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Occurrence of Vicine and Convicine in Seeds of Some Vicia Species and Other Pulses
Occurrence of Vicine and Convicine in Seeds of Some Vicia Species and Other Pulses W.J. Pitz and F. W. Sosulski Department of Crop Science University of Saskatchewan Saskatoon, Saskatchewan S7N OWO and
L.R. Hogge Prairie Regional Laboratory National Research Council Saskatoon, Saskatchewan S7N OW9
Abstract
carried out by the latter workers have shown that the aglycones, divicine and isouramil, derived from vicine and convicine, respectively, have a marked destructive effect on reduced glutathione (GSH) levels of glucose-6-phosphate dehydrogenase deficient red blood cells, a characteristic feature of the disease. Vicine and convicine were first isolated from the seeds of Vicia sativa (Ritthausen and Kreusler, 1870; Ritthausen, 1881) and were later found in Vicia faba (Johnson, 1914; Fisher and Johnson, 1932). Vicine was also reported to be present in beet juice and peas (Bendich and Clements, 1953; Jamalian et al., 1977) and in Lupinus albus (Pompei and Lucisano, 1976). Some investigators (Liener, 1973) have questioned whether vicine and convicine are actually responsible for favism because the disease is only associated with the ingestion of V. faba. Therefore, further information is required on the occurrence of these glycosides in other plant materials. Both vicine and convicine are l3-glycosides of the plant pyrimidines, divicine and isouramil, respectively, (Bendich and Clements, 1953; Bien et aI., 1968) and are hydrolyzed by l3-glucosidases (Herissey and Cheymol, 1931; Mager et aI., 1965). In addition, vicine and convicine form volatile trimethylsilyl derivatives which permit a gas-liquid chromatography-mass spectrometry analysis of the glycosides (Pitz and Sosulski, 1979). Advantage was taken of these properties and used as qualitative criteria in the present work to study the occurrence and distribution of vicine and convicine in the seeds of a number of Vicia species, other pulse crops and beet root.
The occurrence and distribution of vicine and convicine in a number of pulse crops and beet root were studied using gas-liquid chromatography. The presence of the glycosides in the samples was confirmed by retention time studies or co-chromatography on different liquid phases, gas-liquid chromatography-mass spectrometry, and enzyme hydrolyzate studies. Fababean, broad bean, common vetch and Vicia narbonensis contained 0.72, 0.71, 0.75 and 0.06% vicine and 0.27,0.19,0.08 and 0.01 % convicine, respectively. Examination of fababean starch and protein fractions revealed that the glycosides were concentrated in the protein fraction. Neither vicine nor convicine were found in chickpea, soybean, lentil, baby lima bean, blue lupine, sweet white lupine, mung bean, field pea, navy bean, kidney bean, grass pea, pinto bean, cowpea and Great Northern bean or in beet root.
Resume A l'aide de chromatographie en phase gaz-liquide, on a etudie la presence et la distribution de la vicine et de la convicine dans certaines legumineuses et dans la racine de betterave. Il a ete possible de confirmer la presence de glycosides dans les echantilIons par des etudes de temps de retention ou par chromatographie parallele sur differentes phases liquides, en chromatographie en phase gaz-liquide suivie de spectrometrie de masse, et par des etudes d'hydrolysats enzymatiques. La feverole, la feve des Inarais, la vesce commune et Vicia narbonensis contenaient respectivement 0.72, 0.71, 0.75 et 0.06% de vicine et 0.27, 0.19, 0.08 et 0.01 % de convicine. A I' examen des fractions amidon et proteine de la feverole, on a observe que les glycosides se trouvaient concentres dans la fraction proteine. On n'a pas trouve de vicine ni de convicine dans le po is chiche, la feve soja, la lentille, la mini feve lima, le lupin bleu, le lupin blanc de senteur, I'amberique, le pois des champs, le haricot blanc, le haricot Vulgaire, le pois fourrager (Lathyrus sativus), la feve pinto, le dolique de Chine (Vigna sinensis), et la feve du Grand Nord, ou dans la racine de betterave.
Introduction . Vicine and convicine are implicated as the factors In fababeans responsible for an acute hemolytic disease that affects certain susceptible individuals following ingestion of the bean (Lin and Ling, 1962a, 1962b; Mager et al., 1965). In vitro studies Copyright
Materials and Methods Seeds of fababean (V. faba minor L.), broad bean
0315-5463/801010035·05$02.00/0 1980 Canadian Institute of Food Science and Technology
35
(V. faba major L.), common vetch (V. sativaL.), V. narbonensis L. (a possible wild progenitor of fababean), soybean (Glycine max (L.) Merr.), chickpea (Cicer arietinum L.), lentil (Lens culinaris Medic.), baby lima bean (Phaseolus lunatus L.), mung bean (P. aureus Roxb.), field pea (Pisum sativum arvense L.), navy bean (P. vulgaris L.), kidney bean (P. vulgaris L.), grass pea (Lathyrus sativus L.), cowpea (Vigna sinensis Endl.), sweet white lupine (L. albus L.) and blue lupine (L. angustifolius L.) were obtained from plots grown at the University of Saskatchewan or commercial sources. Root of red table beet (Beta vulgaris L.) was obtained from a local supermarket. Vicine was isolated from V. faba minor by the original Ritthausen procedure as described by Bendich and Clements (1953). Purity data obtained on the isolate compared favourably with that obtained on authentic samples supplied by Dr. A. Bendich of the Memorial Sloan-Kettering Cancer Center, New York and Dr. R. Gmelin of the Institute of Pharmacognosy and Phytochemistry, Berlin. Convicine was isolated from both V. faba major and minor by the procedure of Bien et al. (1968) and an authentic sample was also received from Dr. S. Bien of the Technion-Israel Institute of Technology, Israel. I3-Glucosidase (almond extract) was obtained from Sigma Chemical Co., St. Louis, Mo. Sample Preparation: Whole mature seeds of the different pulse crops were finely ground and the meal oven-dried overnight at 60°C. Beet root was shredded, freeze-dried and ground using a mortar and pestle. Crude extracts of vicine and convicine from the ground sample were prepared in duplicate for gas-liquid chromatography (GLC) and gas-liquid chromatography-mass-spectrometry (GC-MS) as described by Pitz and Sosulski (1979). For l3-glucosidase treatment, an aliquot of the final filtrate (pH 4.2) of each species was adjusted to pH
5.25 with O.lN NaOH and a 1 ml volume mixed with 1 ml l3-glucosidase solution (aqueous solution, 1 ml == 1 mg) and incubated overnight at 37°C. Enzyme activity was then terminated by heating and the mixture freeze-dried and silylated for GLC. Other 1 ml volumes were treated with 1 ml suspensions of heat-denatured enzyme and prepared for GLC in a similar manner. Gas-Liquid Chromatography: A Hewlett-Packard Model 402 gas chromatograph equipped with a dual flame ionization detector and glass columns (1.83 m x 3 mm I.D.), one packed with 4% OV-17 on Chromosorb W (HP) 80-100 mesh, and the other with 4% OV-225 on the same support material was used. The column oven operating temperature was isothermal at 230°C in the former case and 195°C in the latter. A third column packed with 3% OV-l on Chromosorb W (HP) was also used at an isothermal column oven temperature of 230°C. The injection port and detector oven were maintained at 280°C and the carrier gas (helium) flow rate was 60 cc/min. Gas Chromatography-Mass Spectrometry: Electron impact ionization (El) mass spectral data of the trimethylsilyl derivatives were obtained using a Finnigan Model 3300 instrument interfaced to an Incos Model 2300 data acquisition system. A glass column (1.83 m x 2 mm I.D.) containing 4% OV-17 on Chromosorb W (HP) 80-100 mesh was employed. The carrier gas (helium) flow rate was 30 cc/min. Operating temperatures were 235°C, oven; 250°C, injection port; 250°C, separator oven. Mass spectral scans were taken every 2 sec at an electron energy of 70 e V.
Results and Discussion Quantitative GLC analysis on the 4% OV-17 column at 230°C (Figure 1) showed that the small fababean, broad bean, common vetch and V. narbo-
Table 1. Qualitative and quantitative vicine data obtained by gas-liquid chromatography on seeds of fababean and other pulses and on beet root.
Species fababean broad bean common vetch
V. narbonensis chickpea soybean lentil baby lima bean blue lupine white lupine mung bean field pea navy bean kidney bean grass pea pinto bean cowpea Northern bean beet root
_________________ ~ ___________ .Qu~it'!tiy~' ____ .~~ _____ ~~_. _______ ~~~ Co-chromatography with Peak hydrolyzed Mass spectral ______aIJthentic...vicine._____ with J3-glucosidase data OV-17 OV-225 (OV-17 column)
+ (lOO) + ( 99)
+ (104) + ( 8)
+ (lOO) + ( 99) + (104)
+
+ ( 8)
+
+ ( 20)
+ +
+ + +
+
+ ( 31) + ( 12) + ( 6)
+(
7)
+ ( 1)
+ ( 4)
+ ( 3) + ( 4)
+ ( 2) + ( 2) + ( 2) + ( 2)
+ ( 2) + ( 2)
N/A N/A N/A N/A N/A N/A N/A N/A N/A
__ Qu~'!!i!ative' (%, dry basis)
__
0.72 0.71 0.75
0.06
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
'( +) sign denotes result identical to authentic vicine and (-) sign a different result. Values in brackets indicate approximate peak area as a percent of that found in the small fababean. N/A indicates that data was unobtainable because of absence or insufficient amounts for analysis.
36/Pitz, Sosulski and Hogge
Can. Inst. Food Sci. Technol. 1. Vo!. 13, No. 1, January 1980
FABABEAN OV-17 LIQUID PHASE
FABABEAN OV-225 LIQUID PHASE
UJ
r/)
z o0-
1
.'.:
r/)
UJ Cl: Cl:
11
•
........ authentic convicine ---- authentic vici"e
o
t
....... authentic convicine ---- authentic vicine
UJ ,.... UJ o
o
2
4
6
10
8
12
0
2
4
6
8
10
12
ELUTION TIME IN MINUTES
Fig. 1. Gas-liquid chromatograms of fababean extract obtained using OV-17 and OV-225 liquid phases. Broad bean, common vetch and V. narbonensis also had components that co-chromatographed with the authentic glycosides on both liquid phases.
nensis contained 0.72, 0.71, 0.75 and 0.06% vicine (Table 1) and 0.27,0.19,0.08 and 0.01 % convicine, respectively (Table 2). The fababean and broad bean values agree with those reported in the literature (Collier, 1976; lamalian et al., 1977; Olsen and Andersen, 1978). Bendich and Clements (1953) isolated 2.75g of high purity vicine from two kg of seeds of common vetch. Confirmation of the peaks as those of vicine and convicine was obtained when they were shown to have identical mass spectra to those of the authentic glycosides. In addition, they co-chromatographed with the authentic glycosides on the OV-225 liquid phase (Figure 1) and were not present in chromatograms of l3-glucosidase hydrolyzates. Their absence in the chromatograms of enzyme hydrolyzates was consistent with their prop-
erties (Pitz and Sosulski, 1979). The quantity of convicine in the V. narbonensis sample was very small and a reliable mass spectrum could not be obtained. The enzyme study and co-chromatography were taken as sufficient evidence of its presence. V. narbonensis appeared to contain much higher levels of the oligosaccharides, raffinose and stachyose (identified in the samples by co-chromatography), than the other Vicia species. The vicine and convicine contents of various fababean fractions obtained by pin-milling and air classification are given in Table 3. The high glycoside contents of the protein fractions could be a constraint on the widespread use of fababean protein obtained by this dry milling process. Collier (1976) and Olsen and Andersen (1978) also found
Table 2. Qualitative and quantitative convicine data obtained by gas-liquid chromatography on seeds of fababean and other pulses and on beet root. ---
- - - -
---
. .-
-
___________ Q"'u=a=lit=ative a
Species
Co-chromatography with authentic convicine OV-17 OV-225
fababean broad bean common vetch
+ (100) + ( 70) + ( 30)
V. narbonensis
+ ( 4) + (218)
chickpea soybean lentil baby lima bean blue lupine white lupine mung bean field pea navy bean kidney bean grass pea pinto bean COWpea Northern bean beet root
+( +(
+
(100)
+ ( 70) + ( 30)
+(
4)
-
... _ - - - - _ . _ ..
Peak hydrolyzed with l3-glucosidase (OV-17 column)
Mass spectral data
+ +
+ +
+ +
+
+
98) 51)
+ ( 29) + ( 19) + ( 23)
+(
1)
N/A N/A N/A N/A N/A N/A N/A N/A N/A
+ ( 30)
__quantita.!i~ (%. dry basis)
0.27 0.19 0.08 0.01
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
"( +) sign denotes result identical to authentic convicine and (-) sign a different result. peak area as a percent of that found in the small fababean. amounts for analysis.
1. l1I8t. Can. Sci. Technol. Aliment. Vo!. 13. No. 1, Janvier 1980
Values in brackets indicate the approximate NI A indicates that data was unobtainable because of absence or insufficient
Pitz, Sosulski and Hogge/37
Table 3. Yicine and convicine contents of fababean fractions obtained by pin-milling and air classification."
Fraction
however, whether their study was done on the sweet white lupine or the bitter high alkaloid white lupine. The aV-17 chromatograms of beet root and the other pulses examined (mung bean, field pea, navy bean, kidney bean, grass pea, pinto bean, cowpea and Great Northern bean) had little or no peaks at retention times corresponding to those of the authentic glycosides (Tables 1 and 2). For this reason, reliable mass spectral data could not be obtained on these pulses. The presence of even trace all!0unts of either glycoside could not be substantiated by analysis on the aV-225 liquid phase or chromatography of the enzyme hydrolyzates (Tables 1 and 2). Jamalian et al. (1977) reported trace amounts of vicine in peas. In summary, vicine and convicine appear to be associated only with Vicia species and are not present in other common legume.s or beet root: Th~s data is consistent with the expenence that fav1sm 1S only associated with the ingestion of V. faba. Also, fresh green beans have been found to contain much higher levels of vicine and convicine than dry mature beans (Jamalian et al., 1977; Pitz and Sosulski, 1979) and the incidence of favism resulting from ingestion of fresh green and dry beans parallels this occurrence (Dacie, 1967; Belsey, 1973). Both these findings are consistent with the role of vicine and convicine as causative agents of favism.
Protein content % glycosidein !!'~!i9_Il__(~!Y basist (N x 6.25) Convicine Yicine Total
Whole seeds (meal) Flour Starch I Starch 11 Protein I Protein 11
28.1
0.26
0.67
0.93
31.3 21.3 12.4 71.5 66.5
0.29 0.19 0.08 0.68 0.63
0.68 0.47 0.23 1.50 1.45
0.97 0.66 0.31 2.18 2.08
'See Yose et al. (1976) for procedures used to obtain starch and protein fractions I and 11. bYalues reported are means of two determinations.
high glycoside contents in the protein fr~cti?ns obtained by mechanical processes, but protem 1S0lates obtained by isoelectric precipitation were virtually free of vicine and convicine. Chickpea, soybean, lentil, baby lima bean, blue lupine and sweet white lupine samples gave significant peaks at retention times corresponding to those of the authentic glycosides when analyzed on the av -17 column (Tables 1 and 2). However, the peaks were still present in the chromatograms of the enzyme hydrolyzates, and the GC-MS data showed that neither peak in any of the samples was vicine or convicine. Furthermore, when the samples were analyzed on the av -225 column, the peaks no longer corresponded in retention time to the authentic glycosides (Figure 2). The aV-225 chromatogram of blue lupine, however, retained small peaks in the convicine and vicine regions, and lentil had a similar peak in the convicine region. These two species were, therefore, further studied using a 3% aV-1 column at 230°C. Authentic convicine and vicine (5 mg of each), added directly to 10 g of meal of each species before extraction, eluted at the 5.7 and 7.0 min mark, respectively. Samples which had no added glycosides did not show peaks at those elution times. The present findings differ from those of Pompei and Lucisano (1976) who reported 6.55% vicine and convicine in white lupine. The authors did not state,
Acknowledgements Financial support from the College of Graduate Studies and Research, University of Saskatchewan, and the National Research Council of Canada is greatly appreciated.
References Belsey, M.A. 1973. The epidemiology of favism. Bull. World Health Organ. 48: I. Bendich. A. and Clements. G.c. 1953. A revision of the structural formulation of vicine and its pyrimidine aglycone, divicine. Biochim. Biophys. Acta 12: 462. Bien, S.• Salemmik. G .. Zamir, L. and Rosenblum, M. 1%8. The structure of convicine. J. Chem. Soc. 1968C: 496. Collier. H.B. 1976. The estimation of vidne in fababeans by an ultraviolet spectrophotometric method. Can. Inst. Food Sci. Technol. J. 9: 155. Dade. J.V. 1967. Favism. The Haemolytic Anaemias, 2nd ed .. Part IV. J. and A. Churchill. Lld .. London. pp. 1061.
CHICKPEA QV-17 L10UID PHASE
CHICKPEA QV- 225 LIOUID PHASE
w
CIl
Z
...... authentic convicine -- -- authentic vicine
...... . authentic convicine ----. authentic vicine
oQ. CIl
W
et: et:
o
t3w ~
w
c
o
2
4
6
8
10
12
0
2
4
6
8
10
12
ELUTION TIME IN MINUTES
Fig. 2. Gas-liquid chromatograms of chickpea extract obtained using QY-17 and QY-225Iiq.uid. phas.es. Soybean, lentil, baby lima bean, blue lupine and white lupine gave similar chromatograms when analyzed on both lIqUId phases.
38/Pitz, Sosulski and Hogge
Can. Inst. Food Sci. Technol. J. Vol. 13, No. I, January 1980
L.R. Hogge Prairie Regional Laboratory National Research Council Saskatoon, Saskatchewan S7N OW9
Abstract
carried out by the latter workers have shown that the aglycones, divicine and isouramil, derived from vicine and convicine, respectively, have a marked destructive effect on reduced glutathione (GSH) levels of glucose-6-phosphate dehydrogenase deficient red blood cells, a characteristic feature of the disease. Vicine and convicine were first isolated from the seeds of Vicia sativa (Ritthausen and Kreusler, 1870; Ritthausen, 1881) and were later found in Vicia faba (Johnson, 1914; Fisher and Johnson, 1932). Vicine was also reported to be present in beet juice and peas (Bendich and Clements, 1953; Jamalian et al., 1977) and in Lupinus albus (Pompei and Lucisano, 1976). Some investigators (Liener, 1973) have questioned whether vicine and convicine are actually responsible for favism because the disease is only associated with the ingestion of V. faba. Therefore, further information is required on the occurrence of these glycosides in other plant materials. Both vicine and convicine are l3-glycosides of the plant pyrimidines, divicine and isouramil, respectively, (Bendich and Clements, 1953; Bien et aI., 1968) and are hydrolyzed by l3-glucosidases (Herissey and Cheymol, 1931; Mager et aI., 1965). In addition, vicine and convicine form volatile trimethylsilyl derivatives which permit a gas-liquid chromatography-mass spectrometry analysis of the glycosides (Pitz and Sosulski, 1979). Advantage was taken of these properties and used as qualitative criteria in the present work to study the occurrence and distribution of vicine and convicine in the seeds of a number of Vicia species, other pulse crops and beet root.
The occurrence and distribution of vicine and convicine in a number of pulse crops and beet root were studied using gas-liquid chromatography. The presence of the glycosides in the samples was confirmed by retention time studies or co-chromatography on different liquid phases, gas-liquid chromatography-mass spectrometry, and enzyme hydrolyzate studies. Fababean, broad bean, common vetch and Vicia narbonensis contained 0.72, 0.71, 0.75 and 0.06% vicine and 0.27,0.19,0.08 and 0.01 % convicine, respectively. Examination of fababean starch and protein fractions revealed that the glycosides were concentrated in the protein fraction. Neither vicine nor convicine were found in chickpea, soybean, lentil, baby lima bean, blue lupine, sweet white lupine, mung bean, field pea, navy bean, kidney bean, grass pea, pinto bean, cowpea and Great Northern bean or in beet root.
Resume A l'aide de chromatographie en phase gaz-liquide, on a etudie la presence et la distribution de la vicine et de la convicine dans certaines legumineuses et dans la racine de betterave. Il a ete possible de confirmer la presence de glycosides dans les echantilIons par des etudes de temps de retention ou par chromatographie parallele sur differentes phases liquides, en chromatographie en phase gaz-liquide suivie de spectrometrie de masse, et par des etudes d'hydrolysats enzymatiques. La feverole, la feve des Inarais, la vesce commune et Vicia narbonensis contenaient respectivement 0.72, 0.71, 0.75 et 0.06% de vicine et 0.27, 0.19, 0.08 et 0.01 % de convicine. A I' examen des fractions amidon et proteine de la feverole, on a observe que les glycosides se trouvaient concentres dans la fraction proteine. On n'a pas trouve de vicine ni de convicine dans le po is chiche, la feve soja, la lentille, la mini feve lima, le lupin bleu, le lupin blanc de senteur, I'amberique, le pois des champs, le haricot blanc, le haricot Vulgaire, le pois fourrager (Lathyrus sativus), la feve pinto, le dolique de Chine (Vigna sinensis), et la feve du Grand Nord, ou dans la racine de betterave.
Introduction . Vicine and convicine are implicated as the factors In fababeans responsible for an acute hemolytic disease that affects certain susceptible individuals following ingestion of the bean (Lin and Ling, 1962a, 1962b; Mager et al., 1965). In vitro studies Copyright
Materials and Methods Seeds of fababean (V. faba minor L.), broad bean
0315-5463/801010035·05$02.00/0 1980 Canadian Institute of Food Science and Technology
35
(V. faba major L.), common vetch (V. sativaL.), V. narbonensis L. (a possible wild progenitor of fababean), soybean (Glycine max (L.) Merr.), chickpea (Cicer arietinum L.), lentil (Lens culinaris Medic.), baby lima bean (Phaseolus lunatus L.), mung bean (P. aureus Roxb.), field pea (Pisum sativum arvense L.), navy bean (P. vulgaris L.), kidney bean (P. vulgaris L.), grass pea (Lathyrus sativus L.), cowpea (Vigna sinensis Endl.), sweet white lupine (L. albus L.) and blue lupine (L. angustifolius L.) were obtained from plots grown at the University of Saskatchewan or commercial sources. Root of red table beet (Beta vulgaris L.) was obtained from a local supermarket. Vicine was isolated from V. faba minor by the original Ritthausen procedure as described by Bendich and Clements (1953). Purity data obtained on the isolate compared favourably with that obtained on authentic samples supplied by Dr. A. Bendich of the Memorial Sloan-Kettering Cancer Center, New York and Dr. R. Gmelin of the Institute of Pharmacognosy and Phytochemistry, Berlin. Convicine was isolated from both V. faba major and minor by the procedure of Bien et al. (1968) and an authentic sample was also received from Dr. S. Bien of the Technion-Israel Institute of Technology, Israel. I3-Glucosidase (almond extract) was obtained from Sigma Chemical Co., St. Louis, Mo. Sample Preparation: Whole mature seeds of the different pulse crops were finely ground and the meal oven-dried overnight at 60°C. Beet root was shredded, freeze-dried and ground using a mortar and pestle. Crude extracts of vicine and convicine from the ground sample were prepared in duplicate for gas-liquid chromatography (GLC) and gas-liquid chromatography-mass-spectrometry (GC-MS) as described by Pitz and Sosulski (1979). For l3-glucosidase treatment, an aliquot of the final filtrate (pH 4.2) of each species was adjusted to pH
5.25 with O.lN NaOH and a 1 ml volume mixed with 1 ml l3-glucosidase solution (aqueous solution, 1 ml == 1 mg) and incubated overnight at 37°C. Enzyme activity was then terminated by heating and the mixture freeze-dried and silylated for GLC. Other 1 ml volumes were treated with 1 ml suspensions of heat-denatured enzyme and prepared for GLC in a similar manner. Gas-Liquid Chromatography: A Hewlett-Packard Model 402 gas chromatograph equipped with a dual flame ionization detector and glass columns (1.83 m x 3 mm I.D.), one packed with 4% OV-17 on Chromosorb W (HP) 80-100 mesh, and the other with 4% OV-225 on the same support material was used. The column oven operating temperature was isothermal at 230°C in the former case and 195°C in the latter. A third column packed with 3% OV-l on Chromosorb W (HP) was also used at an isothermal column oven temperature of 230°C. The injection port and detector oven were maintained at 280°C and the carrier gas (helium) flow rate was 60 cc/min. Gas Chromatography-Mass Spectrometry: Electron impact ionization (El) mass spectral data of the trimethylsilyl derivatives were obtained using a Finnigan Model 3300 instrument interfaced to an Incos Model 2300 data acquisition system. A glass column (1.83 m x 2 mm I.D.) containing 4% OV-17 on Chromosorb W (HP) 80-100 mesh was employed. The carrier gas (helium) flow rate was 30 cc/min. Operating temperatures were 235°C, oven; 250°C, injection port; 250°C, separator oven. Mass spectral scans were taken every 2 sec at an electron energy of 70 e V.
Results and Discussion Quantitative GLC analysis on the 4% OV-17 column at 230°C (Figure 1) showed that the small fababean, broad bean, common vetch and V. narbo-
Table 1. Qualitative and quantitative vicine data obtained by gas-liquid chromatography on seeds of fababean and other pulses and on beet root.
Species fababean broad bean common vetch
V. narbonensis chickpea soybean lentil baby lima bean blue lupine white lupine mung bean field pea navy bean kidney bean grass pea pinto bean cowpea Northern bean beet root
_________________ ~ ___________ .Qu~it'!tiy~' ____ .~~ _____ ~~_. _______ ~~~ Co-chromatography with Peak hydrolyzed Mass spectral ______aIJthentic...vicine._____ with J3-glucosidase data OV-17 OV-225 (OV-17 column)
+ (lOO) + ( 99)
+ (104) + ( 8)
+ (lOO) + ( 99) + (104)
+
+ ( 8)
+
+ ( 20)
+ +
+ + +
+
+ ( 31) + ( 12) + ( 6)
+(
7)
+ ( 1)
+ ( 4)
+ ( 3) + ( 4)
+ ( 2) + ( 2) + ( 2) + ( 2)
+ ( 2) + ( 2)
N/A N/A N/A N/A N/A N/A N/A N/A N/A
__ Qu~'!!i!ative' (%, dry basis)
__
0.72 0.71 0.75
0.06
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
'( +) sign denotes result identical to authentic vicine and (-) sign a different result. Values in brackets indicate approximate peak area as a percent of that found in the small fababean. N/A indicates that data was unobtainable because of absence or insufficient amounts for analysis.
36/Pitz, Sosulski and Hogge
Can. Inst. Food Sci. Technol. 1. Vo!. 13, No. 1, January 1980
FABABEAN OV-17 LIQUID PHASE
FABABEAN OV-225 LIQUID PHASE
UJ
r/)
z o0-
1
.'.:
r/)
UJ Cl: Cl:
11
•
........ authentic convicine ---- authentic vici"e
o
t
....... authentic convicine ---- authentic vicine
UJ ,.... UJ o
o
2
4
6
10
8
12
0
2
4
6
8
10
12
ELUTION TIME IN MINUTES
Fig. 1. Gas-liquid chromatograms of fababean extract obtained using OV-17 and OV-225 liquid phases. Broad bean, common vetch and V. narbonensis also had components that co-chromatographed with the authentic glycosides on both liquid phases.
nensis contained 0.72, 0.71, 0.75 and 0.06% vicine (Table 1) and 0.27,0.19,0.08 and 0.01 % convicine, respectively (Table 2). The fababean and broad bean values agree with those reported in the literature (Collier, 1976; lamalian et al., 1977; Olsen and Andersen, 1978). Bendich and Clements (1953) isolated 2.75g of high purity vicine from two kg of seeds of common vetch. Confirmation of the peaks as those of vicine and convicine was obtained when they were shown to have identical mass spectra to those of the authentic glycosides. In addition, they co-chromatographed with the authentic glycosides on the OV-225 liquid phase (Figure 1) and were not present in chromatograms of l3-glucosidase hydrolyzates. Their absence in the chromatograms of enzyme hydrolyzates was consistent with their prop-
erties (Pitz and Sosulski, 1979). The quantity of convicine in the V. narbonensis sample was very small and a reliable mass spectrum could not be obtained. The enzyme study and co-chromatography were taken as sufficient evidence of its presence. V. narbonensis appeared to contain much higher levels of the oligosaccharides, raffinose and stachyose (identified in the samples by co-chromatography), than the other Vicia species. The vicine and convicine contents of various fababean fractions obtained by pin-milling and air classification are given in Table 3. The high glycoside contents of the protein fractions could be a constraint on the widespread use of fababean protein obtained by this dry milling process. Collier (1976) and Olsen and Andersen (1978) also found
Table 2. Qualitative and quantitative convicine data obtained by gas-liquid chromatography on seeds of fababean and other pulses and on beet root. ---
- - - -
---
. .-
-
___________ Q"'u=a=lit=ative a
Species
Co-chromatography with authentic convicine OV-17 OV-225
fababean broad bean common vetch
+ (100) + ( 70) + ( 30)
V. narbonensis
+ ( 4) + (218)
chickpea soybean lentil baby lima bean blue lupine white lupine mung bean field pea navy bean kidney bean grass pea pinto bean COWpea Northern bean beet root
+( +(
+
(100)
+ ( 70) + ( 30)
+(
4)
-
... _ - - - - _ . _ ..
Peak hydrolyzed with l3-glucosidase (OV-17 column)
Mass spectral data
+ +
+ +
+ +
+
+
98) 51)
+ ( 29) + ( 19) + ( 23)
+(
1)
N/A N/A N/A N/A N/A N/A N/A N/A N/A
+ ( 30)
__quantita.!i~ (%. dry basis)
0.27 0.19 0.08 0.01
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
"( +) sign denotes result identical to authentic convicine and (-) sign a different result. peak area as a percent of that found in the small fababean. amounts for analysis.
1. l1I8t. Can. Sci. Technol. Aliment. Vo!. 13. No. 1, Janvier 1980
Values in brackets indicate the approximate NI A indicates that data was unobtainable because of absence or insufficient
Pitz, Sosulski and Hogge/37
Table 3. Yicine and convicine contents of fababean fractions obtained by pin-milling and air classification."
Fraction
however, whether their study was done on the sweet white lupine or the bitter high alkaloid white lupine. The aV-17 chromatograms of beet root and the other pulses examined (mung bean, field pea, navy bean, kidney bean, grass pea, pinto bean, cowpea and Great Northern bean) had little or no peaks at retention times corresponding to those of the authentic glycosides (Tables 1 and 2). For this reason, reliable mass spectral data could not be obtained on these pulses. The presence of even trace all!0unts of either glycoside could not be substantiated by analysis on the aV-225 liquid phase or chromatography of the enzyme hydrolyzates (Tables 1 and 2). Jamalian et al. (1977) reported trace amounts of vicine in peas. In summary, vicine and convicine appear to be associated only with Vicia species and are not present in other common legume.s or beet root: Th~s data is consistent with the expenence that fav1sm 1S only associated with the ingestion of V. faba. Also, fresh green beans have been found to contain much higher levels of vicine and convicine than dry mature beans (Jamalian et al., 1977; Pitz and Sosulski, 1979) and the incidence of favism resulting from ingestion of fresh green and dry beans parallels this occurrence (Dacie, 1967; Belsey, 1973). Both these findings are consistent with the role of vicine and convicine as causative agents of favism.
Protein content % glycosidein !!'~!i9_Il__(~!Y basist (N x 6.25) Convicine Yicine Total
Whole seeds (meal) Flour Starch I Starch 11 Protein I Protein 11
28.1
0.26
0.67
0.93
31.3 21.3 12.4 71.5 66.5
0.29 0.19 0.08 0.68 0.63
0.68 0.47 0.23 1.50 1.45
0.97 0.66 0.31 2.18 2.08
'See Yose et al. (1976) for procedures used to obtain starch and protein fractions I and 11. bYalues reported are means of two determinations.
high glycoside contents in the protein fr~cti?ns obtained by mechanical processes, but protem 1S0lates obtained by isoelectric precipitation were virtually free of vicine and convicine. Chickpea, soybean, lentil, baby lima bean, blue lupine and sweet white lupine samples gave significant peaks at retention times corresponding to those of the authentic glycosides when analyzed on the av -17 column (Tables 1 and 2). However, the peaks were still present in the chromatograms of the enzyme hydrolyzates, and the GC-MS data showed that neither peak in any of the samples was vicine or convicine. Furthermore, when the samples were analyzed on the av -225 column, the peaks no longer corresponded in retention time to the authentic glycosides (Figure 2). The aV-225 chromatogram of blue lupine, however, retained small peaks in the convicine and vicine regions, and lentil had a similar peak in the convicine region. These two species were, therefore, further studied using a 3% aV-1 column at 230°C. Authentic convicine and vicine (5 mg of each), added directly to 10 g of meal of each species before extraction, eluted at the 5.7 and 7.0 min mark, respectively. Samples which had no added glycosides did not show peaks at those elution times. The present findings differ from those of Pompei and Lucisano (1976) who reported 6.55% vicine and convicine in white lupine. The authors did not state,
Acknowledgements Financial support from the College of Graduate Studies and Research, University of Saskatchewan, and the National Research Council of Canada is greatly appreciated.
References Belsey, M.A. 1973. The epidemiology of favism. Bull. World Health Organ. 48: I. Bendich. A. and Clements. G.c. 1953. A revision of the structural formulation of vicine and its pyrimidine aglycone, divicine. Biochim. Biophys. Acta 12: 462. Bien, S.• Salemmik. G .. Zamir, L. and Rosenblum, M. 1%8. The structure of convicine. J. Chem. Soc. 1968C: 496. Collier. H.B. 1976. The estimation of vidne in fababeans by an ultraviolet spectrophotometric method. Can. Inst. Food Sci. Technol. J. 9: 155. Dade. J.V. 1967. Favism. The Haemolytic Anaemias, 2nd ed .. Part IV. J. and A. Churchill. Lld .. London. pp. 1061.
CHICKPEA QV-17 L10UID PHASE
CHICKPEA QV- 225 LIOUID PHASE
w
CIl
Z
...... authentic convicine -- -- authentic vicine
...... . authentic convicine ----. authentic vicine
oQ. CIl
W
et: et:
o
t3w ~
w
c
o
2
4
6
8
10
12
0
2
4
6
8
10
12
ELUTION TIME IN MINUTES
Fig. 2. Gas-liquid chromatograms of chickpea extract obtained using QY-17 and QY-225Iiq.uid. phas.es. Soybean, lentil, baby lima bean, blue lupine and white lupine gave similar chromatograms when analyzed on both lIqUId phases.
38/Pitz, Sosulski and Hogge
Can. Inst. Food Sci. Technol. J. Vol. 13, No. I, January 1980