Process for obtaining malonyl isoflavone glycosides and obtaining isoflavone glycosides or isoflavone aglycons from malonyl isoflavone glycosides

A malonylisoflavone glycoside present in soybean is obtained efficiently. In addition, the corresponding isoflavone glycoside and aglycone are obtained from the malonylisoflavone glycoside. An aqueous extract of soybean is adsorbed on an adsorbent, and eluted with an aqueous alcohol solution.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a process for obtaining malonylisoflavone 
glycosides from an aqueous extract of soybean, and to a process for 
obtaining isoflavone glycosides as well as isoflavone aglycones from the 
malonylisoflavone glycosides. 
2. Related Art 
It has hitherto been confirmed that soybean contains isoflavone compounds 
such as daidzin, glycitin, genistin, acetyldaidzin and acetylgenistin, or 
their aglycone derivatives such as daidzein, glycitein and genistein, 
which exhibit a variety of pharmacological effects such as estrogenic 
activity, anti-bacterial activity, antioxidizing activity and cancerocidal 
activity. 
Furthermore, it has been recently confirmed that malonylisoflavone 
glycosides such as malonyldaidzin and malonylgenistin represented by the 
formulae: 
##STR1## 
are present in soybean, and it has been proved that these glycoside 
derivatives comprise the main components of isoflavone compounds in 
soybean. These malonylisoflavone glycosides are easily soluble in water 
and exhibit per se anti-oxidizing activity, so that they are anticipated 
to have pharmacological effects described above from the structural 
similarity to the afore-mentioned isoflavone and aglycone compounds. 
The content of malonylisoflavone glycosides in soybean varies depending on 
the kinds and harvesting times of soybean and typically specified in Table 
1 J. Agric. Food Chem., 42, 1674 (1994)!. 
The contents are expressed by microgram per gram of soybean. 
TABLE 1 
______________________________________ 
Soybean (Year) 
Malonyldaidzin 
Malonylgenistin 
______________________________________ 
American 
Vinton 81 (1989) 
410 958 
Vinton 81 (1990) 
300 743 
Vinton 81 (1991) 
237 545 
Pioneer 9111 (1989) 
690 1756 
Pioneer 9202 (1989) 
630 1705 
Prize (1989) 709 1342 
HP204 (1989) 345 915 
LS301 (1989) 752 1558 
KL72 (1989) 198 1042 
Strayer 2233 (1989) 
385 883 
Japanese 
Keburi (1991) 562 1232 
Keburi (1992) 322 670 
Kuro daizu (1991) 
375 1187 
Kuro daizu (1992) 
222 717 
Raiden (1991) 407 1191 
Raiden (1992) 242 723 
______________________________________ 
In addition, the malonylisoflavone glycosides can be easily treated with an 
alkali or by heating to cut the ester linkage between malonyl moiety and 
glucopyranosyl moiety and to form isoflavone glycosides such as daidzin 
and genistin, which are further treated with an acid or an enzyme to form 
isoflavone aglycones. In other words, the malonylisoflavone glycosides can 
also be used as the raw materials for obtaining daidzin or genistin. 
A method for preparing a malonylisoflavone glycoside from soybean is 
described for example in Japanese Patent Application Kokai (Laid-Open) No. 
3-170495, in which ground soybean is extracted with an alcohol, and the 
extract is further extracted with water incompatible organic solvents via 
several steps to give the product. 
This method has however defects of complicated operation, difficulty of 
purification due to the contamination of oleophilic components during the 
alcohol extraction as well as low yields of 5-20%. 
SUMMARY OF THE INVENTION 
In consideration of the current situations, the present inventors have 
studied on the processes for obtaining a malonylisoflavone glycoside from 
soybean in a simple operation. As a result, they have found that the 
extraction of soybean with water makes possible of the selective 
extraction of the malonylisoflavone glycoside relatively easily, that when 
the aqueous extract is directly put into contact with an adsorbent, the 
malonylisoflavone glycoside in the extract is easily adsorbed on the 
adsorbent, which is then rinsed with an aqueous alcohol solution to elute 
the malonylisoflavone glycoside in efficiency, and that the 
malonylisoflavone glycoside may be treated for example with an alkali to 
convert it easily into an isoflavone glycoside or an isoflavone aglycone. 
The present invention has thus been accomplished.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention is described in detail below. 
Raw materials for malonylisoflavone glycosides include whole soybean, 
dehulled soybean, and defatted soybean, and aqueous extracts thereof are 
usually used as the raw material. The extract is the one which is obtained 
by soaking whole soybeans, dehulled soybeans, or defatted soybeans in 
water at 20.degree.-80.degree. C. for 2-30 hours, but dehulled soybeans or 
defatted soybeans are preferably used because of low extraction rate in 
whole soybeans. 
Naturally, waste water (soaked water of soybeans or whey of tofu) resulting 
in the preparation of tofu or soybean milk can also be used effectively. 
As a preferred embodiment for obtaining an aqueous extract of soybean, 
dehulled soybeans are soaked in water which has been adjusted to pH of 
10.0 or less, preferably 7.5-9.0 with sodium hydroxide at a temperature of 
45.degree.-65.degree. C. for 2-4 hours, and the soaked soybeans are 
removed to give the soaking water as the aqueous extract. 
In this case, if the soaking water has pH of 10.0 or more, the yield of a 
malonylisoflavone glycoside is decreased because the malonylisoflavone 
glycoside is decomposed and an isoflavone glycoside is formed therefrom. 
While it is advantageous to soak soybeans in water at a higher temperature 
in order to increase the extraction rate of the soybean components, the 
malonylisoflavone glycoside will be decomposed at a temperature of 
70.degree. C. or more. Thus, it is suitable to conduct a soaking at a 
temperature of 45.degree.-65.degree. C. in view of the balance of the 
extraction rate and the decomposition rate. 
The defatted soybean used as a raw material is appropriately a 
low-denatured defatted soybean, or it may be a soybean whey which is 
obtained by directly soaking the soybean in water for extraction, or by 
extracting the ground low-denatured defatted soybean with water or an 
aqueous alkali to remove insoluble residues and precipitating the extract 
with an acid at pH of about 4.3 with hydrochloric acid to remove the 
separated soybean protein. 
The filtrate of such an extract of the soybean, from which protein has been 
removed with an ultrafiltration membrane according to necessities, or the 
supernatant of the soybean whey, which has been adjusted to pH of about 
4.3 with hydrochloric acid to precipitate proteins dissolved in the 
extract, is put into contact with an adsorbent. 
The filtrate or the supernatant is put into contact with an adsorbent 
directly or after the filtrate or the supernatant is adjusted to pH of 
about 8.0 with sodium hydroxide. 
These methods are advantageous in that the adsorbed amount per adsorbent is 
increased in the former method, and the malonylisoflavone glycoside and 
the isoflavone glycoside are easily separated in the latter method. 
In either of these methods, the adsorbent used includes for example a 
synthetic adsorbent, active carbon or alumina, specifically DIAION HP-20 
(manufactured by Mitsubishi Chemical Corp.), Purified SHIRASAGI ACTIVE 
CARBON (manufactured by Takeda Chemical Industries, Ltd.), Active Alumina 
(manufactured by Wako Pure Chemical Industries, Ltd.), or the like. 
Contact may be carried out by the conventional methods such as a batch 
method or a column method, for example the extract may be put into contact 
with the adsorbent by flowing through a column in which the adsorbent has 
been packed, and thus most of the malonylisoflavone glycoside in the 
extract is adsorbed on the adsorbent. 
Next, the malonylisoflavone glycoside adsorbed on the adsorbent is eluted 
with an aqueous alcohol solution or an aqueous alkaline alcohol solution. 
The solution thus obtained is concentrated under reduced pressure, or 
concentrated under reduced pressure and lyophilized to give a dry powder 
of the malonylisoflavone glycoside. 
The concentrated or the dry crude powder is a mixture of malonyldaidzin and 
malonylgenistin, which can be fractionally separated by reverse phase 
chromatography. By way of example, the concentrate is flown through a 
column in which an ODS resin (manufactured by YAMAMURA KAGAKU) is packed 
to adsorb the malonylisoflavone glycoside, which is eluted with an aqueous 
alcohol solution to fractionate malonyldaidzin and malonylgenistin, and 
these fractions are concentrated under reduced pressure and then 
lyophilized to give dry powders of malonyldaidzin and malonylgenistin, 
respectively. 
In this connection, malonyldaidzin and malonylgenistin are preferably 
obtained efficiently by the following method. That is, soybean extract is 
put into contact with an adsorbent in the same manner as above, elution is 
conducted with various concentrations of an aqueous alcohol solution to 
fractionate roughly malonyldaidzin and malonylgenistin, and these 
fractions are further purified with an ODS resin column. 
According to the present invention, the malonylisoflavone glycoside, or 
malonyldaidzin and malonylgenistin can be obtained fractionatingly from 
the extract of soybean in a simple procedure, and an isoflavone glycoside 
as well as an isoflavone aglycone can also be obtained starting from the 
above described compounds by the alkali treatment or the like. 
By way of example, a solution having the malonylisoflavone glycoside 
dissolved therein is adjusted to pH of 8-13 with an alkali such as 
ammonia, sodium hydroxide and sodium carbonate and left standing for 0.5 
hour or more to convert the malonylisoflavone glycoside into an isoflavone 
glycoside. The treatment with an alkali having a higher pH results in a 
higher conversion. In addition, the malonylisoflavone glycoside is 
converted into the isoflavone glycoside by heating a solution of the 
malonylisoflavone glycoside at 70.degree.-150.degree. C. for 0.5-12 hours. 
Heating at a higher temperature for a longer period results in a higher 
conversion rate. The conversion rate can be further increased with a 
combination of pH of the solution and the heating temperature. 
The isoflavone glycoside thus converted is adsorbed on an ODS resin and 
eluted with an aqueous alcohol solution, and the eluate is concentrated 
under reduced pressure and lyophilized to give the isoflavone glycoside in 
a high purity. 
The isoflavone glycoside thus obtained can be converted into an isoflavone 
aglycone by the further treatment with an acid or an enzyme. 
In the case of the acid treatment, for example, a solution of the 
isoflavone glycoside in a mixture of hydrochloric acid (12N) and methanol 
at a ratio of 1:4.5 is heated under reflux at at least 70.degree. C. for 
about 6 hours, cooled, and diluted with water to deposit the products. The 
products are collected by filtration, washed with water, dissolved in hot 
ethanol, diluted with water in a ratio of 4 to 6 of the ethanol solution. 
The mixture is left standing at room temperature to crystallize the 
corresponding aglycone. 
In this acid treatment, the aglycone is also obtained even if the 
malonylisoflavone glycoside is used as the raw material. 
In the case of the enzyme treatment, the isoflavone glycoside is dispersed 
in a solution of .beta.-glucosidase derived from soybean in 1/10M 
phosphate buffer (pH 5.0). After reaction at 50.degree. C. for about 6 
hours, the reaction mixture is adsorbed on an ODS resin column, eluted 
with an aqueous alcohol solution, concentrated under reduced pressure, and 
lyophilized to give an isoflavone aglycone as a purified product. 
EXAMPLE 
The present invention is specifically described by the following examples. 
Example 1 
Dehulled soybean (3 kg) obtained by heating a commercially available 
American soybean (IOM) to a soybean temperature of about 80.degree. C. 
under the atmosphere of 120.degree. C. or more, dividing in two with a 
rubber roll, cooling the divided soybean, and removing the hulls, was 
soaked into and extracted with 30 liters of water heated to 50.degree. C. 
for 2 hours with adjusting pH to 8.0 to give 25 liters of an extract. The 
extract was adjusted to pH 4.0 with concentrated hydrochloric acid, left 
standing for 2 hours, and decanted to give 20 liters of a supernatant, 
which was next flown through a column (5.times.21.5 cm, 420 ml) having a 
synthetic adsorbent DIAION HP-20 (manufactured by Mitsubishi Kagaku) 
packed therein at a flow rate of 1 liter/hour to adsorb the 
malonylisoflavone glycoside, and washed with 2 liters of distilled water. 
Next, elution was carried out with 2 liters of a 5% aqueous ethanol 
solution, 3 liters of 10%, 20%, 30% and 40% aqueous ethanol solutions, 
respectively, and 2 liters of a 50% aqueous ethanol solution. 
Elutes was collected in 1 liter fractions, analyzed by high performance 
liquid chromatography (HPLC), separated into a malonyldaidzin fraction and 
a malonylgenistin fraction, which were concentrated under reduced pressure 
at a temperature of 50.degree. C. to a volume of about 2 liters to give 
concentrates containing 1.61 g of malonyldaidzin and 1.76 g of 
malonylgenistin, respectively. 
Purification of Malonyldaidzin 
The malonyldaidzin containing concentrate was next adjusted to pH 8.0 with 
2N NaOH, flown through a column (4.times.16 cm, 200 ml) packed with a 
synthetic resin, ODS resin at a flow rate of 30 ml/min, and washed with 
0.5 liter of distilled water. Next, elution was carried out with 2 liters 
of a 5% aqueous ethanol solution, and fractions containing the preferred 
product were collected, concentrated, and lyophilized to give 1.15 g of 
the sodium salt of malonyldaidzin. 
Purification of Malonylgenistin 
Also, the malonyldaidzin containing concentrate was adjusted to pH 8.0 with 
2N NaOH, flown through a column packed with a synthetic resin, ODS resin 
in the similar manner, and washed with 0.5 liter of distilled water. Next, 
elution was carried out with 2 liters of a 10% aqueous ethanol solution, 
and fractions containing the preferred product were collected, 
concentrated, and lyophilized to give 1.15 g of the sodium salt of 
malonylgenistin. 
.sup.1 H- and .sup.13 C-NMR spectra of malonyldaidzin and malonylgenistin 
thus obtained were accorded with those described in Agric. Biol. Chem., 55 
(9), 2227 (1991). In addition, dehulled soybean used as the raw material 
was analyzed on malonylisoflavone glycoside according to the method 
described by Wang et al. in J. Agric. Food Chem., 42, 1666 (1994). 
That is, dehulled soybeans were ground, and 2.0 g of the sample which 
passed through a screen of 40 mesh was mixed with 100 ml of acetonitrile 
and 20 ml of 0.1N HCl. After stirring the mixture at room temperature for 
2 hours, it was filtered through a Toyo Filter Paper No. 2, and the 
filtrate was concentrated to dryness in a rotary evaporator at a 
temperature of 30.degree. C. or less. 
The residue was dissolved in 10 ml of 80% methanol, filtered through a 
membrane filter, and a 20 .mu.l of the filtrate was subjected to HPLC 
analysis. Average values of three runs of the analysis were 68.8 mg for 
malonyldaidzin and 103.3 mg for malonylgenistin per 100 g of the dehulled 
soybeans. 
From these results, the yields of malonyldaidzin and malonylgenistin in 
Example 1 were determined to be 56% and 44%, respectively. 
Example 2 
Warm water extract of dehulled soybeans (20 liters) obtained in the same 
manner as in Example 1 was adjusted to pH 4.0 with hydrochloric acid, left 
standing for 2 hours, mixed with a filtration aid (RADIOLITE # 500, 
manufactured by SHOWA KAGAKU), and filtered under reduced pressure through 
a Buechner funnel. The filtrate was next flown through a column 
(5.times.22 cm, 430 ml) having active carbon (Purified SHIRASAGI, for 
chromatography, manufactured by Takeda Chemical Industries, Ltd.) packed 
therein at a flow rate of 1.5 liter/hour to adsorb the malonylisoflavone 
glycoside, and washed with 3 liters of 1% aqueous ammonia. 
Next, elution was carried out with 5 liters of a 50% aqueous ethanol 
solution containing 1% ammonia, and the elute obtained was concentrated 
under reduced pressure at 50.degree. C. to give 500 ml of a concentrate 
containing 1.23 g of malonyldaidzin and 1.05 g of malonyl genistin. 
The concentrate was adjusted to pH 8.0 with 2N sodium hydroxide, flown 
through a column (4.times.24 cm, 300 ml) packed with an ODS resin at a 
flow rate of 30 ml/min, and washed with 0.5 liter of distilled water. 
Next, elution was carried out with 2 liters of 2%, 5% and 10% aqueous 
ethanol solutions, respectively. The malonyldaidzin fraction was obtained 
from the fractions eluted with the 5% aqueous ethanol solution, and the 
malonylgenistin fraction was obtained from the fractions eluted with the 
10% aqueous ethanol solution. Each of these fractions was concentrated 
under reduced pressure at 50.degree. C., and lyophilized to give 652 mg of 
the sodium salt of malonyldaidzin and 412 mg of the sodium salt of 
malonylgenistin. 
Example 3 
To low-denatured defatted soybeans (manufactured by NISSHIN OIL MILLS LTD., 
3 kg) was added 30 liters of water, and the mixture was adjusted to pH 7.0 
with a 1N NaOH solution, stirred at 25.degree. C. for 2 hours, and 
centrifuged to remove solids. The supernatant was adjusted to pH 4.5 with 
a 1N HCl solution, and centrifuged to give 24 liters of a whey portion 
containing 1.1 g of malonyldaidzin and 0.8 g of malonylgenistin. The whey 
portion was purified in the same manner as in Example 1 to give the sodium 
salts of malonyldaidzin and malonylgenistin in the yields of 0.68 g and 
0.44 g, respectively. 
Example 4 
To low-denatured defatted soybean (manufactured by NISSHIN OIL MILLS LTD., 
3 kg) was added 30 liters of water, and the mixture was adjusted to pH 7.0 
with a 1N NaOH solution, stirred at 25.degree. C. for 2 hours, and 
centrifuged to remove solids. The supernatant was adjusted to pH 4.5 with 
a 1N HCl solution, and centrifuged to give 24 liters of a whey portion. 
The whey portion was flown through a column having a synthetic adsorbent, 
DIAION HP-20, packed therein to adsorb the malonylisoflavone glycoside, 
and elution, fractionation, concentration and purification were conducted 
in the same way as in Example 1 to give the sodium salts of malonyldaidzin 
and malonylgenistin in the yields of 0.68 g and 0.44 g, respectively. 
Example 5 
To low-denatured defatted soybeans (manufactured by NISSHIN OIL MILLS LTD., 
3 kg) was added 30 liters of water, and the mixture was adjusted to pH 8.0 
with a 1N NaOH solution, stirred at 25.degree. C. for 2 hours, and 
centrifuged to remove solids. The supernatant (22.5 liters) thus obtained 
was adjusted to pH 4.5 with a 1N HCl solution, and centrifuged to give 22 
liters of a whey portion. The whey portion was flown through a column 
having a synthetic adsorbent, DIAION HP-20, packed therein to adsorb the 
malonylisoflavone glycoside, and elution, fractionation, concentration and 
purification were conducted in the same way as in Example 1 to give the 
sodium salts of malonyldaidzin and malonylgenistin in the yields of 0.53 g 
and 0.32 g, respectively. 
Example 6 
Each of 1 g samples of the sodium salts of malonyldaidzin and 
malonylgenistin obtained in the same manner as in Example 1 was dissolved 
in 500 ml of distilled water, and heated in a device equipped with a 
condenser in a boiling water for 5 hours. Analysis by HPLC proved that the 
conversion of the salt into the corresponding glycoside was 90% or more. 
The product was purified on a column having an ODS resin packed therein to 
give the corresponding glycosides, daidzin and genistin in the yields of 
0.73 g and 0.67 g, respectively. 
Example 7 
Each of 2 g samples of the sodium salts of malonyldaidzin and 
malonylgenistin obtained in the same manner as in Example 1 was dissolved 
in 1,000 ml of distilled water, and adjusted to pH 10 with 1N NaOH. The 
solution was left standing at room temperature for 24 hours. Analysis by 
HPLC proved that the conversion of the salt into the corresponding 
glycoside was 90% or more. The product was neutralized with hydrochloric 
acid and purified on a column having an ODS resin packed therein in the 
same manner as in Example 4 to give the corresponding glycosides, daidzin 
and genistin in the yields of 1.36 g and 1.30 g, respectively. 
Example 8 
To each of 1 g samples of daidzin and genistin obtained in Example 7 were 
added 45 ml of methanol and 10 ml of 12N HCl, and the glycoside was 
decomposed under reflux for 6 hours. After cooling, the reaction mixture 
was diluted with water, and the solid product was collected by filtration, 
washed with water, dissolved in 90 ml of a 80% ethanol solution, filtered 
through a filter paper to give a supernatant. The supernatant was left 
standing at room temperature to deposit a crystalline product. The mixture 
was left standing overnight, and the crystals were collected and dried to 
give daidzein and genistein in the yields of 410 mg and 440 mg, 
respectively. 
The isoflavone glycosides and the isoflavone aglycones obtained in Examples 
6-8 have the same physical properties as those of the authentic samples 
(available from FUNAKOSHI K.K.). 
According to the present invention, it is possible to obtain efficiently in 
a simple procedure from an aqueous extract of soybean the 
malonylisoflavone glycosides, from which the isoflavone glycosides as well 
as isoflavone aglycones are also obtained by treatments with for example 
an alkali and the like.