Plant Science 148 1999 111 – 119
Immunoanalysis of isoflavonoids in Pisum sati6um and Vigna radiata
Oldrˇich Lapcˇı´k
a,
, Martin Hill
a
, Ivan C erny´
b
, Jaromı´r Lachman
c
, Nawaf Al-Maharik
d
, Herman Adlercreutz
e
, Richard Hampl
a
a
Institute of Endocrinology, Na´rodnı´ trˇı´da
8
,
116 94
Praha
1
, Czech Republic
b
Institute of Organic Chemistry and Biochemistry, Praha, Czech Republic
c
Czech Uni6ersity of Agriculture, Praha, Czech Republic
d
Department of Chemistry, Uni6ersity of Helsinki, Helsinki, Finland
e
Department of Clinical Chemistry, Uni6ersity of Helsinki, Helsinki, Finland Received 19 January 1999; received in revised form 25 June 1999; accepted 25 June 1999
Abstract
Radioimmunoassays RIAs combined with liquid chromatography were used for study of isoflavonoids in seeds of pea Pisum sati6um and mung bean Vigna radiata. Radioimmunoassays with these specifities were used: 1 daidzein and its 4-derivatives e.g.
formononetin; 2 daidzein and its 7-derivatives e.g. daidzin, isoformononetin; 3 genistein and its 4-derivatives e.g. biochanin A; and 4 genistein and its 7-derivatives e.g. genistin, prunetin. Dormant or germinating seeds were extracted with 80 ethanol.
Immunoreactivities were measured either in crude extracts or after chromatographic fractionation by HPLC reversed phase, octadecylsilica. Chromatographic mobilities of immunoreactive fractions were compared to those of daidzein, daidzin, for-
mononetin, isoformononetin, genistein, genistin biochanin A and prunetin standards. Extracts from Vigna radinfa contained daidzein, genistein and their 7-O glucosides, daidzin and genistin, respectively. No immunoreactivity was recorded in HPLC
fractions corresponding to glycosides in extracts from P. sati6um, but the methods sensitive to 7-derivatives of daidzein and genistein showed peaks with chromatographic mobilities identical to those of the 7-methoxyderivatives, isoformononetin and
prunetin, respectively. In additional experiments, the pea extracts were fractionated either by thin layer chromatography TLC on silica or by ion-exchange TLC on aminosilica. Identity of the daidzein-7 and genistein-7 immunoreactive entities with
isoformononetin and prunetin, respectively, was confirmed by the identical chromatographic behavior in all these different chromatographic systems. © 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Vigna radiata; Pisum sati6um; Isoflavonoid; 7-methoxy isoflavonoid; Isoformononetin; Prunetin; Immunoassay www.elsevier.comlocateplantsci
1. Introduction
Isoflavonoids are found characteristically in the Viciaceae sub-family of the Fabaceae. They were
found in different parts of leguminous plants roots, leaves, seeds. Isoflavonoids are typically
stored as 7-O glycosides, whilst secretion of agly- cones is elicited by different physiologic or patho-
logic situations [1]. In addition to their presence in legumes, isoflavonoids were found in genera Iris
Iridaceae, Prunus Rosaceae, Podocarpus Podo- carpaceae,
Maclura Moraceae
and Iresine
Amaranthaceae [2,3]. Low concentrations of cer- tain isoflavonoids were found recently in numer-
ous ‘non-classical’ plants, e.g. barley, wheat, sunflower seeds or tea [4,5].
Isoflavonoids, present in food of plant origin, were shown to influence the endocrine system in
animals by interaction with hormone receptors andor steroid metabolizing enzymes [5]. Recently,
we have developed radioimmunoassays RIA for measurement of daidzein and genistein in human
body fluids and in beer [6 – 9] Fig. 1. These
Corresponding author. Tel.: + 420-2-24905-275; fax: + 420-2- 24905-325.
E-mail address
:
olapcikendo.cz O. Lapcˇı´k 0168-945299 - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 1 2 5 - 9
methods are now being used for screening of isoflavonoids in dormant and germinating seeds.
Here we present evidence of daidzein, for- mononetin, isoformononetin and prunetin in P.
sati6um and daidzin, daidzein, genistin and genis- tein in V. radiata.
2. Experimental
2
.
1
. Chemicals Daidzin was a generous gift of Dr Takaaki
Yasuda from Tohoku College of Pharmacy, Miyagi, Japan. Equol, O-desmethylangolensin,
dihydrodaidzein and carboxymethyl derivatives of daidzein and genistein were synthesized as de-
scribed elsewhere [6,7].
Diethyl ether was from Synthesia Pardubice, Czech Republic, all other organic solvents were
from Merck Darmstadt, Germany. Daidzein, genistein, genistin, luteolin, quercetin, and api-
genin were from Sigma St. Louis, Missouri, USA, dextran T-70 from Pharmacia Uppsala,
Sweden. All other substances were of analytical grade, from Lachema Brno, Czech Republic.
2
.
2
. NMR
1
H NMR spectra were taken on a Varian- UNITY-200 200 MHz and Varian UNITY-500
500 MHz
spectrometers at
23°C in
hex- adeuteroacetone and hexadeuterodimethyl sulfox-
ide, being referenced to acetone d 2.20 ppm and dimethyl sulfoxide d 2.50 ppm, respec-
tively.
2
.
3
. Mass spectrometry Mass spectrum of prunetin preparation was
recorded on a VG Analytical ZAB-EQ spectrom- eter energy of ionizing electrons 70 eV, ion
source temperature 170°C.
2
.
4
. Synthesis and characterization of isoformononetin and prunetin
Isoformononetin was
synthesized from
daidzein as described by Ingham [10], using dia- zomethane as a methylation agent. Prunetin was
prepared from genistein in the same way. Ethe- real diazomethane 0.5 ml, approximately 250
m mol was added to an ice cooled solution of the
parent isoflavone approximately 50 mmol in dimethyl sulfoxide 0.1 ml and methanol 0.5
ml. The reaction vessel was stopped, the cooling was removed, and the mixture was stirred for 25
min. at room temperature. Then acetic acid was added 1 drop, the solvents were removed in
vacuum, and the residue was coevaporated with toluene 2 × 1 ml. The product was separated
from unreacted isoflavone by column chromatog- raphy on silica gel 5 ml in a mixture of chloro-
form-methanol 50:1. Crystallization of the main fraction from hot methanol gave the correspond-
ing 7-O-methyl isoflavone in the yield of about 50.
7-O-methyl daidzein isoformononetin, m.p. 224 – 225°C Ref. [11] gives 224 – 225°C.
1
H NMR spectrum in d
6
-acetone was in accord with results in the literature [10]. 7-O-methylgenistein
prunetin, m.p. 238 – 239°C Ref. [12] gives 233 – 235°C from ethanol.
1
H NMR spectrum 500 MHz, d
6
-dimethylsulfoxide: 12.96 s, 1 H 5- OH, 9.60 s, 1 H 4-OH, 8.42 s, 1 H H-2,
6.83 d, 2 H J = 8.5, H-3 and H-5, 7.39 d, 2 H J = 8.5, H-2 and H-6, 6.67 d, 1 H J = 2.3,
H-8, 6.42 d, 1 H J = 2.3, H-6, 3.87 s, 3 H OCH
3
. Mass spectrum mz 284 100, M
+
. In addition, both preparations displayed im-
munochemical and chromatographic behavior ex- pected for 7-methoxy derivatives, namely: high
crossreactivity in
the 7-position
based im-
munoassays and low crossreactivity in the 4-po- sition
based ones,
different chromatographic
Fig. 1. Analytes: Daidzein, R
1
= R
2
= R
3
= H; Daidzin, R
1
= glucose, R
2
= R
3
= H; Formononetin, R
1
= R
3
= H, R
2
= CH
3
; Isoformononetin, R
1
= CH
3
, R
2
= R
3
= H; Genistein,
R
1
= R
2
= H, R
3
= OH; Genistin, R
1
= glucose, R
2
= H,
R
3
= OH;
Biochanin A:
R
1
= H,
R
2
= CH
3
, R
3
= OH;
Prunetin: R
1
= CH
3
, R
2
= H, R
3
= OH. Haptens used for
developing of the immunoassays: 1 Daidzein 4-car- boxymethylether, R
1
= H, R
2
= CH
2
COOH, R
3
= H; 2
Daidzein 7-carboxymethylether, R
1
= CH
2
COOH, R
2
= H,
R
3
= H; 3 Genistein 4-carboxymethylether, R
1
= H, R
2
= CH
2
COOH, R
3
= OH;
and 4
Genistein 7-car-
boxymethylether, R
1
= CH
2
COOH, R
2
= H, R
3
= OH.
mobility on aminosilica. Fluorescence of the TLC spots
in UV,
typical for
daidzein and
formononetin, was absent in isoformononetin preparation.
2
.
5
. Plant material Mung beans V. radiata and pea P. sati6um
var. Kapucin were purchased in Country Life Hostivice, Czech Republic. Pea P. sati6um var.
Lantra was obtained in Czech University of Agriculture, Prague, Czech Republic.
Seeds were placed into a polypropylene beaker on a cellulose sheet wet with tap water 1 ml1 g
of seeds and the top was covered partly with a glass plate. One beaker was harvested each day,
the others were wet with several additional drops of water. Seeds were disintegrated by an Eta
1042 mixer Electro-Praga, Hlinsko, Czech Re- public in 80 ethanol 10 mlg of dry weight
and left to extract at 4°C for at least 72 h with several shakings every day. Finally, the debris
was left to settle and the supernatant was cen- trifuged to complete the sedimentation of solid
particles. Aliquots of the extracts were analyzed by radioimmunoassays either directly, or after
chromatographic
fractionation. Before
chro- matography the extracts were evaporated on a
speedvac and reconstituted in water. Lipids were extracted with petroleum ether,
and the water phase evaporated again The dry residue was then dissolved in 80 ethanol a
quarter of the original volume. Typically 20 m1 of this solution corresponding to an extract
from 8 mg of seeds were injected to the HPLC, 5 ml of this solution were applied to a TLC
plate.
2
.
6
. Hydrolysis For some experiments, the water-ethanol ex-
tracts 2.0 ml were evaporated, reconstituted in 1.0 ml of water, and non polar substances were
extracted to light petroleum ether. The lipids de- pleted water phase was then hydrolyzed by
adding hydrochloric acid final volume 2 ml, final concentration 1.0 M, 90°C, 2 h. After hy-
drolysis, the mixture was cooled, and the agly- cones were extracted by 2 ml of diethyl ether.
The dry ether extract was dissolved in original volume of ethanol again. These conditions were
previously shown to hydrolyze glycosides of daidzein and genistein, whilst the aglycones, in-
cluding the methoxy derivatives, were preserved [4].
2
.
7
. Radioimmunoassay RIA methods were used, based on polyclonal
rabbit antibodies to bovine serum albumin con- jugates
with daidzein-4-carboxymethyl
ether Dai-4-, daidzein-7-carboxymethyl ether Dai-7,
genistein-4-carboxymethyl ether Gen-4- and genistein-7-carboxymethyl
ether Gen-7.
Ho- mologous conjugates of respective isoflavone
with [
125
I] tyrosine methyl ester were used as ra- dioligands. Parameters of the assays are summa-
rized in Table 1 [6 – 8].
2
.
8
. TLC Direct phase TLC: Alugram Nano-Sil GUV
254
aluminium sheets Art. 818 142 from Macherey Nagel Duren, Germany were developed in
dichloromethane — isopropanol 95:5. Ion-ex- change TLC: TLC aluminium sheets NH
2
F
254S
Art. 1.05533 from Merck Darmstad, Germany were developed twice in the same system.
After developing the plates were cut with scis- sors to 30 strips 6 mm of width, and each strip
was eluted with 2 ml of ethanol. Aliquots 200 m
l of the eluate were pipetted to glass tubes, evaporated on a speedvac, dissolved in the assay
buffer and analyzed by the appropriate RIA.
2
.
9
. HPLC The HPLC system consisted of LC-6A pump
Shimadzu, Japan, column oven LCO100 and UV detector LCD 2082 Ecom, Czech Republic,
fraction collector FC-203B Gilson, France. An ET 2504 Nucleosil 100-5 C18 Macheray-Nagel,
Germany column was used.Mobile phase A: 40 methanol in water; mobile phase B: 100
methanol. Gradient all steps linearly: 0 min, A = 100, B = 0; 10 min, B = 20; 25 min,
B = 50; 30 min, B = 100, next 10 min. B = 100, then step to A = 100, reconditioning of
the column for 10 min. Flow rate: 1.0 mlmin, temperature 40°C. UV at 254 nm was recorded.
A total of 0.5 ml fractions were collected.
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
