Table 1 Parameters of the radioimmunoassays used in this study. The sensitivities were expressed as the lowest levels of daidzein methods
1, 2 and genistein methods 3, 4, distinguishable from zero with 95 probability. The sensitivity to other compounds detected by the above methods may differ in a similar manner as do the cross-reactivities. The cross-reactivities were expressed as ratios
of 50 intercept of the main analytes to 50 intercepts of the cross-reactants. The cross-reactivities of very weak cross-reactants below 0.5 were calculated as a ratio of the signal elicited by 10 ng of the respective cross-reactant to the analyte
Method 2. daidzein-7- Method 3. genistein-4-
Method 4. genistein-7- Method 1. daidzein-4-
Sensitivity pgtube 1.3
1.4 1.4
2.3 Cross-reactivity
Daidzein 100.0
100.0 5.50
6.10 21.6
100.0 1.30
100.0 Genistein
59.7 Formononetin
1.36 3.90
0.036 0.68
172.90 Biochanin A
0.73 1.48
474.1 0.02
0.22 13.9
Isoformononetin 0.06
Prunetin 137.4
4.54 149.8
B 0.01
Daidzin 52.40
B 0.01
0.24 5.60
B 0.01
B 0.01
26.4 Genistin
2.39 Dihydrodaidzein
2.00 0.36
0.36 0.25
B 0.01
Equol B
0.01 1.57
0.04 B
0.01 B
0.01 B
0.01 Apigenin
B 0.01
Luteolin 0.04
B 0.01
B 0.01
0.02 B
0.01 B
0.01 Quercetin
B 0.01
B 0.01
0 01 B
0. 01 B
0.01 O-desmethylango-
lensin
3. Results
3
.
1
. Isofla6onoid immunoreacti6ity in crude extracts
Daidzein- and genistein- like immunoreactivi- ties were found by RIAs in non-fractionated wa-
ter-ethanolic extracts from dormant seeds of P. sati6um and Vigna radiata, and the signal in-
creased during germination. In both species, the immunoassays
sensitive to
7-derivatives of
daidzein and genistein measured mostly higher levels than the 4-derivatives sensitive ones Fig.
2.
Fig. 2. Isoflavonoid immunoreactivities in ethanolic extracts from P. sati6um and V. radiata: 1 daidzein-4-specific RIA; 2 daidzein-7-specific RIA; 3 genistein-4-specific RIA; and 4 genistein-7-specific RIA. Immunoreactivity was measured using
daidzein 1, 2 and genistein 3, 4 for the calibration of the respective method. The levels were expressed in nanograms per gram of original dry weight of seeds.
Fig. 3. Immunoreactivity in HPLC fractions of ethanolic extracts from dormant and germinating seeds of V. radiata. Extract from 8 mg of seeds was chromatographed, the fractions were divided to ten aliquots and analyzed by RIA. The number assigns the type
of immunoassay used in the same set of samples. Daidzein 1, 2 and genistein 3, 4 were used for construction of calibration curves, respectively. Arrows indicate position of standards on a control chromatogram. One segment on the Y-axis corresponds
to the signal of 10 pg of the respective standard.
3
.
2
. HPLC of V. radiata extracts Immunoassay
of individual
fractions after
HPLC on octadecyl silica showed analogous pat- terns in extracts from dormant seeds and after 3
days of germination. Germinating seeds con- tained higher amounts of all detected immunore-
activities.
According to
the chromatographic
behavior of the immunoreactive peaks, V. radi- ata contained daidzin, daidzein, genistin and
genistein Fig. 3. No signal was detected in that part of chromatogram, where methoxy deriva-
tives of daidzein and genistein would occur. The genistein-7 sensitive method showed two addi-
tional peaks, more polar than genistin, in ex- tracts from germinating V. radiata. It is known,
that isoflavone glycosides may occur malonylated or acetylated at the glucose moiety [13]. The ad-
ditional genistein-7 immunoreactive peaks in the ‘glucoside part’ of chromatogram most probably
reflect formation of conjugates of this type in germinating V. radiata.
3
.
3
. HPLC of P. sati6um extracts The extracts from P. sati6um contained im-
munoreactivities in
the positions
on chro-
matogram corresponding
to daidzein,
formononetin, isoformononetin,
and prunetin
Fig. 4. The immunoreactivity corresponding to
Fig. 4. Immunoreactivity in HPLC fractions of ethanolic extracts from dormant and germinating seeds of pea. Extract from 8 mg of seeds was chromatographed, the fractions were divided to ten aliquots and analyzed by RIA. The number assigns the type of
immunoassay used in the same set of samples. Daidzein 1, 2 and genistein 3, 4 were used for construction of calibration curves, respectively. Arrows indicate position of standards on a control chromatogram. One segment on the Y-axis corresponds to the
signal of 10 pg of the respective standard.
Table 2 Isoflavonoids detected in chromatographic fractions by radioimmunoassays. The individual levels were estimated from the analysis
of corresponding HPLC fractions and expressed in nanograms per gram of original dry weight of seeds
a
Vigna angularis Pisum sati6um
Chromatographic system Compound
II. Rf×100 I. Retention
III. Rf×100 Dormant
3 day germi- Dormant
3 day germi- nation
nation time min
– More polar,
– 3.50
n.d. ++
n.d. n.d.
genistin-like 4.85
Daidzin +
7 n.d.
n.d Genistin
+++ 2
7.15 n.d.
n.d. 24
14.02 Daidzein
+ 7
+ 3
– 15.50
– Unknown
n.d. n.d.
+ 4
daidzein-4-like Genistein
+ 18.45
22 39
n.d. n.d.
23.04 52
49 Isoformononetin n.d.
n.d. +++
2 4
Formononetin n.d.
52 n.d.
23.13 ++
3 68
29.47 50
Prunetin n.d.
n.d. ++
++ 3
Biochanin A 31.61
n.d. 68
n.d. Detected occasionally
a
Dormant seeds, n.d.: not detected; +: 10–50 ngg; ++: 50–200 ngg; +++: 200–500 ngg. 3 day germination: n.d.: not detected; N: increase of the signal during germination N-fold. Chromatographic systems: I. C18 RP HPLC, II. TLC on silica,
III. TLC on aminosilica.
biochanin A was detected only in some samples, as the concentration was low. The daidzein-4
sensitive method always detected one additional peak,
not detected
by the
complementary daidzein-7 sensitive method, moving between
daidzein and genis-tein on the RP HPLC. The signal of this unknown cross-reactant was ap-
proximately equal to that of daidzein, regardless of the strain of pea or germination status.
No immunoreactivity
was found
in that
part of chromatogram, where glycosides would appear.
Genistein was
not clearly
detected either.
3
.
4
. TLC of P. sati6um extracts Chromatographic mobilities of all detected im-
munoreactivities were identical to those of the authentic standards in direct phase TLC on sil-
ica. Chromatographic mobilities of the daidzein- 7
and genistein-7
immunoreactivities were
identical to
those of
isoformononetin and
prunetin standards, respectively, in ion exchange TLC on aminosilica. TLC on aminosilica sepa-
rates both 7-methoxy isoflavones from the other forms 7-glucoside, 4-methoxy and free due to
absence of the 7-OH group.
3
.
5
. Hydrolysis of P. sati6um extracts All immunoreactivities found in P. sati6um al-
most completely resisted hydrolysis in hydrochlo- ric acid 1 M, 2 h, 90°C and were extractable
with diethyl ether.
3
.
6
. Conclusion In conclusion, by combination of different
types of liquid chromatography and radioim- munoassay we have identified several isoflavone
derivatives in dormant and germinating seeds of P. sati6um and V. radiata as summarized in
Table 2.
4. Discussion
