Journal of Applied Botany and Food Quality 89, 264 - 269 (2016), DOI:10.5073/JABFQ.2016.089.034 1Department of Chemistry of Natural Compounds, Faculty of Food and Biochemical Technology, University of Chemistry and Technology Prague, Prague, Czech Republic 2 Forensic Laboratory of Biologically Active Substances, University of Chemistry and Technology Prague, Prague, Czech Republic 3 Department of Quality of Agricultural Products, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Prague, Czech Republic 4Department of Soil Science and Soil Protection, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Prague, Czech Republic 5Department of Food Analysis and Nutrition, Faculty of Food and Biochemical Technology, University of Chemistry and Technology Prague, Prague, Czech Republic 6School of Agriculture and Food Technology, Faculty of Business and Economy, The University of the South Pacific, Independent State of Samoa 7Department of Nutritive Substances, Food Research Institute Prague, Prague, Czech Republic 8 Department of Crop Sciences and Agroforestry, Faculty of Tropical AgriSciences, Czech University of Life Sciences Prague, Prague, Czech Republic Fatty acids, minerals, phenolics and vitamins in the seeds of Inocarpus fagifer, a Pacific Island underutilized legume Lukas Huml1, Petra Miksatkova1,2, Pavel Novy3, Ondrej Drabek4, Monika Sabolova5, Mohammed Umar6, Vaclav Tejnecky4, Barbora Pohorela5, Lenka Kourimska3, Eva Maskova7, Anabella Tulin6, Oldrich Lapcik1, Ladislav Kokoska8,* (Received August 16, 2016) * Corresponding author Summary Recently, Pacific nations have faced to alarming increase in pre- valence of noncommunicable diseases connected with consumption of non-traditional processed food. It is believed that re-introduction of native diet may mitigate these negative trends. One of the tradi- tional staple food of Pacific region are seeds of underutilized legu- minous tree Inocarpus fagifer. Nevertheless, information on their chemical composition and nutritional properties are missing. There- fore we decided to analyze this crop for the presence of fatty acids, minerals, phenolics and vitamins. Performed analyses revealed a slightly predominating portion of unsaturated (e.g. 18:2 n-6; 18:1 n-9 and 18:3α n-3) over saturated (e.g. C18 and C16) fatty acids. Consi- dering minerals, the substantial concentrations of copper, magne- sium, manganese, potassium and zinc (19.32; 1823.21; 8.44; 23308.41 and 77.99 mg kg-1 of dry matter respectively) were recorded. Ferulic and coumaric acids were the most abundant phenolics (3.23 and 1.48 mg kg-1 of dry matter respectively), whereas flavonoids, iso- flavonoids and coumestrol were also present. Regarding vitamins, niacin and riboflavin were found in respective concentrations 131.80 and 4.47 mg kg-1 of dry matter. Our findings suggest I. fagifer seeds as a prospective food source of several health-beneficial constituents which might contribute to the well-being of Pacific islanders. Introduction Globalization of processed, carbohydrate rich diets with lack of micronutrients leads to a higher vulnerability to overweight and non-communicable diseases (NCDs) not only in the developed world but also in low- and middle- income countries where almost three quarters of the world‘s NCD deaths currently occur. Despite the remote location, Pacific islanders have also been facing severe changes in dietary habits, whereas the current prevalence of raised blood glucose and obesity is dramatically increasing in certain re- gions. For example in Samoa, the levels of both above mentioned factors are more than two-fold higher than the global average (Cur- tis, 2014; WHO, 2014; WHO, 2002; Win Tin et al., 2015). Since many underutilized crops are being recognized for their dietetic value due to the presence of specific health-beneficial constituents, their re-introduction to the diet has a potential to reverse the nega- tive impact of recently occurring trends in the nutrition and health of the local populations (Ebert, 2014; Gibson et al., 2015; Thies, 2000). Nevertheless, before any recommendations can be made on consumption of novel plant foods, it is necessary to assess their di- etetic and toxicological properties by detailed analysis (Nasi et al., 2009). Edible seeds of leguminous plants are considered as a valuable source of nutrition and functional compounds. Besides the basic nutritional proteins and oligosaccharides they also provide other es- sential factors such as unsaturated omega-3 and omega-6 fatty acids, minerals and vitamins, which play an irreplaceable role in the hu- man metabolism and health (Combs, 2001; Schieber et al., 2001; Yildiz, 2010). Characteristic secondary metabolites of legumes are isoflavonoids (Veitch, 2013), which exert various biological effects. Some of them possess an ability to bind to the estrogen receptor (Andres et al., 2015), while it might lead to e.g. relief of menopau- sal symptoms, inhibition of intestinal glucose-uptake (Vedavanam et al., 1999) or prevention of steroid hormone-dependent cancers (Perabo et al., 2008). Other biologically active phenolic deriva- tives found in leguminous plants are flavonoids and phenolic acids (Dewick, 2009). Their consumption is connected with a lower risk of oxidative-stress related diseases due to their antioxidant potential (Gordon, 1996). Inocarpus fagifer (Parkinson) Fosberg, known as Tahitian or Poly- nesian chestnut, is a medium-sized tree belonging to the Legumi- nosae family (Lewis et al., 2005) and naturalized in the lowland tropical forests of Melanesia, Micronesia and Polynesia. Its inde- hiscent pod contains a kidney-shaped seed with an edible kernel which when prepared raw or cooked is considered to be among the most important cash crops and culinary nut species of the Pacific (Lim, 2012), however, as a food, it is practically unknown in the global context. In Samoa, this multipurpose tree called “ifi” has played an important role in the livelihood of local inhabitants and even nowadays its seed is considered a staple food. It has been also consumed in other Pacific regions, where it is traditionally prepared in many different ways, including grilling, roasting, baking, boiling or mashed in pudding. Moreover, significance of this tree also relies Pioneering study on nutritional components of Inocarpus fagifer 265 on the medicinal use of various tree parts and its timber is suitable for light constructions, tool handles, furniture and canoes manu- facturing (Pauku, 2006; Whistler, 2002). For above mentioned reasons, I. fagifer was designated as one of the priority crops for the Pacific region (APAARI, 2011). Despite the well recorded traditional food use of I. fagifer in many Pacific island nations, the plant has received little attention from research and extension services and the data on chemical composi- tion and nutritional properties of this species are totally missing. Therefore we decided to determine the presence of certain basic nu- tritional components of its seeds collected in Samoa. Materials and methods Plant material The fruits of Inocarpus fagifer were collected from a wild popula- tion of trees (13°30.743´ S, 172°47.828´ W) growing in the coastal area near to the village of Falealupo (Vaisigano district) located at Savai’i island of the Independent State of Samoa in August 2014. During randomized collection, 30 fruits were gathered from three different trees (ten fruits from each tree). The plant was authenticated by Prof. Ladislav Kokoska and voucher specimen (No. 03015KBFR) is deposited in the herbarium of the Department of Botany and Plant Physiology of the Faculty of Agrobiology, Food and Natural Re- sources of the Czech University of Life Sciences Prague (Czech Republic). Sample preparation and dry matter determination A fleshy mesocarp and testa, which are inedible for humans, were removed and hulled seeds were lyophilized in the Free-Zone 1 freeze dry system (Labconco, Kansas city, MO, USA). Immediately after lyophilization the seeds were pulverized using an IKA A11 analy- tical mill (IKA Werke GmbH & Co. KG, Staufen, Germany). Homogenized material was stored under -20 °C until analysis. The residual moisture of lyophilized samples was determined gravi- metrically at 130 °C for 60 min by the Scaltec SMO 01 analyzer (Scaltec Instruments, Gottingen, Germany) according to the Official Methods of Analysis of AOAC International (2012). Chemicals and reagents Bacto Niacin Assay Medium was purchased from Becton, Dickson and Company (Franklin Lakes, NJ, USA), coumestrol, daidzein, daidzin, 7,4-dimethoxyisoflavone, formononetin, genistein, genistin, glycitein, glycitin, kaempferol, liquiritigenin, luteolin, naringenin, naringenin-7-O-glucoside, prunetin, rutin, sissotrin from Indofine (Hillsborough, NJ, USA), chloroform, nitric acid, sodium carbo- nate, sodium hydroxide, sodium sulfate from Lach-Ner (Nerato- vice, Czech Republic), ethanol, Folin-Ciocalteu reagent, formic acid, gallic acid, methanol from Merck (Darmstadt, Germany), acetic acid, dichlormethan, hydrogen peroxide, n-hexane, sodium acetate, sulfuric acid, potassium permanganate from Penta (Prague, Czech Republic), tectoridin from PhytoLab (Vestenbergsgreut, Germany), apigenin-7-O-glucoside, boron trifluoride-methanol solution, bromo- thymol blue indicator, caffeic acid, p-coumaric acid, (-)-epicatechin, ferulic acid, gallic acid, isoquercitrin, nicotinic acid, salicylic acid, sinapic acid and takadiastase (EC 3.2.1.1) from Aspergillus oryzae from Sigma-Aldrich (Saint Luis, MO, USA), standard 37 component FAME mixture was obtained from Supelco (Bellefonte, PA, USA). Minerals analysis Determination of minerals was carried out after acid digestion of samples performed according to the method previously described by vanek et al. (2010). Initially, a 0.5 g of sample was kept overnight at laboratory temperature in a closed, but not sealed, Teflon vessel (Savillex, Eden Prairie, MN, USA) with 10 mL of 65 % nitric acid. Then the Teflon vessel was sealed and the mixture was heated at 120 °C on a hot plate for 2 hours. The digested solution was then quantitatively transferred to the 50 mL volumetric flask and filled up to the mark with deionized water. The solution was filtered through 0.45 μm Nylon disk filters (Cronus, Gloucester, United Kingdom) prior to analysis. An inductively coupled plasma optical emission spectrometer DUO iCap 7000 (Thermo Scientific, Waltham, MA, USA) operating at 61.15 kW with respective nebulizer and auxiliary gas flow rates of 0.5 and 1 L/min was used for contents determination of selected elements, which were monitored at the following spectral lines (wavelength in nm): boron (B, 208.959), calcium (Ca, 317.933), copper (Cu, 224.700), iron (Fe, 238.204), magnesium (Mg, 279.079), manganese (Mn, 257.610), phosphorus (P, 177.495), potassium (K, 766.490), sodium (Na, 589.592), sulfur (S, 180.731), and zinc (Zn, 202.548). Quality of digestion and analysis were controlled using blanks and the standard reference materials (NIST SRM 1575a Pine Needles and NCS DC 73351 Tea). Fatty acids analysis The oil was obtained from the sample (about 3 g) by multiple extrac- tion with 180 mL n-hexane:dichlormethan 80:20 v/v in a Soxhlet apparatus for 6 h. The fat was recovered under vacuum and dried over anhydrous sodium sulfate and transferred to a capped bottle and stored at 4 °C until used for analysis. Derivatization of fatty acids was based on the base-catalyzed re- action using sodium hydroxide-methanol as a reagent with boron trifluoride as a catalyst. Fatty acid methyl esters (FAMEs) were then extracted to hexane. FAMEs were analyzed by gas-liquid chroma- tography using a SP-2560 fused silica capillary column (100 m × 0.25 mm i.d., 0.2 μm film thickness) from Supelco (Bellefonte, PA, USA) in Agilent 6890 gas chromatograph (Agilent, Palo Alto, CA, USA) equipped with a flame ionization detector. The oven tempera- ture was 175 °C for 30 min, then it was increased by 1 °C min-1 to 210 °C and this temperature was maintained for 140 min. Detec- tor and injection port temperatures were 220 °C and the nitrogen carrier gas flow was 1 mL min-1. For the identification of FAMEs, standard FAME mixtures were analyzed. To confirm the identi- fication of some FAMEs, GC/MS analysis was carried out in the GC/MSD system Agilent 5975 (Agilent, Palo Alto, CA) with the same column and temperature conditions as above, except for the helium flow, which was 0.6 mL/min and the detector temperature was 250 °C. GC-FID chromatogram of fatty acids composition in I. fagifer seed extract is available in supplementary information (Fig. S1). UHPLC-MS/MS analysis of phenolic compounds Prior to analysis, the plant material (2 g) was extracted in a Soxhlet- like fex IKA 50 extractor (IKA Werke GmbH & Co. KG, Staufen, Germany) in 70 % ethanol in 1/20 (w/v) proportion during three 7-min cycles at 130 °C followed by cooling to 50 °C. The extracts were evaporated on a rotary evaporator, dissolved in 5 mL of 40 % methanol and filtered through a Teflon (PTFE) syringe filter (0.45 μm) (Labicom, Olomouc, Czech Republic). Standard stock solutions of coumestrol, daidzein, daidzin, 7,4-dimethoxyisoflavone, formononetin, genistein, genistin, glycitein, glycitin, kaempferol, liquiritigenin, luteolin, naringenin, naringenin-7-O-glucoside, pru- netin, rutin, sissotrin, tectoridin, apigenin-7-O-glucoside, caffeic acid, p-coumaric acid, (-)-epicatechin, ferulic acid, gallic acid, iso- quercitrin, salicylic acid and sinapic acid were prepared in methanol at a concentration of 1 mg mL-1 and were subsequently diluted in 40 % (v/v) methanol-water solution at concentrations ranging from 0.1 to 1000 ng mL-1. 266 L. Huml, P. Miksatkova, P. Novy, O. Drabek, M. Sabolova, M. Umar, V. Tejnecky, B. Pohorela, L. Kourimska, E. Maskova, A. Tulin, O. Lapcik, L. Kokoska UHPLC-MS/MS analysis of polyphenolic compounds was carried out using an Agilent 1290 Infinity instrument (Agilent, Santa Clara, CA, USA) equipped with a binary pump (G4220B), an autosampler (G4226A), an autosampler thermostat (G1330B) and a column com- partment thermostat (G1316C), coupled to an Agilent triple quadru- pole mass spectrometer (6460A) with a Jet Stream ESI ion source. A Kinetex PFP column (2.6 μm, 100A, 150 × 3.00 mm) from Phe- nomenex (Torrance, CA, USA) was used for the chromatographic separation of the extracts. Column temperature was set at 35 °C and the injection volume was 3 μL. 10 mM formic acid (eluent A) and methanol (eluent B) were used as mobile phases for a gradient elu- tion. The linear gradient solvent system started in 40 % of eluent B and increased to 100 % of eluent B in 10 min. This was maintained for 4 min and in 1 min the conditions returned to the initial ratio, which was maintained for a further 4 min. The flow rate was set at 0.3 mL min-1. The MS/MS apparatus was operating in positive and negative mode in the same analysis. The applied conditions of Jet Stream Ion Source were: drying gas temperature 290 °C; drying gas flow 4 L min-1; sheath gas temperature 380 °C; sheath gas flow 10 L min-1; nebulizer pressure 35 psi; capillary voltage was set at 3500 and 5000 V in positive and negative acquisitions, respectively. Multiple reaction monitoring (MRM) mode was used for the de- tection. Except for p-coumaric acid, the two most abundant transi- tions per analyte were used. The transitions and specific MS/MS parameters used for each compound are available in supplementary information (Tab. S1). An Agilent Mass Hunter (Agilent, Santa Clara, CA, USA) was used for data acquisition and quantification of samples. Extracted ion chromatograms of phenolic compounds detected in I. fagifer seed extract in MRM mode can be found also in supplementary information (Fig. S2). Total phenolic content Total phenolic content was measured using slightly modified me- thod previously developed by singleton et al. (1998). Firstly, each sample in volume of 100 μL was added to a 96-well microtiter plate. Thereafter, 25 μL of pure Folin-Ciocalteu reagent was added. The plate was inserted in an orbital shaker at 400 rpm for 5 min. Reac- tion was started by adding 75 μL of 12 % sodium carbonate. The mixture was kept in dark at 37 °C for 1 h. Absorbance was measured at 760 nm by Infinite M200 PRO (Tecan, Grödig, Austria). Results were expressed as gallic acid equivalents. Vitamin B2 and B3 content The content of vitamin B2 (riboflavin) was evaluated by fluorimetric method after its conversion to lumiflavin according to the Czech technical standard method CSN 560054 (1972). The riboflavin was extracted after hydrolysis with 0.05M sulfuric acid at 100 °C for 30 min followed by dephosphorylation using an enzymatic treat- ment (Takadiastase from Aspergillus oryzae) at 37 °C for 16 h after pH adjustment to 4.5 by 2.5M sodium acetate solution. The sample was filtrated and the aliquot of the filtrate was shaken with chloro- form. Concentrated sodium hydroxide solution (7M) was added to the clear aqueous layer; the solution was then illuminated with a 40 W fluorescent tube and riboflavin in the sample converted to lu- miflavin. The solution was acidified with a few drops of acetic acid and interfering substances of reaction were inactivated by oxidation with 4 % potassium permanganate solution. After 1 minute of expo- sure the decolorization of the solution by 3 % of hydrogen peroxide was performed. Lumiflavin was then extracted by chloroform and measured fluorimetrically (excitation 435 nm, emission 525 nm) at Spekol 11 (Carl Zeiss, Oberkochen, Germany). The microbiological determinations of vitamins B3 (niacin) was performed according to the Czech technical standards CSN 56 0051 (1987). The sample was hydrolyzed with 0.5M sulfuric acid at 121 °C for 60 min and then cooled to room temperature. The pH of the sample was adjusted to 4.5, filtered and diluted to a concentra- tion suitable for the assay calibration range. For determination of vitamin B3, pH was adjusted by 1M sodium hydroxide solution to 6.8. The diluted filtrate, appropriate medium (Bacto Niacin Assay Medium), standard solution of nicotinic acid and deionized water were pipetted into assay tubes. Tubes were sterilized by autoclaving (116 °C, 15 min) and after cooling to room temperature inoculated with Lactobacillus plantarum ATCC 8014 (The Czech National Collection of Type Cultures, The National Institute of Public Health, Prague, Czech Republic). After 72 hours of incubation at 30 °C, the content of B3 was determined by titration with 0.1M sodium hy- droxide and bromothymol blue indicator. The growth response of the tested microorganism was compared quantitatively to that of known standard solutions. Statistical analysis and calculations All analytical experiments were performed in triplicate and results are expressed as mean and standard deviation. For comparison of our results with contents of minerals and vitamins reported by the USDA (2014), the conversion of nutrient values (NV) from fresh weight (FW) to dry matter (DM) basis was performed according to the formula NV in g kg-1 of DM = (100 × NV in g kg-1 of FW)/(100 – water content in g kg-1 of FW) as recommended in FAO/INFOODS guidelines (2012). Results and discussion A complex set of analysis has revealed substantial contents of several minerals together with the presence of various fatty acids, phenolic compounds and B vitamins. The complete results are shown in the Tab. 1-3. The determined oil content of I. fagifer seed (2.2 ± 0.2 % w/w) cor- relates with oil content of other legumes such as peas, lentils and beans (USDA, 2015). Within the oil fraction, overall 14 unsaturated fatty acids (UFAs) and 21 saturated fatty acids (SFAs) were detected while the quantity of each compound was expressed as a relative percentage (Tab. 1). The most abundant UFAs determined in our study were linoleic (18:2 n-6; represented by 26.72 %), followed by oleic (18:1 n-9; 18.49 %); α-linolenic (18:3α n-3; 4.42 %) and vac- cenic acid (18:1 n-7; 1.27 %). The rest of UFAs were represented by less than 1 %. Among SFAs, palmitic (C16; 22.73 %), stearic (C18; 7.35 %), myristic (C14; 4.41 %) and lignoceric (C24; 4.20 %) acids were the most predominant. Behenic (C22), arachidonic (C20) and cerotic acid (C26) were detected in respective amounts of 1.70, 1.33 and 1.17 %. Remaining SFAs were found in lower concentration than 1 %. The complete results of fatty acids (FAs) analysis showed that UFAs were slightly predominating (53.57 %) over SFAs (46.22 %). The overall profile also demonstrated a greater portion of poly- unsaturated FAs (31.14 %) over monounsaturated FAs (22.06 %). Recent nutritional studies are focusing on the omega-6/omega-3 polyunsaturated FAs proportion as one of the keystones to prevent health complications caused by an unhealthy diet. It is proposed that their high ratio (1:15-25) occurring in the western diet is connected with the development of NCDs (Simopoulos, 2012). In the case of I. fagifer seed, the results showed a relatively desirable proportion of omega-6/omega-3 FAs (1:6.05) whereas this ratio is comparable to those of other legumes such as soybean (Rusnikova et al., 2013). These findings may be interpreted as indicating the potential of this traditional crop to be an alternative to current diets with an undesir- able UFAs ratio. The results of mineral elements analysis (Tab. 2) showed that cal- cium (Ca), potassium (K), magnesium (Mg), phosphorus (P) and Pioneering study on nutritional components of Inocarpus fagifer 267 Tab. 2: Minerals and vitamins content of Inocarpus fagifer seed Minerals Content (mg kg-1 dry matter) SD a boron 37.77 19.45 calcium 1536.39 151.27 copper 19.32 10.11 iron 44.84 42.30 magnesium 1823.21 86.32 manganese 8.44 0.69 phosphorus 3904.02 34.48 potassium 23308.41 570.80 sodium 348.35 98.01 sulfur 2179.05 41.31 zinc 77.99 7.01 Vitamins niacin 131.80 2.67 riboflavin 4.47 1.62 a standard deviation (n=3) sulfur (S) were the most abundant entities detected in the I. fagi- fer seeds; while boron (B), copper (Cu), iron (Fe), manganese (Mn), sodium (Na) and zinc (Zn) were found in lower concentrations. K was present in the highest amount of 23.31 g kg-1, exceeding those of soybean and peanut (19.65 and 7.54 g kg-1 respectively) (USDA, 2015). According to the minimum recommended dietary allow- ances (RDA; 1.6-2 g day-1 per healthy adult) this study indicates I. fagifer as a valuable dietary source of this element (NRC, 1989a). Considering the importance of K in the human diet, this finding could boost its availability and intake for people from Pacific island nations. Relatively high Na/K ratio (1:67) with low Na concentration (348.35 mg kg-1) also suggests a possible beneficial effect of seed consumption in persons suffering from hypertension (NRC, 1989b; Perez and Chang, 2014). Taking into the account the RDA of other micronutrients, the notable concentrations of Cu (19.32 mg kg-1), Mg (1823.21 mg kg-1), Mn (8.44 mg kg-1) and Zn (77.99 mg kg-1) were detected. These results indicate that 100 g of the dry matter edible portion of I. fagifer seed contains approximately half of the RDA of mentioned micro-elements (Cu = 1.5-3 mg day-1; Mg = 200- 350 mg day-1; Mn = 2-5 mg day-1; Zn = 11-15 mg day-1 per healthy adult) (NRC, 1989a) whereas analyzed material has higher content of Zn than soybean (53.47 mg kg-1) (USDA, 2015). In order to evaluate the presence of phenolic compounds in I. fagi- fer seed, the total phenolic content (TPC) was determined (63.75 ± 4.62 mg of gallic acid equivalents kg-1 of dry matter). Furthermore, 27 phenolic entities with a total sum 6.81 mg kg-1 were detected by detailed analysis (Tab. 3). The most abundant phenolic compounds found in the seed of I. fagifer were hydroxycinnamic acids, namely ferulic and coumaric, in respective amounts of 3.23 and 1.48 mg kg-1 while other phenolic acids were determined in much lower concen- trations. The analysis also revealed the presence of isoflavonoids. From this class of compounds, the most abundant were glycitein, its glycoside glycitin and formononetin (568.64; 71.28 and 179.73 μg kg-1 respectively). Daidzein and genistein were identified along with their glycosides daidzin and genistin in concentrations ranging from 2.46 to 14.48 μg kg-1. It is known that quantities of isoflavonoids may vary in underutilized legumes significantly (Leuner et al., 2013). Although the amounts of isoflavonoids detected in I. fagifer seeds are rather low, the presence of this metabolic pathway within this species is in agreement with authors studying other members of the Dalbergieae tribe (Hegnauer and Hegnauer, 2001). Coumestrol was the only coumestan found (530.06 μg kg-1). Among flavonoids, the highest concentration was recorded in the case of liquiritigenin and rutin (152.92 and 123.68 μg kg-1respectively). The determined TPC of I. fagifer seed was by several orders of magnitude lower in the comparison with other pulses e.g. soybean and peanut (Djordjevic et al., 2011). Although it is obvious that Tab. 1: Fatty acids composition of Inocarpus fagifer seed oil Fatty acid shorthand designation Content (%) SDa Saturated fatty acids (SFA) C 6:0 0.15 0.02 C 8:0 0.15 0.02 C 10:0 0.10 0.02 C 12:0 0.91 0.07 C 14:0 4.41 0.27 C 15:0 0.22 0.02 C 16:0 22.73 0.57 C 17:0 0.23 0.02 C 18:0 7.35 0.26 C 19:0 0.16 0.02 C 20:0 1.33 0.11 C 21:0 0.07 0.02 C 22:0 1.70 0.14 C 23:0 0.49 0.06 C 24:0 4.20 0.38 C 25:0 0.43 0.06 C 26:0 1.17 0.12 C 27:0 0.09 0.02 C 28:0 0.20 0.04 C 29:0 0.05 0.02 C 30:0 0.08 0.02 Total SFA 43.22 Unsaturated acid (UFA) C14:1 (cis 9) tr.b C16:1 (cis 7) 0.29 0.03 C16:1 (cis 9) 0.88 0.07 C16:1 (cis 11) 0.60 0.05 C17:1 (cis 9) 0.03 n.a.c C18:1 (trans isomers) tr. C18:1 (cis 9) 18.49 0.46 C18:1 (cis 11) 1.27 0.10 C18:1 (cis isomers) tr. C18:2 (cis trans isomers) 0.19 n.a. C18:2 (cis 9, cis 12, n-6) 26.72 0.67 C18:3 (cis trans isomers) 0.18 n.a. C18:3 (cis 9, cis12, cis15, n-3) 4.42 0.27 C20:1 (cis 11) 0.50 0.05 Total UFA 53.57 astandard deviation (n=3), btraces (below 0.01 %), cnot applicable 268 L. Huml, P. Miksatkova, P. Novy, O. Drabek, M. Sabolova, M. Umar, V. Tejnecky, B. Pohorela, L. Kourimska, E. Maskova, A. Tulin, O. Lapcik, L. Kokoska this crop represents a rather weak source of phenolics, as long as it serves as a staple food in certain regions, it would appear that the cumulative amount consumed on a daily bases might contribute to the prevention of oxidative stress-related diseases. Because the de- termined value of TPC and the sum of quantified phenolics do not match, it seems that a considerable amount of other phenolic entities might be present within the examined material. Possible candidates include condensed tannins which are found in numerous legumes (Coda et al., 2015; Curiel et al., 2015). Considering the vitamins content (Tab. 2), representatives of group B, niacin and riboflavin were found in respective concentrations of 131.80 and 4.47 mg kg-1. Niacin was present in the seed of I. fagifer in a higher concentration than in soybean or peanut (USDA, 2015), while a 100 g dry matter edible portion along with sufficient intake of tryptophan (200 mg) should cover daily recommended minimum consumption (NRC, 1989a). Administration of niacin increases adi- ponectin secretion and may therefore contribute to the reduction of obesity via lipid-lowering effect (Westphal et al., 2010). In the case of riboflavin, a 100 g dry matter edible portion represents ca. 1/3 of re-commended minimum intake (1.2 mg day-1 per healthy adult) (NRC, 1989a). Conclusion This study, to our best knowledge, is a pioneering investigation on the chemical composition of I. fagifer seed highlighting the pres- ence of minerals, fatty acids, phenolics and B vitamins. Of great significance is the presence of micronutrients such as Cu, Mg, Mn and Zn in substantial amounts. These micronutrients together with K are essential to the human metabolism. Appropriate to the human diet seem to be also concentrations of niacin and riboflavin together with determined ratios of sodium/potassium and omega-6/omega-3 UFAs, indicating its potential as a valuable dietary source. Consid- ering the obtained results, it seems that this crop possesses several health-beneficial attributes and therefore might potentially play a positive role in the lowering of prevalence of obesity and NCDs mainly in Pacific island nations where it is traditionally consumed as a staple food. However, further studies on chemical composition, biological activities and toxicology of this underutilized crop which has received little attention from researches in the past, are neces- sary to verify its potential as a healthy food. Acknowledgements This research was supported by the Internal Grant Agency of the Czech University of Life Sciences Prague (project No. IGA 20155012) and by Specific University Research Financial Support (MSMT No 20/2015). References Andres, S., Hansen, U., Niemann, B., Palavinskas, R., Lampen, A., 2015: Determination of the isoflavone composition and estrogenic activity of commercial dietary supplements based on soy or red clover. Food Funct. 6, 2017-2025. AOAC (Association of Official Analytical Chemists) INTERNATIONAL, 2012: Official Methods of Analysis of AOAC INTERNATIONAL, Official Method 925.10 (19th Ed.), Locator number: 31.1.03. Gaithersburg, MD, USA: AOAC INTERNATIONAL. APAARI (Asia-Pacific Association of Agricultural Research Institutions), 2011: Strengthening of plant genetic resources for food and agriculture: Conservation and utilization in the Pacific, 2-52. Bangkok, Thailand: Asia-Pacific Association of Agricultural Research Institutions. Coda, R., Melama, L., Rizzello, C.G., Curiel, J.A., Sibakov, J., Holo- painen, U., Pulkkinen, M., Sozer, N., 2015: Effect of air classification and fermentation by Lactobacillus plantarum VTT E-133328 on faba bean (Vicia faba L.) flour nutritional properties. Int. J. Food Microbiol. 193, 34-42. Combs, G.F. Jr., 2012: The Vitamins (4th ed.), 140-269. San Diego, USA: Academic Press. CSN 56 0051 (Czech Technical Standard), 1987: Determination of niacin (nicotinic acid). Prague, CZ: Czech Standards Institute. CSN 56 0054 (Czech Technical Standard), 1972: Determination of ribo- flavin (vitamin B2). Prague, CZ: Czech Standards Institute. Curiel, J.A., Coda, R., Centomani, I., Summo, C., Gobbetti, M., Riz- zello, C.G., 2015: Exploitation of the nutritional and functional cha- racteristics of traditional Italian legumes: The potential of sourdough fermentation. Int. J. Food Microbiol. 196, 51-61. Curtis, M., 2004: The obesity epidemic in the Pacific Islands. J. Dev. Social Transformation 1, 37-42. Dewick, P.M., 2009: The shikimate pathway: aromatic amino acids and phenylpropanoids. In: Medicinal natural products: a biosynthetic ap- proach (3rd Ed.), 137-186. Chichester, UK: John Wiley & Sons. Tab. 3: Phenolic compounds content of Inocarpus fagifer seed Phenolics acids Content (μg kg-1 dry matter) SDa Caffeic acid 97.89 1.52 p-Coumaric acid 1478.47 15.11 Ferulic acid 3229.08 32.90 Gallic acid tr.b Salicylic acid 88.75 0.90 Sinapinic acid 65.80 0.29 Isoflavonoids Daidzein 13.87 0.07 Daidzin 2.46 0.03 7,4´-dimethoxyisoflavon 64.99 0.49 Formononetin 179.73 1.58 Genistein 13.53 0.16 Genistin 14.48 0.11 Glycitein 568.64 7.84 Glycitin 71.28 0.89 Prunetin 7.78 0.05 Sissotrin 3.31 0.05 Tectoridin 15.11 0.10 Flavonoids Apigenin-7-glu tr. Epicatechin tr. Isokvercitrin 21.53 0.21 Kaempferol 46.91 0.39 Liquiritigenin 152.92 2.74 Luteolin 7.05 0.05 Naringenin 13.75 0.26 Naringenin-7-glu tr. Rutin 123.68 0.17 Coumestans Coumestrol 530.06 8.61 astandard deviation (n=3), btraces (below 0.01%) Pioneering study on nutritional components of Inocarpus fagifer 269 Djordjevic, T.M., Šiler-Marinkovic, S.S., Dimitrijevic-Brankovic, S.I., 2011: Antioxidant activity and total phenolic content in some cere- als and legumes. Int. J. Food Prop. 14, 175-184. Ebert, A.W., 2014: Potential of underutilized traditional vegetables and le- gume crops to contribute to food and nutritional security, income and more sustainable production systems. Sustainability 6, 319-335. FAO (Food and Agriculture Organization of the United Nations)/INFOODS, 2012: Guidelines for converting units, denominators and expressions, version 1.0. Retrieved from http://www.fao.org/fileadmin/templates/ food _composition /documents/1nutr ition /Conversion_Guidelines- V1.0.pdf gibson, R.S., Carriquiry, A., Gibbs, M., 2015: Selecting desirable micro- nutrient fortificants for plant-based complementary foods for infants and young children in low-income countries. J. Sci. Food Agric. 95, 221-224. Gordon, M.H., 1996: Dietary antioxidants in disease prevention. Nat. Prod. Rep. 13, 265-273. hegnauer, p.r., hegnauer, M., 2001: Dalbergieae. In: Chemotaxonomie der Pflanzen, 116-160. Basel, CH: Birkhäuser. Leuner, O., Havlik, J., Hummelova, J., Prokudina, E., Novy, P., Kokoska, L., 2013: Distribution of isoflavones and coumestrol in neg- lected tropical and subtropical legumes. J. Sci. Food Agric. 93, 575- 579. Lewis, G., Schrire, B., MacKinder, B., Lock, M., 2005: Legumes of the world, 318. London, UK: Kew Royal Botanic Gardens. Lim, T.K., 2012: Edible medicinal and non-medicinal plants, Vol. 2. Fruits, 726-728. Dordrecht, DE: Springer. Nasi, A., Picariello, G., Ferranti, P., 2009: Proteomic approaches to study structure, functions and toxicity of legume seeds lectins. Perspec- tives for the assessment of food quality and safety. Plant Proteomics 72, 527-538. NRC (National Research Council), 1989a: Recommended dietary allow- ances (10th Ed.), 44-231. Washington, D.C., USA: National academy press. NRC (National Research Council), 1989b: Hypertension. In: Diet and health: Implications for reducing chronic disease risk, 549-562. Washington, D.C., USA: National academy press. Pauku, R.L., 2006: Inocarpus fagifer (Tahitian chestnut). In: Elevitch, C.R. (ed.), Traditional trees of Pacific islands: Their culture, environment, and use, (version 2.1), Holualoa, HI, USA: Permanent agriculture re- sources. Perabo, F.G.E., Löw, E.C.V., Ellinger, J., Rücker, A., von Müller, S.C., Bastian, P.J., 2008: Soy isoflavone genistein in prevention and treat- ment of prostate cancer. Prostate Cancer Prostatic Dis. 11, 6-12. Perez, V., Chang, E.T., 2014: Sodium-to-potassium ratio and blood pres- sure, hypertension, and related factors. Adv. Nutr. Int. Rev. J. 5, 712- 741. Rusnikova, L., Strakova, E., Suchy, P., 2013: Assessment of oils of se- lected legumes and oil plants with regard to animal nutrition. Acta Vet. Brno 82, 283-288. Schieber, A., Stintzing, F.C., Carle, R., 2001: By-products of plant food processing as a source of functional compounds – recent developments. Trends in Food Sci. Technol. 12, 401-413. Simopoulos, A., 2012: The importance of the ratio of omega-6/omega-3 es- sential fatty acids. Biomed. Pharmacother. 56, 365-379. Singleton, V.L., orthofer, R., Lamuela-Raventos, R.M., 1998: Analy- sis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol. 299, 152-178. Thies, E., 2000: Promising and underutilized species, crops and breeds, 3-19. Eschborn, Germany: Deutsche Gesellschaft für technische Zusam- menarbeit GmbH. USDA (United States Department of Agriculture), 2015: National nutrient database for standard reference (Release 27). Nutrient data laboratory. Retrieved from http://www.ars.usda.gov/nutrientdata. Vanek, A., Komarek, M., Chrastny, V., Becka, D., Mihaljevic, M., Sebek, O., Panuskova, G., Schusterova, Z., 2010: Thallium uptake by white mustard (Sinapis alba L.) grown on moderately contaminated soils-Agro-environmental implications. J. Hazard. Mat. 182, 303-308. Vedavanam, K., Srijayanta, S., O’Reilly, J., Raman, A., Wiseman, H., 1999: Antioxidant action and potential antidiabetic properties of an iso- flavonoid-containing soyabean phytochemical extract (SPE). Phytother. Res. 13, 601-608. Veitch, n.c., 2013: Isoflavonoids of the Leguminosae. Nat. Prod. Rep. 30, 988-1027. Westphal, S., Borucki, K., Taneva, E., Makarova, R., Luley, C., 2010: Adipokines and treatment with niacin. Metabolism 55, 1283-1285. Whistler, W.A., 2000: Plants in Samoan culture: The ethnobotany of Samoa, 35. Honolulu, HI, USA: Isle Botanica. WHO (World Health Organization), 2014: Global status report on noncom- municable diseases, 9-272. Geneva, Switzerland: WHO. WHO (World Health Organization), 2002: Obesity in the Pacific: too big to ignore, 3-18. Noumea, New Caledonia: Regional Office for the West- ern Pacific, Secretariat of the Pacific Community, International Obesity Taskforce, Food and Agriculture Organization of the United Nations and International Life Sciences Institute. Win Tin, S.T., Lee, C.M.Y., Colagiuri, R., 2015: A profile of diabetes in Pacific Island countries and territories. Diabetes Res. Clin. Pract. 107, 233-246. Yildiz, F., 2010: Advances in Food Biochemistry, 250-267. Boca Raton, USA: CRC Press. Addresses of the authors: 1Department of Chemistry of Natural Compounds, Faculty of Food and Biochemical Technology, University of Chemistry and Technology Prague, Technicka 5, 166 28, Praha 6 - Dejvice, Czech Republic 2Forensic Laboratory of Biologically Active Substances, University of Chemistry and Technology Prague, Technicka 5, 166 28, Praha 6 - Dejvice, Czech Republic 3 Department of Quality of Agricultural Products, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamycka 129, 165 21, Prague 6 - Suchdol, Czech Republic 4Department of Soil Science and Soil Protection, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamycka 129, 165 21, Prague 6 - Suchdol, Czech Republic 5Department of Food Analysis and Nutrition, Faculty of Food and Biochemi- cal Technology, University of Chemistry and Technology Prague, Technicka 5, 166 28, Praha 6 - Dejvice, Czech Republic 6School of Agriculture and Food Technology, Faculty of Business and Eco- nomy, The University of the South Pacific, Independent State of Samoa 7Department of Nutritive Substances, Food Research Institute Prague, Ra- diova 7, 102 31 Prague 10 - Hostivar, Czech Republic 8Department of Crop Sciences and Agroforestry, Faculty of Tropical Agri- Sciences, Czech University of Life Sciences Prague, Kamycka 129, 165 21, Prague 6 - Suchdol, Czech Republic E-mail: kokoska@ftz.czu.cz © The Author(s) 2016. This is an Open Access article distributed under the terms of the Creative Commons Attribution Share-Alike License (http://creative- commons.org/licenses/by-sa/4.0/). I Supplementary material Fig. S1: GC-FID chromatogram of Inocarpus fagifer seed extract. Peak identification: 1 = caprylic acid, 2 = capric acid, 3 = lauric acid, 4 = myristic, 5 = pentade- cylic acid, 6 = palmitic acid, 7 = hexadecenoic acid (cis 7), 8 = hexadecenoic acid (cis 9), 9 = hexadecenoic acid (cis 11), 10 = heptadecanoic acid, 11 = heptadecenoic acid (cis 9), 12 = stearic acid, 13 = oleic acid (cis 9), 14 = octadecenoic acid (cis 11), 15 = nonadecanoic acid, 16 + 17 = octadecadienoic acid (cis trans isomers), 18 = linoleic acid, 19 = arachidic acid, 20 = eicosenoic acid (cis 11), 21 = α-linolenic, 22 = heneicosanoic acid, 23 = unidenti- fied fatty acid, 24 = behenic acid, 25 = tricosanoic acid, 26 = lignoceric acid, 27 = pentacosanoic acid, 28 = cerotic acid, 29 = heptacosanoic acid, 30 = octacosanoic acid, 31 = nonacosanoic acid, 32 = triacontanoic acid. Fig. S2: Extracted ion chromatograms of phenolic compounds detected in Inocarpus fagifer seed extract. Supplementary material II2    T ab le S 1. T h e tr an si ti o n s an d s p ec if ic M S /M S p ar am et er s u se d f o r an al y si s o f p h en o li c co m p o u n d s 13   io n is at io n m o d e re te n ti o n ti m e (m in )a fr ag m en to r (V ) p re cu rs o r io n ( m /z ) p ro d u ct i o n (m /z ) L O D (n g /m L )c L O Q (n g /m L )d q u an ti fi ca ti o n tr an si ti o n C E (e V )b co n fi rm at io n tr an si ti o n C E (e V )b 7 .4 ´- d im et h o x y is o fl av o n e E S I + 9 .0 5 ( 0 .5 ) 1 1 7 2 8 3 .1 0 1 7 1 .3 4 2 1 7 6 .0 1 8 0 .4 1 .4 ap ig en in -7 -O -g lu co si d e E S I + 5 .5 4 ( 0 .5 ) 1 0 9 4 3 3 .1 2 2 7 1 .0 1 3 1 5 3 .0 6 0 0 .2 0 .8 ca ff ei c ac id E S I - 3 .6 0 ( 0 .4 ) 8 1 1 7 9 .0 0 8 9 .0 3 0 1 3 5 .1 1 3 2 .3 7 .7 ch lo ro g en ic a ci d E S I - 3 .0 1 ( 1 .0 ) 8 1 3 5 3 .0 9 1 9 1 .1 9 0 .6 1 .9 p- co u m ar ic a ci d E S I + 4 .3 7 ( 0 .5 ) 6 0 1 6 5 .0 5 1 4 7 .1 9 0 .9 3 .0 co u m es tr o l E S I + 7 .8 8 ( 0 .5 ) 1 2 1 2 6 9 .0 5 1 1 5 .0 5 3 1 5 7 .0 3 3 0 .5 1 .6 d ai d ze in E S I - 6 .0 1 ( 0 .5 ) 1 2 6 2 5 3 .0 5 1 3 2 .1 4 2 9 1 .0 3 4 0 .1 0 .3 d ai d zi n E S I + 3 .6 6 ( 0 .5 ) 8 5 4 1 7 .1 2 2 5 5 1 4 1 5 2 .0 7 8 0 .1 0 .2 ep ic at ec h in E S I - 3 .1 0 ( 0 .5 ) 1 1 1 2 8 9 .0 7 1 0 9 1 3 2 4 5 .1 5 0 .7 2 .4 fe ru li c ac id E S I + 4 .6 5 ( 0 .5 ) 6 3 1 9 5 .0 7 1 4 5 .0 1 3 1 7 7 .0 5 1 .0 3 .2 g al li c ac id E S I - 2 .4 3 ( 0 .6 ) 7 5 1 6 9 .0 1 1 2 4 .9 9 1 6 9 .0 5 0 .7 2 .2 g en is te in E S I - 7 .2 1 ( 0 .5 ) 1 2 3 2 6 9 .0 4 1 3 3 .0 3 0 6 3 .1 3 4 0 .1 0 .4 g en is ti n E S I - 4 .6 2 ( 0 .5 ) 1 6 2 4 3 1 .1 0 2 6 9 .0 1 0 2 3 9 .1 4 6 0 .1 0 .4 g ly ci te in E S I - 6 .3 2 ( 0 .5 ) 9 9 2 8 3 .0 6 2 4 0 .0 1 8 2 6 8 .0 1 0 0 .1 0 .2 g ly ci ti n E S I + 3 .9 6 ( 0 .5 ) 8 2 4 4 7 .1 3 2 8 5 .0 1 4 2 7 0 .0 4 6 0 .1 0 .1 is o q u er ci tr in E S I - 4 .8 7 ( 0 .5 ) 1 5 0 4 6 3 .0 9 3 0 0 .3 1 8 2 7 1 .0 4 2 0 .4 1 .2 k ae m p fe ro l E IS + 7 .5 5 ( 0 .6 ) 1 6 1 2 8 7 .0 6 1 5 3 .0 4 1 6 9 .1 5 3 1 .1 3 .6 li q u ir it ig en in E S I - 6 .0 6 ( 0 .5 ) 9 0 2 5 5 .0 6 1 3 5 .0 9 1 1 9 .1 2 1 0 .1 0 .3 lu te o li n E S I - 7 .2 3 ( 0 .8 ) 1 2 8 2 8 5 .0 4 1 3 3 .0 3 3 1 5 1 .0 2 0 0 .4 1 .4 n ar in g en in E S I - 7 .0 5 ( 0 .5 ) 9 3 2 7 1 .0 6 1 1 9 .0 2 1 1 5 1 .0 9 0 .1 0 .1 n ar in g en in -7 -O -g lu co si d e E S I - 4 .7 6 ( 0 .5 ) 1 1 7 4 3 3 .1 1 2 7 1 .1 1 0 1 1 9 .0 5 0 0 .1 0 .4 p ru n et in E S I - 8 .8 2 ( 0 .3 ) 1 1 1 2 8 3 .0 6 2 6 8 .0 1 3 2 3 9 .0 2 5 0 .3 0 .9 ru ti n E S I - 4 .6 9 ( 0 .5 ) 1 6 3 6 0 9 .1 4 2 7 1 .0 6 1 3 0 0 .0 6 5 0 .3 0 .9 sa li cy li c ac id E S I - 5 .2 2 ( 0 .6 ) 7 2 1 3 7 .0 2 9 3 .1 1 3 6 5 .1 2 9 0 .5 1 .8 si n ap ic a ci d E S I - 4 .8 0 ( 0 .4 ) 8 1 2 2 3 .0 6 2 0 8 .1 9 1 4 9 .0 1 7 0 .1 0 .2 si ss o tr in E S I - 6 .5 5 ( 0 .5 ) 1 6 2 4 4 5 .1 1 2 8 3 .1 1 0 2 6 8 .0 3 0 0 .1 0 .3 te ct o ri d in E S I - 4 .7 4 ( 0 .5 ) 1 3 8 4 6 1 .1 1 2 9 9 .0 1 3 2 8 3 .0 2 9 0 .1 0 .4 Ta b. S 1: T he tr an si tio ns a nd s pe ci fic M S/ M S pa ra m et er s us ed fo r a na ly si s of p he no lic c om po un ds a R et en tio n tim e w in do w (m in ) i s gi ve n in b ra ck et s, b C ol lis io n en er gy , c L im its o f d et ec tio n (s ig na l- to -n oi se ra tio o f 3 ), d L im its o f q ua nt ifi ca tio n (s ig na l- to -n oi se ra tio o f 1 0)