Article pubs.acs.org/JAFC
Ultraperformance Liquid Chromatography Tandem Mass Spectrometry Determination of Cyanogenic Glucosides in Trifolium Species Tamar Muzashvili,†,‡ Barbara Moniuszko-Szajwaj,† Lukasz Pecio,† Wieslaw Oleszek,† and Anna Stochmal*,† †
Department of Biochemistry, Institute of Soil Science and Plant Cultivation−State Research Institute, Czartoryskich 8, 24-100 Pulawy, Poland ‡ Iovel Kutateladze Institute of Pharmacochemistry, Tbilisi State Medical University, P. Sarajishvili Street 36, 0159 Tbilisi, Georgia S Supporting Information *
ABSTRACT: Cyanogenic glucosides were analyzed by ultra-high-performance liquid chromatography combined with mass spectrometry in 88 Trifolium species grown at the same site. On the basis of the occurrence of cyanogenic glucosides and the linamarin/lotaustralin ratio species could be grouped into five clusters. Cluster C1 included 37 species, which did not contain cyanogens. Cluster C2 (22 species) included plants containing only lotaustralin. In clusters C3 (14 species), C4 (13 species), and C5 (2 species) both linamarin and lotaustralin were present but at different ratios. In C3 and C4 the linamarin/lotaustralin ratio was below 1, whereas in cluster C5 the ratio was much higher. Generally, the total content of cyanogens was below 500 μg/g dry matter. Only in Trifolium repens var. biasoletti and Trifolium montanum extremely high cyanogen concentrations were observed. There was no general rule of occurrence of cyanogens. Samples of the same species from different countries accumulated cyanogens or could be free of these compounds. KEYWORDS: Trifolium species, cyanogenic glucosides, UPLC-MS/MS
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method.15 Besides, the sensitivity of such detectors is low, and we deemed it of interest to develop the protocol based on mass spectrometric detection, which is very sensitive and more reliable. Using this method 88 Trifolium species have been analyzed for their cyanogenic composition. Obtained data together with our previous work on isoflavones, phenolic acids, flavonoids, and saponins5 in a similar set of species can be a useful tool for breeding purposes or for extensive studies of the biosynthesis, transport, and degradation of cyanogenic glucosides in the genus Trifolium.
INTRODUCTION Genus Trifolium (Papilionoidae−Trifolieae) includes about 250−300 species distributed in temperate and subtropical regions of both hemispheres.1 Several species of the genus are extensively cultivated because of their nitrogen-fixing and nutritive properties.2 These species are also a rich source of different secondary metabolites. Cyanogenic glycosides, phenolic compounds, and saponins have been reported from the aerial and subterranean parts of Trifolium cultivars.3−7 To the isoflavonoids of Trifolium subterraneum, Trifolium pannonicum, and Trifolium pratense the estrogenic, anti-inflammatory, and antitumoral effects are attributed.2,8,9 Cyanogenic glucosides are present in more than 2500 plant species. These compounds are known to have an important role in plant defense against pathogens10 and herbivores. Their role as storage compounds of reduced nitrogen and sugars that may be mobilized when demanded for use in primary metabolism has been demonstrated.11 From a nutritional point of view they are recognized as antinutritional factors lowering clover pasture quality.12,13 Two major cyanogenic glycosides, linamarin, 1, and lotaustralin, 2 (Figure 1), have been reported in Trifolium species. For determination of cyanogenic glycosides a highperformance liquid chromatograph equipped with the refractive index detection method has been developed with purification of extracts on a C18 support prior to analysis. In eight cultivars of Polish origin seasonal changes of their occurrence have been investigated, and analytical liquid chromatography has been used for their detection for the first time.14,15 Cyanogenic glycosides have often been detected on the basis of their refractometric index, which is a rather nonspecific © 2014 American Chemical Society
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MATERIALS AND METHODS
Chemicals and Reagents. Milli-Q water was obtained by passing deionized and distilled water through a Simplicity 185 Milli-Q water system (Millipore SAS, Molsheim, France) prior to use. HPLC-grade acetonitrile, formic acid, acetic acid, methanol, ethyl acetate, and chloroform were purchased from J.T. Baker (Deventer, The Netherlands). Plant Material. Seeds of 88 Trifolium species were obtained from Genebank, Leibniz Institute of Plant Genetics and Crop Plant Research (Gatersleben, Germany). Seeds were planted in early spring 2011 (1 m × 1 m plots) in an experimental field of the Institute of Soil Science and Plant Cultivation in Pulawy, Poland. Aerial parts of plants were harvested at the beginning of flowering, frozen, and lyophilized. Since the collection included both annual and perennial species, the dynamics of their growth and flowering period differed significantly, Received: Revised: Accepted: Published: 1777
September 16, 2013 January 22, 2014 January 29, 2014 January 29, 2014 dx.doi.org/10.1021/jf4056659 | J. Agric. Food Chem. 2014, 62, 1777−1782
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Figure 1. Structures of standards, linamarin (1) and lotaustralin (2), isolated from Trifolium repens aerial parts. μL) and strong wash (300 μL), respectively. A 1 μL sample was injected for analysis. For MS detection, negative ESI was used as the ionization mode. Nitrogen was used as the desolvation and cone gas with a flow rate of 800 and 80 L/h, respectively. Argon was used as the collision gas at a flow rate of 0.2 mL/min. The optimal MS parameters were as follows: capillary, 3.0 kV; extractor and radiofrequency voltage were fixed to 3.0 and 0.1 V, respectively; source temperature, 140 °C; desolvation temperature, 350 °C; cone voltage varied from 19 to 23 V; collision energies varied from 13 to 15 eV. Quantitation was performed using the selected reaction monitoring (SRM) mode; parent > daughter transitions were m/z 292 > 161 and m/z 306 > 161 for linamarin and lotaustralin, respectively. Statistical Analysis. Statistical and cluster analyses (K-means clustering) were performed using the XLSTAT 2012 program.
and they were harvested on July 8 (23 species), 18 (25 species), and 29 (14 species) and August 18th (26 species). A list of examined Trifolium species, their origins, and voucher specimens is reported in Table 1. Extraction and Purification. Powdered aerial parts of Trifolium species were defatted with chloroform in Soxhlet apparatus. After removal of chlorophyll the samples (100 mg each) were extracted in an automated accelerated solvent extractor ASE 200 (Dionex, Sunnyvale, CA) using 80% aqueous MeOH (23 mL) at 40 °C and 1500 psi working pressure. Extracts were evaporated to dryness (at 40 °C). Residues were dissolved in Milli-Q water and purified by solidphase extraction (SPE) using water preconditioned 1 mL, 360 mg C18 Sep-Pak cartridges (Waters Corp., Milford, MA). After loading the samples, cartridges were washed with water (1 mL) to remove carbohydrates; cyanogenic glycosides were eluted with 20% (v/v) MeOH (6 mL). Fractions were concentrated under reduced pressure and dissolved in 1 mL of 20% (v/v) MeOH for analyses. Three independent extraction and purification procedures were performed for each species, and data were presented as mean values with standard deviations (Table 2). Isolation of Standards. Linamarin, 1, and lotaustralin, 2 (Figure 1), used as standards for analysis, were isolated from T. repens L. cultivar ‘Haifa’. The specimen (Voucher number 7/2010) has been deposited at the Department of Biochemistry and Crop Quality, Institute of Soil Science and Plant Cultivation, State Research Institute, Pulawy, Poland. Plant material was defatted with chloroform and extracted three times with 75% (v/v) MeOH at room temperature for 24 h using an ultrasonic bath. Extracts were concentrated under reduced pressure and lyophilized. Then they were dissolved in water and loaded onto a 100 × 60 mm LiChroprep RP-18, 40−63 μm vacuum liquid chromatography column (Merck, Darmstadt, Germany). The column was washed with water, and cyanogenic glycosides were eluted with 20% (v/v) MeOH. Cyanogenic glycosides fraction was chromatographed with a semipreparative high-performance chromatograph Gilson HPLC equipped with a PrepELS II evaporative light-scattering detector and RP-18 column (10 × 250 mm; 5 μm; Kromasil C18, Eka Chemicals AB, Sweeden) at a flow rate of 5.0 mL/min using a 0.5% (v/v) HOAc−MeCN gradient as the mobile phase. Fractions containing pure compounds were precipitated in ethyl acetate. Single compounds were identified by NMR analysis, which was consistent with reported data. Purity of the standards linamarin, 1, and lotaustralin, 2, was 99.0 and 99.8%, respectively. UPLC-MS/MS Analysis. A Waters ACQUITY UPLC system equipped with a binary solvent manager and coupled to a Waters ACQUITY TQD (tandem quadruple mass detector) with an electrospray ionization (ESI) source was used for quantitation. All data were acquired and processed using Waters MassLynx 4.1 and QuanLynx software. A Waters BEH C18 column (1 × 100 mm, 1.7 μm particles) was used to separate analytes. The elution program used for chromatographic separation consisted of solvents A (Milli-Q water containing 0.1% (v/v) HCOOH) and B (MeCN containing 0.1% (v/ v) HCOOH) delivered as follows: 0.0−0.5 min (7% B), 8.0 min (25% B), 11.5 min (60% B), 12.0 min (80% B), 13.0 min (80% B), 13.1 min (7% B), and finished at 15.0 min. The flow rate was 0.15 mL/min, and the column temperature was 50 °C. The injection wash solvents were MeCN/H2O (5:95, v/v) and MeOH/H2O (5:95, v/v) for weak (900
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RESULTS AND DISCUSSION Analysis of cyanogenic glucosides in plant material creates some analytical problems. Older methods of analysis were based on sodium picrate or Feigl-Anger reagent.16 Both tests depended on the release of HCN by enzymatic or nonenzymatic hydrolysis of cyanogenic glucosides. They were very sensitive but not always proper due to the fact that some other plant components (glucosinolates) could produce misleading information. Improvement in cyanogenic glucosides analysis was the application of high-performance liquid chromatography with refractive index detection.15 The method provided more objective data, but its sensitivity was low, and some plants with trace amounts of compounds could be classified as noncyanogenic. In the present research HPLC combined with the mass detector was applied in selected reaction monitoring (SRM) mode. This gave high sensitivity and precision of analysis, and both compounds could be detected even at very low concentration in plants (Figure 2). Analysis of 88 species, originating from different parts of the globe but grown at the same site, provided interesting results. All analyzed species with respect to the content of cyanogenic glycosides could be grouped in five clusters (Table 2). As a criteria for clustering the presence of linamarin and lotaustralin and the linamarin/lotaustralin ratio were considered. The majority of 37 species belonged to cluster C1, which was characterized by the absence of cyanogenic glucosides. The second cluster C2 of 22 species grouped those, which contained only lotaustralin as a sole cyanogenic glucoside ranging from trace amounts of 0.85 (T. alexandrinum) to 121.47 μg/g (T. incarnatum) of dry weight. The third cluster C3 of 14 species was characterized by the presence of both linamarin and lotaustralin, but the linamarin occurred at a very low level; the linamarin/lotaustralin ratio ranged from 0.01 to 0.08, and the total concentration ranged from 13.26 (T. ochroleucon Huds.) to 424.77 μg/g (T. resupinatum L. var. majus Boiss.). Similarly, the fourth cluster C4 of 13 species also contained both linamarin and lotaustralin, but the ratio of linamarin/ lotaustralin ranged from 0.11 to 0.61, and the total 1778
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Table 1. List of Examined Trifolium Species no.
species (subspecies, variety)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
T. alexandrinum L. T. alexandrinum L. T. alexandrinum L. T. alexandrinum L. T. alexandrinum L. T. alexandrinum L. T. alexandrinum L. T. alpestre L. T. ambiguum M. Bieb. T. angustifolium L. T. apertum Bobrov. T. argutum Sol. T. arvense L. T. bocconei Savi T. campestre Schreb. T. cernuum Brot. T. cherleri Jusl. T. clussii Godr. & Green. var. clussii T. clypeatum L. T. dubium Sibth. T. echinatum M.Bieb. var. carmeli (Boiss.) Gibelli & Belli T. echinatum M.Bieb. var. echinatum T. f ragiferum L. T. f ragiferum L. ssp. bonanni (Presl) Sojak T. f ragiferum L. ssp. Fragiferum T. f ragiferum L. ssp. bonanni (Presl) Sojak T. f ragiferum L. ssp. Fragiferum T. glomeratum L. T. heldreichianum Hausskn. T. hirtum All. T. hybridum L. T. hybridum L. T. hybridum L. T. hybridum L. T. hybridum L. var. hybridum T. hybridum L. var. hybridum T. incarnatum L. T. incarnatum L. T. incarnatum L. T. incarnatum L. T. incarnatum L. T. incarnatum L. var. Molinerii (Balb. ex Hornem.) Ser. T. isthmocarpum Brot. T. lappaceum L. T. leucanthum M. Bieb.
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
origin
herbarium voucher
unknown India unknown unknown Albania Italy Greece unknown unknown France unknown unknown unknown Portugal Sweden Portugal unknown unknown Israel Germany Israel
TRIF 10 TRIF 30 TRIF 45 TRIF 47 TRIF 269 TRIF 312 TRIF 1237 TRIF 201 TRIF 178 TRIF 139 TRIF 44 TRIF 80 TRIF 40 TRIF 81 TRIF 99 TRIF 52 TRIF 74 TRIF 57 TRIF 129 TRIF 103 TRIF 100
Romania Bulgaria Ukraine
TRIF 104 TRIF 1151 TRIF 197
United States Hungary
TRIF 1241 TRIF 208
unknown Morocco unknown unknown unknown United States Finland Denmark Norway Canada Czechoslovakia Poland United States Italy United States France
TRIF 37 TRIF 107 TRIF 149 TRIF 213 TRIF 6 TRIF 155 TRIF 194 TRIF 1177 TRIF 1242 TRIF 1243 TRIF 82 TRIF 86 TRIF 247 TRIF 1141 TRIF 1189 TRIF 280
Portugal unknown Israel
TRIF 77 TRIF 55 TRIF 131
no.
species (subspecies, variety)
46 47 48 49 50
T. ligusticum Balb. T. medium L. T. medium L. var. medium T. medium L. var. medium T. medium L. var. sarosiense (Hazsl.) Savul. & Rayss T. michelianum Savi T. michelianum Savi var. balansae (Boiss.) Azn. T. miegeanum Maire T. montanum L. T. nigrescens Viv. ssp. Nigrescens T. obscurum Savi T. ochroleucon Huds. T. palidum Waldst. et Kit. T. pannonicum Jacq. T. pannonicum Jacq. T. phleoides Pourr. ex. Willd. T. pratense L. T. pratense L. T. pratense L. T. pratense L. T. pratense L. T. pratense L. T. purpureum Lois. var. desvauxii (Boiss. & Blanche) Post T. repens L. T. repens L. T. repens L. T. repens L. var. biasoletti (Stend. & Hochst.) Asch. & Graebn. T. repens L. gigantem Lagr.-Foss. T. repens L. var. biasoletti (Steud. & Hochst.) Asch. & Graebn. T. resupinatum L. var. majus Boiss. T. resupinatum L. var. majus Boiss. T. resupinatum L. var. resupinatum T. resupinatum L. var. resupinatum T. resupinatum L. var. majus Boiss. T. resupinatum L. var. resupinatum T. scabrum L. T. spumosum L. T. squarrosum L. T. stellatum L. T. striatum L. T. subterraneum L. ssp. Subterraneum T. tomentosum L. var. tomentosum T. tomentosum L. var. curvisepalum (Täckh.) Thiebaut
51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88
concentration varied from 4.97 (T. repens L.) to 1877.33 μg/g (T. montanum L.). Finally, the fifth cluster C5 contained only two species: T. repens L. and T. repens L. giganteum Lagr.-Foss with a relatively low total concentration of both cyanogenic glucosides (27.73 and 68.27 μg/g, respectively), but in these plants linamarin was the dominating compound and the linamarin/lotaustralin ratio was 3.25 and 3.75, respectively. Considering the total concentration of cyanogenic glucosides it should be noticed that all samples were collected at the beginning of flowering, the period when most of the secondary
origin
herbarium voucher
Portugal unknown unknown unknown unknown
TRIF TRIF TRIF TRIF TRIF
113 281 32 35 179
unknown Bulgaria
TRIF 79 TRIF 145
Portugal unknown Portugal Denmark Slovakia Italy unknown unknown unknown Poland United States Sweden Austria Belgium Netherlands United States
TRIF 116 TRIF 147 TRIF 117 TRIF 133 TRIF 173 TRIF 253 TRIF 8 TRIF 9 TRIF 132 TRIF 1482 TRIF 1491 TRIF 1745 TRIF 2016 TRIF 2536 TRIF 2561 TRIF 143
unknown Poland United States France
TRIF 15 TRIF 1156 TRIF 1157 TRIF 254
Hungary France
TRIF 229 TRIF 255
United States Germany United States Turkey Iran unknown Portugal unknown unknown Croatia France United States Portugal unknown
TRIF 224 TRIF 1135 TRIF 59 TRIF 1134 TRIF 61 TRIF 43 TRIF 120 TRIF 67 TRIF 122 TRIF 215 TRIF 70 TRIF 259 TRIF 218 TRIF 53
metabolites are most intensively biosynthesized.17 On the other hand, plants were harvested during the summer period (July− August) when, as shown with white clover varieties, cyanogenic glucoside content was the lowest,13 which corresponded with the general view that cyanogen concentration increases at low temperatures.18 For white clover in Europe the Swiss cultivar ‘Milkanova’ was accepted as a reference variety as regards the cyanogen content to be safe for grazing animals. This cultivar in Swiss conditions synthesizes about 370 mg of HCN/kg of dry matter. In these circumstances the concentration of cyanogens 1779
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Table 2. Concentrations (μg/g dm ± SD) of Cyanogenic Glucosides in 88 Trifolium Species species cluster cluster 2 23 4 20 48 58 51 6 61 15 16 45 9 10 5 63 46 14 47 56 12 38 cluster 57 39 25 52 67 49
linamarin C1a C2 NDb ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND C3 1.03 ± 0.20 ± 1.23 ± 0.40 ± 2.53 ± 0.87 ±
0.34 0.00 0.05 0.10 0.24 0.17
lotaustralin
total
0.85 0.93 1.33 1.57 2.47 3.60 5.63 7.50 8.53 11.10 14.03 16.50 17.60 18.17 28.17 28.23 33.93 42.17 71.60 78.67 99.57 121.47
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.05 0.21 0.31 0.48 0.31 0.29 0.29 0.16 0.69 0.22 0.37 0.50 0.56 0.34 0.90 0.12 0.79 0.57 1.15 1.73 0.37 0.68
0.85 0.93 1.31 1.57 2.47 3.60 5.63 7.50 8.53 11.10 14.03 16.50 17.60 18.17 28.17 28.23 33.93 42.17 71.60 78.67 99.57 121.47
12.23 24.30 25.97 40.97 49.50 53.60
± ± ± ± ± ±
0.12 0.24 0.37 0.23 0.65 0.73
13.26 24.50 27.20 41.37 52.03 54.47
species
Lin/Lot ratio
cluster 26 87 13 83 18 22 43 75 cluster 71 53 29 44 30 35 11 60 40 74 55 72 54 cluster 69 73
0.08 0.01 0.05 0.01 0.05 0.02
linamarin C3 1.13 ± 0.42 6.43 ± 0.21 10.33 ± 0.21 1.17 ± 0.12 12.07 ± 0.72 7.47 ± 0.30 21.17 ± 1.60 22.00 ± 0.64 C4 1.40 ± 0.08 6.37 ± 0.25 2.87 ± 0.29 9.20 ± 0.08 4.73 ± 0.53 13.77 ± 0.25 22.87 ± 0.33 36.33 ± 0.45 35.63 ± 1.54 90.00 ± 0.79 59.07 ± 3.27 571.00 ± 2.48 593.00 ± 0.71 C5 21.20 ± 0.43 53.90 ± 0.57
lotaustralin
total
Lin/Lot ratio
108.47 113.37 139.30 152.20 204.73 212.17 338.47 402.77
± ± ± ± ± ± ± ±
1.74 0.88 0.65 0.29 1.84 0.47 13.65 1.09
109.60 119.80 149.63 153.37 216.80 219.64 359.64 424.77
0.01 0.06 0.07 0.01 0.06 0.04 0.06 0.05
4.97 21.63 25.70 23.87 30.60 24.93 106.47 255.50 195.53 252.83 443.27 932.30 1284.33
± ± ± ± ± ± ± ± ± ± ± ± ±
0.09 0.26 0.08 0.84 0.64 0.54 0.99 2.24 1.31 1.93 10.17 3.48 8.81
6.37 28.00 28.57 33.07 35.33 38.70 129.34 291.83 340.76 342.83 502.34 1503.30 1877.30
0.28 0.29 0.11 0.38 0.15 0.55 0.21 0.14 0.18 0.36 0.13 0.61 0.46
27.73 68.27
3.25 3.75
6.53 ± 0.12 14.37 ± 1.51
a
In cluster C1 (species 3, 7, 8, 17, 19, 21, 24, 27, 28, 31, 32, 33, 34, 36, 37, 41, 42, 50, 59, 62, 64, 65, 66, 68, 70, 76, 77, 78, 79, 80, 81, 82, 84, 85, 86, 88), no linamarin, 1, or lotaustralin, 2, was detected. bND − not detected.
g (72). This finding correlates with previous reports, showing that in the Mediterranean region the frequency of cyanogenesis in white clover populations was 100%, while in other European regions it was decreasing when moving to the northeast, and in Central Russia the populations were noncyanogenic.21 This was correlated with the mean temperature of January and preferential grazing of noncyanogenic plantlets by snails. Only the sample originating from Poland (70) was free of cyanogens. This finding confirmed previous data showing that old Polish cultivar ‘Podkowa’ bred from local population was noncyanogenic.14 Similar trends were observed in the case of T. incarnatum. Three of the five samples were free of cyanogens, one (38) contained only lotaustralin, and one (39) contained both linamarin and lotaustralin. In contrast, white clover T. incarnatum originating from France was free of cyanogens, while the sample originating from Poland had the highest content of these glycosides. In the case of T. hybridum, out of six samples five did not contain cyanogens and only the sample originating from Norway (35) possessed linamarin and lotaustralin at a relatively low level of 38.70 μg/g of dry matter. The species T. resupinatum were differentiated in cyanogen content as well. Out of six tested samples five were noncyanogenic and one originating from the United States contained both linamarin and lotaustralin at a relatively high level of 424.77 μg/g.
in plants of clusters C2, C3, and C5 should be considered as rather low. Cluster C4 contained species with concentrations comparable to cited ‘Milkanova’ cultivar. Only two species, T. repens var. biasoletti (Stend & Hochst.) Asch. & Graebn. and T. montanum showed extremely high cyanogen concentration (1505.30 and 1877.30 μg/g of dry matter, respectively). This has a concentration comparable to the content of linamarin and lotaustralin in cassava tubers where they accumulate up to 1.5 g/kg dry weight.19,20 However, in cassava tubers the linamarin/ lotaustralin ratio was approximately 97:3, whereas in most of the Trifolium species lotaustralin has been the dominant glucoside. There was no regularity observed regarding the genusdependent occurrence of cyanogenic glucosides. For these genera, which were represented by several species or subspecies of different origin, their distribution between different clusters was observed. Out of the six samples of widely cultivated red clover (T. pratense), four were free of cyanogenic glucosides, one had only lotaustralin, and one possessed both linamarin and lotaustralin. It is interesting to notice that T. pretense originating from Belgium (66) was free of cyanogens, while the same species originating from Netherlands (67) contained both linamarin and lotaustralin. White clover (T. repens) was also represented by six samples of different origin, and five of them contained both linamarin and lotaustralin. The total cyanogen concentration in these samples ranged between 6.37 and 1503.30 μg/g. The highest concentration was observed for samples originating from France: 342.83 (74) and 1503.30 μg/ 1780
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Figure 2. UPLC ESI-MS/MS ion chromatograms of linamarin (1) and lotaustralin (2) quantified in T. repens var. biasoletti (Steud. & Hochst.) Asch. & Graebn. were obtained with selected reaction monitoring (SRM).
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The presence or absence of both glycosides is regulated by alleles of a single gene, designated Ac, whereas the presence or absence of the enzyme linamarase is governed by alleles or another, independently inherited gene (Li). Only plants possessing dominant functional alleles of both genes liberate HCN when damaged.13,22 Biosynthesis of linamarin, 1, and lotaustralin, 2, the valine and isoleucine-derived cyanogenic glucosides has been recently extensively studied. The biosynthetic pathway that includes enzymes and corresponding genes has been fully identified in Sorghum bicolor, synthesizing aromatic cyanogenic glucoside dhurrin.23 The CYP79 enzymes converting amino acids into oximes have been known in several plant species. For some species (great millet and cassava) the enzymes converting oximes to cyanohydrins have also been identified.24−26 In particular, identification of CYP71E1 and CYP71E7 in cassava synthetizing aliphatic cyanogenic glucoside may aid as a probe for isolation of their paralogs in Trifolium species. Our present findings may help in selection of species/ subspecies for these studies. The full recognition of the enzymes involved in biosynthesis, transport, and degradation of these compounds has been of crucial value for breeding purposes. It is experimentally demonstrated that the acyanogenic plants have a competitive advantage over the cyanogenic ones (the relative fitness, i.e., biomass production and the number of flowers per plant are higher).13 Screening, which was carried out, revealed the cyanogenic and acyanogenic species. Obtained data can be used for their future safe nutritive use or cloverbreeding programs, and they can have a chemotaxonomic significance.
ASSOCIATED CONTENT
S Supporting Information *
NMR data and structures of standards (linamarin and lotaustralin) used for analysis; ESI-MS/MS spectra of linamarin and lotaustralin obtained with selected reaction monitoring (SRM). This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +48 8188 63421, ext 205. Fax: +48 8188 63421, ext 295. E-mail:
[email protected]. Funding
This work was supported by the European Community seventh Framework Program project PROFICIENCY (Contract No. 245751) and OSCAR (Contract No. 289277). Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Bisby, F. A.; Buckingham, J.; Harborne, J. B. In Phytochemical Dictionary of Leguminosae; Chapman and Hall: London, 1994. (2) Smetham, M. A review of subterranean clover (Trifolium subterraneum L.) its ecology, and use in Australasia. Adv. Agron. 2003, 79, 303−350. (3) Drenin, A. A.; Botirov, E.Kh.; Petrulyak, E. V. Two new isoflavonoid monogalactosides from Trifolium pratense roots. Chem. Nat. Compd. 2008, 44, 24−27. (4) Foo, L. Y.; Lu, Y.; Molan, A. L.; Woodfield, D. R.; McNabb, W. C. The phenols and prodelphinidins of white clover flowers. Phytochemistry 2000, 54, 539−548.
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Journal of Agricultural and Food Chemistry
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dx.doi.org/10.1021/jf4056659 | J. Agric. Food Chem. 2014, 62, 1777−1782
