the presence of SDS significantly improves the efficiency of extraction (66%). It could indicate that copper creates the most complexes with hydrophobic proteins. For Tris–HCl and ammonium acetate extracts (respectively), the efficiency of copper extraction was exactly the same (45%). In selenium for instance, the results were obtained on the very low concentrations and with relative high RSD (about 15%). The buffer extraction leads to relatively low extraction efficiency. The best results were obtained after extraction with SDS (48%), what could indicate the presence of insoluble selenium species, such as selenoproteins. It should be pointed out that the yield of extracted metals was the same even when bigger excess of solvents was used in comparison to investigated samples of berries. Enzymatic method improved extraction efficiency of all the investigated elements. The simulation of gastric digestion leads to the extraction yield above 90%, for copper and selenium. Such results indicate that proteins could be the main ligands binding investigated elements in Physalis Peruviana . In cobalt investigation, both steps of digestion method were necessary to obtain satisfactory extraction yield. For that reason, polysaccharides can be proposed as compounds interacting with cobalt because pancreatin used during intestinal digestion improved efficiency of extracted metal in comparison to pepsin. During enzymatic digestion, the metal complexes with low molecular weight compounds were created with high efficiency. 3.2. SEC-ICP MS characteristics of ammonium acetate, Tris and SDS extracts containing metal species Coupling of size exclusion chromatography with inductively coupled plasma mass spectrometry allows the element specific detection of eluted compounds. The eluate from the column was fed directly into the ICP MS. Both buffer and enzymatic extracts of Physalis Peruviana were analysed. In cobalt case the chromatogram obtained for ammonium acetate extract consisted of two, poorly separated peaks (Fig. 2a). First after 25.8 min and second after 27 min of chromatographic process, what can correspond to fraction of cobalt complexes with organic acids or other small molecular weight compounds suspected to reveal ability to bind
cobalt. Chromatograms obtained for Tris–HCl and SDS extracts consisted of one, major peak at about 26.5 min which can correspond to cobalt complexes with molecular mass 17 ± 10 kDa. Additionally, one small peak was observed for SDS extract at 11 min corresponding to compounds with molecular mass ≥ 500 kDa. In C. gooseberry cobalt is complexed mostly by bioligands extracted by means of ammonium acetate. For the comparison the results obtained for cobalt and copper were chosen to show in this study. Copper is another important trace element which is necessary for human beings. Fig. 2b shows chromatograms of copper compounds extracted from C. gooseberry by different extraction media. The chromatograms obtained for copper after extraction with both Tris–HCl and SDS contained one major peak eluted at about 26.7 min, which correspond to compounds with relative molecular weight 17 ± 10 kDa. In the case of SDS extract two peaks were additionally observed in the chromatogram. The first, small peak was observed at 11 min and it was obtained at column’s exclusion limit corresponding to high molecular weight compounds ≥ 670 kDa. The presence of high molecular weight complexes can be explained by ability of metal ions to create agglomerates even with low molecular weight bioligands which was already reported [16]. The second, more intense peak was observed at about 34 min and it can correspond to low molecular weight complexes with molecular mass lower than 1.4 kDa. Because of much more higher intensity than peak at 11 min, it is possible that SDS solution is responsible for partial decomposition of agglomerates. In chromatogram obtained after extraction with ammonium acetate one peak was observed at 27.2 min, which correspond to compounds with relative molecular weight 17 ± 10 kDa. It should be pointed out that different retention time than for Tris–HCl and SDS solutions suggests extraction of copper complexes with another group of bioligands. In contrast to cobalt, copper forms complexes mostly with compounds extracted by means of sodium dodecyl sulphate, dedicated to hydrophobic proteins, which were suspected to reveal ability to bind investigated elements. Chromatograms of buffer extracts of selenium are not shown because they do not contain any peak. However, it should be stressed that a lack of signals in chromatograms of buffer extracts does not mean that selenium compounds do not exist in Physalis Peruviana. 3.3. SEC-ICP MS profiling of gastric and gastrointestinal extracts Gastric and gastrointestinal extracts of Physalis Peruviana were examined by SEC-ICP MS using the same elution conditions as for buffer extracts. The chromatograms obtained for cobalt consisted of two peaks in the case of gastric extract and three peaks for gastrointestinal extract (Fig. 3a), which can correspond to cobalt complexes with digestion products of proteins. It was already reported that cobalt ions have a high affinity for binding to proteins and amino acids [17]. The high molecular weight peak (tr = 19.5 min) can be observed due to cobalt binding by enzymatic proteins. Their ability to interact with cobalt species and improving bioaccessibility was already reported in the case of cobalamins [18]. The chromatograms obtained for copper after analysis both pepsin and pancreatin extracts were quite similar and consisted of two peaks in low molecular weight region (Fig. 3b). However, in relation to gastric extract the second peak was significantly lower. Both signals correspond probably to digestion products of proteins or other high molecular weight compounds present in Physalis Peruviana. These main peaks of gastric extract belong to the region with molecular mass between 44 and 17 kDa which can indicate on copper connections with enzymatic proteins or their self-digestion products. It should be mentioned that copper ions were earlier reported to be bound by pepsin even in the acidic media as well
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Table 2 The amounts of investigated elements in the buffer and enzymatic extracts of Physalis Peruviana.
30 mM Tris–HCl pH 7.4 2% of SDS in water pH 7.4 10 mM CH3 COONH4 pH 5.5 Gastric digestion Gastric and gastrointestinal digestion
Co g g−1 (%)
Cu g g−1 (%)
Se g g−1 (%)
0.16 ± 0.01 (60%) 0.13 ± 0.01 (50%) 0.15 ± 0.01 (56%) 0.17 ± 0.01 (64%) 0.17 ± 0.01 (63%)
3.3 ± 0.7 (45%) 4.8 ± 0.7 (66%) 3.3 ± 0.6 (45%) 6.7 ± 1.0 (92%) 2.1 ± 0.4 (29%)
0.035 ± 0.014 (39%) 0.043 ± 0.019 (48%) 0.032 ± 0.001 (36%) 0.086 ± 0.004 (90%) 0.028 ± 0.001 (30%)
Results represent an average amount established for 3 samples; each measured 3 times;% of total concentration of element.
Fig. 2. SEC-ICP MS chromatograms obtained for Tris–HCl (green dotted line), SDS (blue dots) and ammonium acetate (violet solid line) extracts of Physalis Peruviana for (a) cobalt, (b) copper. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. SEC-ICP MS chromatograms of gastric (black, solid line) and gastrointestinal (red, dotted line) of Physalis Peruviana for (a) cobalt, (b) copper. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
as iron or nickel [19]. After the end of the simulation of digestion process, the peak at tr = 27 min was observed and it corresponds to copper binding by low molecular weight compounds created during digestion. The peak obtained at tr = 31.5 min, present only for gastrointestinal extract could indicate the presence of copper complexes with digestion products of saccharides due to the action of amylase (a part of pancreatin), which is responsible for digestion of those compounds. No signals were observed in chromatograms for buffer extracts of selenium, but after digestion with both pepsin and pancreatin the peaks were noticeable (Fig. 4b). It could be evidence that the insoluble selenoproteins present in the sample are hydrolysed to form hydrophilic selenium species, such as selenoamino acids or inorganic species (selenate or selenite). The lower molecular weight selenium species tends to be dissolved into the aqueous phase. In the chromatograms of enzymatic extracts of C. gooseberry, two peaks are present in the case of gastric extract of selenium. After examination of gastrointestinal extract, only one peak with small intensity was observed in the chromatogram. On the basis of the column calibration, this region represents selenium species
with molecular weight less than 5 kDa, which can correspond to selenopeptides. 3.4. Operationally defined species characterization of cobalt and selenium Information about chemical status of trace elements in C. gooseberry is still very limited. It is believed that metal species created during digestion are accessible by human organism. In reference to this hypothesis, characterization of species of cobalt and selenium was performed in the present investigation by SEC-ICP MS in extracts obtained by simulation of gastrointestinal digestion. Analysis of available standards of cobalt (cyanocobalamin–vitamin B12 ) and selenium (selenomethionine) were carried out. Compounds were identified by matching retention times of peaks in enzymatic extracts with those of standard compounds. As for cobalt, it was able to confirm the presence of vitamin B12 in both gastric and gastrointestinal extracts of Physalis Peruviana (Fig. 4a). The peak at tr = 29.2 min occurs at the same retention time as standard of vitamin B12 . Knowledge about the bioavailability of vitamin B12 from various food sources is rather limited, despite its importance
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Fig. 4. SEC-ICP MS chromatograms of gastric (black, solid line) and gastrointestinal (red, dotted line) with standards used in this study (black, dotted lines) of Physalis Peruviana for (a) cobalt, (b) selenium. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
for human metabolism [20]. The presence of vitamin B12 in fraction bioaccessible for human body (obtained during simulation of gastrointestinal digestion) was confirmed in this study. The detection of low molecular weight fraction of selenium species in enzymatic extracts could be the proof of the presence of selenoproteins in the sample. For the speciation of selenium, the selenoproteins originally present in the berries sample are decomposed into hydrophilic selenopeptides during digestion process, which can be detected by SEC-ICP MS. After analysis of gastric extract the peak at tr = 28 min matches the retention time of selenomethionine standard (Fig. 4b), what could be the evidence on presence of this compound as the major organic compound of selenium found in enzymatic extracts of C. gooseberry. It could also indicate that in enzymatic extracts selenium is present in the form of small seleno-compounds. It could be also concluded that selenium is accessible in human organism in the form of chelates with amino acids [21]. It was reported in earlier studies that organic forms of selenium are better accessible by human organism than inorganic forms and that selenomethionine is the most bioavailable source of selenium [22–24]. It should be noted that the presence of small seleno-compounds (probably selenomethionine) in C. gooseberry was confirmed for the first time. 4. Conclusions In the present study, ICP MS and SEC-ICP MS method was used for the first time for the speciation studies of cobalt, copper and selenium in C. gooseberry (P. Peruviana L.). The combination of SEC and ICP MS allows fractionation of metal-containing species in berries according to their size and shape. Determination of total concentration of metals in food does not provide information about their bioavailability by human organism, bioavailability depends on their chemical forms, which are absorbed and metabolized differently. For that reason, the second aim of the study was to estimate bioaccessible elements in Physalis Peruviana by in vitro simulation of gastrointestinal digestion. Elution profiles of elements were different from each other and also differed depending on the extraction medium used in most of the cases. SEC demonstrates that metals are distributed mostly in medium and low molecular mass range between 44000 and 1000 Da. After simulation of gastrointestinal digestion, copper complexes with digestion products of proteins or with self-digestion products of enzymatic proteins were detected. The presence of vitamin B12 and small seleno-compounds in the enzymatic extracts was confirmed and compared with reference standards. Vitamin B12 is well-known to be the sole vitamin that is absent from plantderived food sources [25]. Physalis Peruviana is a natural plant product and it is suitable vitamin B12 source presently available for vegetarians. High molecular weight selenium species, such as selenoproteins, are hydrolyzed to lower molecular and soluble species (selenopeptides) during gastric and gastrointestinal diges-
tion. Selenomethionine is probably the major organic compound of selenium found in enzymatic extracts of C. gooseberry. It could be concluded that vitamin B12 and selenomethionine are the forms of elements available for human organism. The method described in this study may be used to study the metal species in other plants. Future investigations with using more advanced mass spectrometry techniques (such as ESI QqQ MS/MS or ESI QTOF MS) will be needed to provide a more detailed characterisation and identification of the investigated elements species. Conflict of interest The authors declare no conflict of interest. Acknowledgement Authors are thankful to Warsaw University of Technology for financial support of presented study. References [1] N.P. Seeram, Berries and human health research highlights from the fifth biennial berry health benefits symposium, J. Agric. Food Chem. 62 (2014) 3839–38341. [2] L. Chen, X. Xin, Q. Yuan, D. Su, W. Liu, Phytochemical properties and antioxidant capacities of various colored berries, J. Sci. Food Agric. 94 (2014) 180–188. [3] K. Bravo, S. Sepulveda-Ortega, O. Lara-Guzman, A.A. Navas-Arboleda, E. Osorio, Influence of cultivar and ripening time on bioactive compounds and antioxidant properties in Cape gooseberry (Physalis Peruviana L.), J. Sci. Food Agric. 95 (2015) 1562–1569. [4] M. Yilmaztekin, Characterization of potent aroma compounds of Cape gooseberry (Physalis Peruviana L.) fruits grown in Antalya through the determination of odor activities values, Int. J. Food Prop. 17 (2014) 469–480. [5] M.F. Ramadan, A.H. Nesma, R.M. Elsanhoty, M.Z. Sitohy, Goldenberry (Physalis Peruviana L.) juice Rich in health-beneficial compounds suppresses high-cholesterol diet-Induced hypercholesterolemia in rats, J. Food Biochem (37 2013) 708–722. [6] M.F. Ramadan, Bioactive phytochemicals, nutritional value and functional properties of Cape gooseberry (Physalis Peruviana): an overview, Food Res. Int. 44 (2011) 1830–1836. [7] M.F. Ramadan, J.T. Moersel, Impact of enzymatic treatment on chemical composition, physicochemical properties and radical scavenging activity of goldenberry (Physalis Peruviana L.) juice, J. Sci. Food Agric. 87 (2007) 452–460. [8] M.F.R. Hassanien, Physalis Peruviana: a rich source of bioactive phytochemicals for functional foods and pharmaceuticals (review), Food Rev. Int. 27 (2011) 259–273. [9] H. Popenoe, S.R. King, J. Leon, L.S. Kalinowski, Goldenberry (Cape gooseberry), in: N.R. Council (Ed.), Lost Crops of the Incas: Little-known Plants of the Andes With Promise for Worldwide Cultivation, National Academy Press, Washington DC, 1990, pp. 241–252. [10] N. Izli, G. Yıldız, H. Ünal, E. Is¸ık, V. Uylas¸er, Effect of different drying characteristics, colour, total phenolic content and antioxidant capacity of Goldenberry (Physalis Peruviana L.), Int. J. Food Sci. Technol. 49 (2014) 9–17. [11] M.R. Salazar, J.W. Jones, B. Chaves, A. Cooman, A model for the potential production and dry matter distribution of Cape gooseberry (Physalis Peruviana L.), Sci. Hortic.-Amsterdam 115 (2008) 142–148. ˜ [12] L.A. Puente, C.A. Pinto-Munoz, E.S. Castro, M. Cortés, Physalis Peruviana Linnaeus, the multiple properties of a highly functional fruit: a review, Food Res. Int. 44 (2011) 1733–1740.
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Journal of Trace Elements in Medicine and Biology journal homepage: www.elsevier.com/locate/jtemb
Analytical methodology
Operationally defined species characterization and bioaccessibility evaluation of cobalt, copper and selenium in Cape gooseberry (Physalis Peruviana L.) by SEC-ICP MS Justyna Wojcieszek, Lena Ruzik ∗ Chair of Analytical Chemistry, Warsaw University of Technology, Poland
a r t i c l e
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Article history: Received 21 August 2015 Received in revised form 28 October 2015 Accepted 1 December 2015 Keywords: Bioaccessibility Physalis Peruviana SEC-ICP MS Selenomethionine Vitamin B12
a b s t r a c t Physalis peruviana could attract great interest because of its nutritional and industrial properties. It is an excellent source of vitamins, minerals, essential fatty acids and carotenoids. Physalis Peruviana is also known to have a positive impact on human health. Unfortunately, still little is known about trace elements present in Physalis Peruviana and their forms available for the human body. Thus, the aim of this study was to estimate bioaccessibility and characterization of species of cobalt, copper and selenium in Physalis Peruviana fruits. Total and extractable contents of elements were determined by mass spectrometer with inductively coupled plasma (ICP MS). In order to separate the different types of metal complexes Physalis peruviana fruits were treated with the following solvents: Tris–HCl (pH 7.4), sodium dodecyl sulfate (SDS) (pH 7.4) and ammonium acetate (pH 5.5). The best efficiency of extraction of: cobalt was obtained for ammonium acetate (56%) and Tris–HCl (60%); for copper was obtained for SDS (66%), for selenium the best extraction efficiency was obtained after extraction with SDS (48%). To obtain information about bioaccessibility of investigated elements, enzymatic extraction based on in vitro simulation of gastric (pepsin) and intestinal (pancreatin) digestion was performed. For copper and selenium the simulation of gastric digestion leads to the extraction yield above 90%, while both steps of digestion method were necessary to obtain satisfactory extraction yield in the case of cobalt. Size exclusion chromatography (SEC) coupled to on-line ICP MS detection was used to investigate collected metal species. The main fraction of metal compounds was found in the 17 kDa region. Cobalt and copper create complexes mostly with compounds extracted by means of ammonium acetate and SDS, respectively. Cobalt, copper and selenium were found to be highly bioaccessible from Physalis Peruviana. Investigation of available standards of cobalt and selenium allows confirming the presence of vitamin B12 and probably selenomethionine in the fraction bioaccessible by human body (obtained during enzymatic extraction). It should be noted that the presence of small seleno-compounds in Cape gooseberry was performed for the first time. The results show that the combination of SEC and ICP MS could provide a simple method for separating of soluble element species. © 2015 Elsevier GmbH. All rights reserved.
1. Introduction In recent years berries are becoming more popular due to their nutritional value and health benefits [1,2]. Cape gooseberry (Physalis peruviana L.), also known as goldenberry is a promising exotic fruit that could be a subject of many novel foods. It belongs to
∗ Corresponding author at: Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland. E-mail address: [email protected] (L. Ruzik). http://dx.doi.org/10.1016/j.jtemb.2015.12.001 0946-672X/© 2015 Elsevier GmbH. All rights reserved.
the Solanaceae family and originated in the South America. Nowadays Colombia is the largest Cape gooseberry exporter in the world [3–5]. Physalis Peruviana forms a dome-shaped shrub that can grow to 1 m and the fruits with an approximate weight of 4–5 g are covered by a shiny yellow peel. The flowers, produced in winter, are yellow with purple blotches [5,6]. The pulp is nutritious, containing particularly high levels of carotenoids, minerals and vitamin C [7]. The golden berry is also an excellent source of the vitamin B-complex and essential fatty acids. The protein content is exceptionally high for a fruit [8,9]. Physalis Peruviana is becoming more and more popular, it has increased interest worldwide
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because of its nutritional composition and the presence of biologically active compounds that provide health benefits and reduce the risk of various diseases such as cancer, malaria, asthma, hepatitis, dermatitis and rheumatism [10–13]. Although a lot of available publications focus on C. gooseberry and its properties there is a lack of information about speciation analysis of metals present and their bioavailability by human organism. Carrying out further studies, Physalis Peruviana could become a fruit of particular interest to the world’s up-scale food industry. The aim of the study was to separate complexes of selected elements with different bioligands and characterization of species of chosen metal. Determination of total concentration of metals in food does not provide information about their bioavailability in the human organism. Knowledge about content of element in the bioaccessible fraction is necessary to evaluate bioavailability. The information on the bioaccessibility of important nutrients in food and food supplements seems to be essential. For that reason the second objective of the study was to estimate elements bioaccessibility in C. gooseberry by in vitro simulation of gastrointestinal digestion using two-step model with pepsin as a gastric juice and pancreatin as an intestinal juice. To the best of our knowledge this is the first attempt to carry out characterization of species of chosen metal and bioaccessibility evaluation of metals in C. gooseberry by SEC-ICP MS.
2. Experimental 2.1. Reagents and chemicals Sodium chloride, ammonium acetate, sodium dodecyl sulphate, Tris(hydroxymethyl) aminomethane and hydrochloric acid were purchased from Sigma–Aldrich and were of analytical reagent grade. Selenomethionine and vitamin B12 standards (assay ≥ 98%) were also purchased from Sigma–Aldrich. Pepsin from porcine gastric mucosa and pancreatin were of biological grade (Sigma-Aldrich, Buchs, Switzerland). Deionized water (18 M cm) prepared with a Milli-Q system (Millipore Elix 3, Millipore, Saint–Quentin, France) was used throughout. The SEC column was calibrated using size exclusion standard (BIO-RAD, Warsaw, Poland).
2.2. Instrumentation The screening for the metal complexes was carried out by means of size exclusion chromatography coupled to ICP MS. Prepared samples were analysed on a Superdex200 10/300GL (GE Healthcare Life Sciences) exclusion column with bed volume of 24 mL. Before the analysis the column was calibrated with a mixture of thyroglobulin (670 kDa), ␥-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), vitamin B12 (1,35 kDa). Chromatographic separations were performed using Agilent 1100 gradient HPLC pump (Agilent Technologies, Waldbronn, Germany) as the sample delivery system. Agilent 7500a ICP MS (Tokyo, Japan) was used as on-line HPLC detector. All connections were made with PEEK tubing (0.17 mm i.d.). Operational parameters are summarized in Table 1. The determination of concentration of elements in C. gooseberry samples (after mineralization, buffer and enzymatic extraction) was carried out by Agilent 7500a ICP MS as an element-specific detector. Ni/Cu-skimmer was installed in the interface, the position of torch and nebulizer gas flow was adjusted daily with special emphasis to decrease the level of CsO+ below 0.2% with the aim to minimize the risk of polyatomic interferences caused by oxides. The working conditions were optimized daily using a 10 g L−1 solution of 7 Li+ , 89 Y+ and 209 Bi+ in 2% HNO3 , with a dwell time of 0.1 ms for each isotope.
Table 1 Operational parameters for SEC and ICP MS. Settings SEC separation Pump Column Mobile phase Elution program Flow Injection volume Column temperature ICP-MS RF Power Plasma, auxiliary, nebulizer gas flow Cones Monitored isotopes Dwell time
Agilent 1100 Superdex 200 (10 × 300 mm × 10 m)-GE Healthcare Life Sciences 10 mM ammonium acetate buffer (pH 7.4) Isocratic 0.7 mL min−1 100 L 24 ◦ C Agilent 7500a 1350 W 15.0, 1.0 and 1.05 L min−1 Sampler–Pt, Skimmer–Ni 55 Mn, 59 Co, 63 Cu, 65 Cu, 66 Zn, 67 Zn, 68 Zn, 82 Se, 95 Mo 0.1 ms
A Bandelin Sonorex Model 1210 ultrasonic bath (Germany) and MPW Model 350R centrifuge (MPW Warsaw, Poland) were used for extraction procedures. 2.3. Sample preparation 2.3.1. Determination of total content of elements and buffer extraction method The dried C. gooseberry was obtained from Kenay (Poland, imported from Peru) and stored at 4 ◦ C until analysis. For determination of total amount of elements in Physalis Peruviana, samples (0.3 g of dry mass) were digested by microwaveassisted mineralization with a mixture of 5 mL of HNO3 and 3 mL of H2 O2 . After cooling down the digests were diluted with MQ water to the volume of 25 mL and then diluted before ICP MS analysis. Samples and standard solutions were prepared with addition of 89 Y as the internal standard. The external calibration curves were linear in the investigated range from 2 g L−1 to 150 g L−1 with r2 above 0.997. Samples of C. gooseberry (0.07 g) were extracted during 1 h in ultrasonic bath, using 1 mL of the following buffers: (1) 10 mM ammonium acetate (pH 5.5), (2) 30 mM Tris–HCl (pH 7.4), (3) 2% sodium dodecyl sulphate (SDS) in water (pH 7.4). After extraction, the obtained solutions were centrifuged for 20 min at 15 000 rpm at 15 ◦ C. The final supernatants were filtered with 0.45 m syringe filter (Sigma–Aldrich, Bellefonte, PA, USA), two first drops were discarded and only the remaining part of the filtrates was injected on the size exclusion column. 2.3.2. In vitro simulation of gastrointestinal digestion The in vitro digestion method was based on Luten et al. [14] modified to the studied berries. 2.5 mL of gastric juice (6% w/v pepsin in 0.15 M NaCl, acidified to pH 1.8 by means of HCl) was added to 0.07 g of C. gooseberry and then shaken and sonicated for 10 min in ultrasonic bath. In the next step the mixture was incubated in the thermostatic water bath for 3.5 h at 37 ◦ C. After incubation the mixture was centrifuged at 4 ◦ C for 20 min at 15 000 rpm. The supernatants were filtered through 0.45 m syringe filters (Sigma–Aldrich, Bellefonte, PA, USA) and analyzed. For intestinal digestion, NaHCO3 solution was added to the remaining part of sample to obtain the neutral pH. After that 2.5 mL of intestinal juice (1.5% w/v pancreatin in 0.15 M NaCl) was added and the mixture was incubated for 2 h at 37 ◦ C. Following centrifugation and filtration of supernatant, the gastrointestinal extract was analyzed by SEC-ICP MS. To collect the information about the bioaccessibility of important nutrients from Physalis Peruviana by human organism, the
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Fig. 1. The scheme of analytical procedure for buffer extraction and enzymatic digestion of cobalt, copper and selenium analysis.
efficiency of enzymatic extraction was estimated by establishing the amounts of elements in digestion extracts against total content of elements in mineralized samples. The procedure of samples preparation is presented on Fig. 1. 2.3.3. Preservation of element species Preparation of the sample is a primary concern in speciation analysis of elements. It is important to preserving the original species or chemical form of element and obtaining a high extraction efficiency from the sample matrix. Some selenium species are particularly susceptible to oxidation, which could lead to false identification of selenium species [15]. During the investigation, non-volatile species have been collected by commonly practiced solid–liquid extraction (leaching). During this procedure sonication assisted forms had been used to remove copper, cobalt and selenium compounds from the sample matrix. During enzymatic
extractions (with pepsin and pancreatin) pH and temperature were controlled and maintained in the optimum range of activity of enzymes used during simulation of digestion. During speciation analysis non-oxidizing acid was used. The deep-freezing of samples was used in order to minimize any bacterial or enzymatic degradation or loss from volatility. 3. Results and discussion 3.1. Total content of elements in Physalis Peruviana C. gooseberry is known as an important source of trace elements. Cobalt, copper and selenium were selected for monitoring during samples analysis. Despite great progress made in the analytical instrumentation, the speciation analysis of cobalt and copper in the food is largely limited. Those elements were chosen due to the
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fact that to the best of our knowledge there was no previous studies of their speciation in analyzed fruits. Additionally, all described elements belong to the group “challenging” for analysts but the speciation process can provide information vital for human health. The total concentration of selected elements was determined after microwave-assisted digestion of berries by means of ICP MS. The results were obtained from three independent experiments. As the interest of this study the following total amount of elements -was identified: cobalt (0.27 ± 0.09 g g−1 ), copper (7.3 ± 0.3 g g−1 ) and selenium (0.09 ± 0.03 g g−1 ). Alongside of those elements, total content of other microelements was determined: manganese (16.0 ± 0.9 g g−1 ), zinc (15.4 ± 0.4 g g−1 ) and molybdenum (0.26 ± 0.13 g g−1 ). The results are not in good agreement with the earlier studies reported by other authors but it should be pointed that to our knowledge, this is the first study when the total content of trace elements was determined in dried fruits of Physalis Peruviana, not in the pulp or juice. Concentrations of the same elements in buffer and enzymatic extracts were also determined in C. gooseberry in order to evaluate their dietary intake. The efficiency of extraction of buffer and digestion methods was estimated by comparison the amounts of elements in extracts against total amount of elements obtained by mineralization. The yields of extraction of investigated elements obtained by different extraction methods and enzymatic digestion are presented in the Table 2. In cobalt case, the best efficiency of extraction was obtained for ammonium acetate (56%) and Tris–HCl (60%), what indicate that cobalt is present mostly in the form of water soluble compounds. For copper, the presence of SDS significantly improves the efficiency of extraction (66%). It could indicate that copper creates the most complexes with hydrophobic proteins. For Tris–HCl and ammonium acetate extracts (respectively), the efficiency of copper extraction was exactly the same (45%). In selenium for instance, the results were obtained on the very low concentrations and with relative high RSD (about 15%). The buffer extraction leads to relatively low extraction efficiency. The best results were obtained after extraction with SDS (48%), what could indicate the presence of insoluble selenium species, such as selenoproteins. It should be pointed out that the yield of extracted metals was the same even when bigger excess of solvents was used in comparison to investigated samples of berries. Enzymatic method improved extraction efficiency of all the investigated elements. The simulation of gastric digestion leads to the extraction yield above 90%, for copper and selenium. Such results indicate that proteins could be the main ligands binding investigated elements in Physalis Peruviana . In cobalt investigation, both steps of digestion method were necessary to obtain satisfactory extraction yield. For that reason, polysaccharides can be proposed as compounds interacting with cobalt because pancreatin used during intestinal digestion improved efficiency of extracted metal in comparison to pepsin. During enzymatic digestion, the metal complexes with low molecular weight compounds were created with high efficiency. 3.2. SEC-ICP MS characteristics of ammonium acetate, Tris and SDS extracts containing metal species Coupling of size exclusion chromatography with inductively coupled plasma mass spectrometry allows the element specific detection of eluted compounds. The eluate from the column was fed directly into the ICP MS. Both buffer and enzymatic extracts of Physalis Peruviana were analysed. In cobalt case the chromatogram obtained for ammonium acetate extract consisted of two, poorly separated peaks (Fig. 2a). First after 25.8 min and second after 27 min of chromatographic process, what can correspond to fraction of cobalt complexes with organic acids or other small molecular weight compounds suspected to reveal ability to bind
cobalt. Chromatograms obtained for Tris–HCl and SDS extracts consisted of one, major peak at about 26.5 min which can correspond to cobalt complexes with molecular mass 17 ± 10 kDa. Additionally, one small peak was observed for SDS extract at 11 min corresponding to compounds with molecular mass ≥ 500 kDa. In C. gooseberry cobalt is complexed mostly by bioligands extracted by means of ammonium acetate. For the comparison the results obtained for cobalt and copper were chosen to show in this study. Copper is another important trace element which is necessary for human beings. Fig. 2b shows chromatograms of copper compounds extracted from C. gooseberry by different extraction media. The chromatograms obtained for copper after extraction with both Tris–HCl and SDS contained one major peak eluted at about 26.7 min, which correspond to compounds with relative molecular weight 17 ± 10 kDa. In the case of SDS extract two peaks were additionally observed in the chromatogram. The first, small peak was observed at 11 min and it was obtained at column’s exclusion limit corresponding to high molecular weight compounds ≥ 670 kDa. The presence of high molecular weight complexes can be explained by ability of metal ions to create agglomerates even with low molecular weight bioligands which was already reported [16]. The second, more intense peak was observed at about 34 min and it can correspond to low molecular weight complexes with molecular mass lower than 1.4 kDa. Because of much more higher intensity than peak at 11 min, it is possible that SDS solution is responsible for partial decomposition of agglomerates. In chromatogram obtained after extraction with ammonium acetate one peak was observed at 27.2 min, which correspond to compounds with relative molecular weight 17 ± 10 kDa. It should be pointed out that different retention time than for Tris–HCl and SDS solutions suggests extraction of copper complexes with another group of bioligands. In contrast to cobalt, copper forms complexes mostly with compounds extracted by means of sodium dodecyl sulphate, dedicated to hydrophobic proteins, which were suspected to reveal ability to bind investigated elements. Chromatograms of buffer extracts of selenium are not shown because they do not contain any peak. However, it should be stressed that a lack of signals in chromatograms of buffer extracts does not mean that selenium compounds do not exist in Physalis Peruviana. 3.3. SEC-ICP MS profiling of gastric and gastrointestinal extracts Gastric and gastrointestinal extracts of Physalis Peruviana were examined by SEC-ICP MS using the same elution conditions as for buffer extracts. The chromatograms obtained for cobalt consisted of two peaks in the case of gastric extract and three peaks for gastrointestinal extract (Fig. 3a), which can correspond to cobalt complexes with digestion products of proteins. It was already reported that cobalt ions have a high affinity for binding to proteins and amino acids [17]. The high molecular weight peak (tr = 19.5 min) can be observed due to cobalt binding by enzymatic proteins. Their ability to interact with cobalt species and improving bioaccessibility was already reported in the case of cobalamins [18]. The chromatograms obtained for copper after analysis both pepsin and pancreatin extracts were quite similar and consisted of two peaks in low molecular weight region (Fig. 3b). However, in relation to gastric extract the second peak was significantly lower. Both signals correspond probably to digestion products of proteins or other high molecular weight compounds present in Physalis Peruviana. These main peaks of gastric extract belong to the region with molecular mass between 44 and 17 kDa which can indicate on copper connections with enzymatic proteins or their self-digestion products. It should be mentioned that copper ions were earlier reported to be bound by pepsin even in the acidic media as well
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Table 2 The amounts of investigated elements in the buffer and enzymatic extracts of Physalis Peruviana.
30 mM Tris–HCl pH 7.4 2% of SDS in water pH 7.4 10 mM CH3 COONH4 pH 5.5 Gastric digestion Gastric and gastrointestinal digestion
Co g g−1 (%)
Cu g g−1 (%)
Se g g−1 (%)
0.16 ± 0.01 (60%) 0.13 ± 0.01 (50%) 0.15 ± 0.01 (56%) 0.17 ± 0.01 (64%) 0.17 ± 0.01 (63%)
3.3 ± 0.7 (45%) 4.8 ± 0.7 (66%) 3.3 ± 0.6 (45%) 6.7 ± 1.0 (92%) 2.1 ± 0.4 (29%)
0.035 ± 0.014 (39%) 0.043 ± 0.019 (48%) 0.032 ± 0.001 (36%) 0.086 ± 0.004 (90%) 0.028 ± 0.001 (30%)
Results represent an average amount established for 3 samples; each measured 3 times;% of total concentration of element.
Fig. 2. SEC-ICP MS chromatograms obtained for Tris–HCl (green dotted line), SDS (blue dots) and ammonium acetate (violet solid line) extracts of Physalis Peruviana for (a) cobalt, (b) copper. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. SEC-ICP MS chromatograms of gastric (black, solid line) and gastrointestinal (red, dotted line) of Physalis Peruviana for (a) cobalt, (b) copper. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
as iron or nickel [19]. After the end of the simulation of digestion process, the peak at tr = 27 min was observed and it corresponds to copper binding by low molecular weight compounds created during digestion. The peak obtained at tr = 31.5 min, present only for gastrointestinal extract could indicate the presence of copper complexes with digestion products of saccharides due to the action of amylase (a part of pancreatin), which is responsible for digestion of those compounds. No signals were observed in chromatograms for buffer extracts of selenium, but after digestion with both pepsin and pancreatin the peaks were noticeable (Fig. 4b). It could be evidence that the insoluble selenoproteins present in the sample are hydrolysed to form hydrophilic selenium species, such as selenoamino acids or inorganic species (selenate or selenite). The lower molecular weight selenium species tends to be dissolved into the aqueous phase. In the chromatograms of enzymatic extracts of C. gooseberry, two peaks are present in the case of gastric extract of selenium. After examination of gastrointestinal extract, only one peak with small intensity was observed in the chromatogram. On the basis of the column calibration, this region represents selenium species
with molecular weight less than 5 kDa, which can correspond to selenopeptides. 3.4. Operationally defined species characterization of cobalt and selenium Information about chemical status of trace elements in C. gooseberry is still very limited. It is believed that metal species created during digestion are accessible by human organism. In reference to this hypothesis, characterization of species of cobalt and selenium was performed in the present investigation by SEC-ICP MS in extracts obtained by simulation of gastrointestinal digestion. Analysis of available standards of cobalt (cyanocobalamin–vitamin B12 ) and selenium (selenomethionine) were carried out. Compounds were identified by matching retention times of peaks in enzymatic extracts with those of standard compounds. As for cobalt, it was able to confirm the presence of vitamin B12 in both gastric and gastrointestinal extracts of Physalis Peruviana (Fig. 4a). The peak at tr = 29.2 min occurs at the same retention time as standard of vitamin B12 . Knowledge about the bioavailability of vitamin B12 from various food sources is rather limited, despite its importance
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Fig. 4. SEC-ICP MS chromatograms of gastric (black, solid line) and gastrointestinal (red, dotted line) with standards used in this study (black, dotted lines) of Physalis Peruviana for (a) cobalt, (b) selenium. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
for human metabolism [20]. The presence of vitamin B12 in fraction bioaccessible for human body (obtained during simulation of gastrointestinal digestion) was confirmed in this study. The detection of low molecular weight fraction of selenium species in enzymatic extracts could be the proof of the presence of selenoproteins in the sample. For the speciation of selenium, the selenoproteins originally present in the berries sample are decomposed into hydrophilic selenopeptides during digestion process, which can be detected by SEC-ICP MS. After analysis of gastric extract the peak at tr = 28 min matches the retention time of selenomethionine standard (Fig. 4b), what could be the evidence on presence of this compound as the major organic compound of selenium found in enzymatic extracts of C. gooseberry. It could also indicate that in enzymatic extracts selenium is present in the form of small seleno-compounds. It could be also concluded that selenium is accessible in human organism in the form of chelates with amino acids [21]. It was reported in earlier studies that organic forms of selenium are better accessible by human organism than inorganic forms and that selenomethionine is the most bioavailable source of selenium [22–24]. It should be noted that the presence of small seleno-compounds (probably selenomethionine) in C. gooseberry was confirmed for the first time. 4. Conclusions In the present study, ICP MS and SEC-ICP MS method was used for the first time for the speciation studies of cobalt, copper and selenium in C. gooseberry (P. Peruviana L.). The combination of SEC and ICP MS allows fractionation of metal-containing species in berries according to their size and shape. Determination of total concentration of metals in food does not provide information about their bioavailability by human organism, bioavailability depends on their chemical forms, which are absorbed and metabolized differently. For that reason, the second aim of the study was to estimate bioaccessible elements in Physalis Peruviana by in vitro simulation of gastrointestinal digestion. Elution profiles of elements were different from each other and also differed depending on the extraction medium used in most of the cases. SEC demonstrates that metals are distributed mostly in medium and low molecular mass range between 44000 and 1000 Da. After simulation of gastrointestinal digestion, copper complexes with digestion products of proteins or with self-digestion products of enzymatic proteins were detected. The presence of vitamin B12 and small seleno-compounds in the enzymatic extracts was confirmed and compared with reference standards. Vitamin B12 is well-known to be the sole vitamin that is absent from plantderived food sources [25]. Physalis Peruviana is a natural plant product and it is suitable vitamin B12 source presently available for vegetarians. High molecular weight selenium species, such as selenoproteins, are hydrolyzed to lower molecular and soluble species (selenopeptides) during gastric and gastrointestinal diges-
tion. Selenomethionine is probably the major organic compound of selenium found in enzymatic extracts of C. gooseberry. It could be concluded that vitamin B12 and selenomethionine are the forms of elements available for human organism. The method described in this study may be used to study the metal species in other plants. Future investigations with using more advanced mass spectrometry techniques (such as ESI QqQ MS/MS or ESI QTOF MS) will be needed to provide a more detailed characterisation and identification of the investigated elements species. Conflict of interest The authors declare no conflict of interest. Acknowledgement Authors are thankful to Warsaw University of Technology for financial support of presented study. References [1] N.P. Seeram, Berries and human health research highlights from the fifth biennial berry health benefits symposium, J. Agric. Food Chem. 62 (2014) 3839–38341. [2] L. Chen, X. Xin, Q. Yuan, D. Su, W. Liu, Phytochemical properties and antioxidant capacities of various colored berries, J. Sci. Food Agric. 94 (2014) 180–188. [3] K. Bravo, S. Sepulveda-Ortega, O. Lara-Guzman, A.A. Navas-Arboleda, E. Osorio, Influence of cultivar and ripening time on bioactive compounds and antioxidant properties in Cape gooseberry (Physalis Peruviana L.), J. Sci. Food Agric. 95 (2015) 1562–1569. [4] M. Yilmaztekin, Characterization of potent aroma compounds of Cape gooseberry (Physalis Peruviana L.) fruits grown in Antalya through the determination of odor activities values, Int. J. Food Prop. 17 (2014) 469–480. [5] M.F. Ramadan, A.H. Nesma, R.M. Elsanhoty, M.Z. Sitohy, Goldenberry (Physalis Peruviana L.) juice Rich in health-beneficial compounds suppresses high-cholesterol diet-Induced hypercholesterolemia in rats, J. Food Biochem (37 2013) 708–722. [6] M.F. Ramadan, Bioactive phytochemicals, nutritional value and functional properties of Cape gooseberry (Physalis Peruviana): an overview, Food Res. Int. 44 (2011) 1830–1836. [7] M.F. Ramadan, J.T. Moersel, Impact of enzymatic treatment on chemical composition, physicochemical properties and radical scavenging activity of goldenberry (Physalis Peruviana L.) juice, J. Sci. Food Agric. 87 (2007) 452–460. [8] M.F.R. Hassanien, Physalis Peruviana: a rich source of bioactive phytochemicals for functional foods and pharmaceuticals (review), Food Rev. Int. 27 (2011) 259–273. [9] H. Popenoe, S.R. King, J. Leon, L.S. Kalinowski, Goldenberry (Cape gooseberry), in: N.R. Council (Ed.), Lost Crops of the Incas: Little-known Plants of the Andes With Promise for Worldwide Cultivation, National Academy Press, Washington DC, 1990, pp. 241–252. [10] N. Izli, G. Yıldız, H. Ünal, E. Is¸ık, V. Uylas¸er, Effect of different drying characteristics, colour, total phenolic content and antioxidant capacity of Goldenberry (Physalis Peruviana L.), Int. J. Food Sci. Technol. 49 (2014) 9–17. [11] M.R. Salazar, J.W. Jones, B. Chaves, A. Cooman, A model for the potential production and dry matter distribution of Cape gooseberry (Physalis Peruviana L.), Sci. Hortic.-Amsterdam 115 (2008) 142–148. ˜ [12] L.A. Puente, C.A. Pinto-Munoz, E.S. Castro, M. Cortés, Physalis Peruviana Linnaeus, the multiple properties of a highly functional fruit: a review, Food Res. Int. 44 (2011) 1733–1740.
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