JPH0770170A - New isoflavone derivative and its production - Google Patents

Abstract

PURPOSE:To obtain a new compound expected to have application to medicines, healthy foods, etc., usable for elucidation of action mechanism occurring in a fermentation process of soybeans obtained by extracting the compound from natto (fermented soybeans). CONSTITUTION:A compound of the formula (R1 is H or methoxyl; R2 is H or OH) such as 7-(6-succinyl-beta-glucopyranosyl)-3-(4-hydroxyphenyl)-4H-1- benzopyran-4-one. The compound, for example, is obtained by extracting soybeans fermented with Bacillus natto with an alcohol such as methanol, dissolving the extract in water, successively subjecting the solution to adsorption column chromatography and gel column chromatography, purifying the prepared compound of the formula by reversed phase system column chromatography.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel isoflavone derivative and a method for producing the same.

[0002]

2. Description of the Related Art Conventionally, isoflavone derivatives have a physiological activity such as antioxidant activity, antibacterial activity, anticholesterol activity, tyrosine kinase inhibitory activity, estrogen activity, and bone resorption inhibitory activity. It is reported to present.

For example, regarding the antioxidative effect, Dan E. P.
ratt, et al., "Source of Antioxidant Activity of So
ybeans and Soy Products ", J. of Food Science, 44,
p.1720 (1979) ； About the antibacterial action, Martin Weidenb
orner, et al., "Antifungal Activityof Isoflavonoid
s Against Storage Fungi of The Genus Aspergillu
s ", Phytochemistry, 28, pp.3317-3319 (1989); For the anticholesterol effect, see RD Sharma," Isoflavo.
nes and Hyper-choresterolemia in Rats ", Lipids, 14
(6), p.535 (1978); Regarding the inhibitory effect on tyrosine kinase, K. Higashi, et al., "Mechanism of action of isoflavones in the cell cycle", Cell Science, 8 (8), 33 (1992); Is Bickoff EM, et al., "R
elative Potenciesof SeveralEstrogen-like Compounds
found in Forages ", J. of Agricultureand Food Chem
istry, 10, 410 (1962); and the effect of suppressing bone resorption, M. Tsuda, et al., "The Effect of Tpriflavone.
(TC-80) on Bone Resorption in Tissue Culture ", J.
of Bone and Mineral Research, 1 (2), p.207 (1986),
checking ...

[0004] Isoflavone derivatives, especially isoflavone derivatives contained in soybeans, have a total of 12 kinds of aglycones having different basic skeletons and three kinds of glycosides having different sugar chain structures. Have been reported (eg Shigemitsu, et al., "Malonyl Isoflavone
Glucosides in Soybean Seeds ", Agric. Biol. Chem, 5
5 (9), pp.2227-2233 (1991))
At present, the mechanism of action of the isoflavone derivative contained in soybean during the soybean fermentation process has not been clarified.

[0005]

In view of the above-mentioned problems in the technical field of the present invention, the present inventors have conducted many years of research on isoflavone derivatives in various soybean fermented foods, and as a result, It was invented by discovering an isoflavone derivative having a completely novel structure which does not exist in soybean before fermentation.

That is, according to the present invention, a compound having the following structural formula is provided.

[0007]

[Chemical 2]

In the above formula, R ₁ is hydrogen or a methoxyl group, and R ₂ is hydrogen or a hydroxyl group.

When this compound was analyzed by a high performance liquid chromatogram method for 10 typical natto commercially available in Japan, it was detected in all natto. However, since this compound was not detected in other fermented soybean foods (miso, soy sauce, tempeh, etc.), it is considered to be a component peculiar to natto.

Further, the novel isoflavone derivative of the present invention has higher solubility in water and alcohols such as methanol or dilute methanol, as compared with the conventionally known isoflavone derivative contained in soybean. It is expected that the compound of the present invention has a good absorption rate into the body when the isoflavone derivative of the present invention which is a physiologically active substance is ingested, and that the compound of the present invention is applied to medicines and health foods.

Furthermore, the present inventors have conducted studies on the novel isoflavone derivative production method of the present invention and have constructed the following two types of methods. That is, (1) Bacillus natto (common name: Miura, Takahashi, Yayoi, etc.)
The alcohol extract of soybeans fermented in step 1 is purified by adsorption column chromatography and gel column chromatography in this order to obtain a mixture containing the novel isoflavone derivative.

Then, this mixture is further purified by reverse phase column chromatography to obtain the three novel isoflavone derivatives of the present invention.

(2) Natto natto was added to a hot water extract of soybeans concentrated 2 to 10 times, or to a medium in which soy isoflavone glycosides were added to the hot water extract in an amount of 2 to 10 times the amount of isoflavones. The bacterium is cultured, and the culture is terminated when the concentration of the novel isoflavone derivative of the present invention reaches the maximum.

Next, the culture solution from which the bacterial cells have been removed is purified by adsorption column chromatography and gel column chromatography in this order to obtain a mixture containing the novel isoflavone derivative.

Then, this mixture is further purified by reverse phase column chromatography to obtain the three novel isoflavone derivatives of the present invention.

[0016]

[Examples] Hereinafter, preferred methods for preparing the novel isoflavone derivatives of the present invention will be described, but the following examples are for illustrative purposes and should not be construed as limiting the present invention.

Example 1 Commercially available natto 3.5 kg ("Konbu Mineral Natto" (trade name):
(Fujicco Co., Ltd.) was freeze-dried, crushed, defatted with chloroform at room temperature, and then extracted with methanol.

135 g of the methanol extract was dissolved in distilled water and adsorbed on a column packed with DIAION HP-20 ((trade name): Mitsubishi Kasei Co., Ltd.), 30% methanol, 70%.
Elution was performed sequentially with% methanol.

A 70% methanol eluate was separated by Sephadex LH-20 column chromatography ((trade name): Pharmacia Co.) using methanol as an elution solvent, and a fraction containing a large amount of novel isoflavone derivative 555 mg.
It was obtained (yield from raw material natto: 0.016%).

This fraction was further separated by ODS column using high performance liquid chromatography to obtain three novel isoflavone derivatives (purity: 99% or more).
The purity of the isoflavone derivative was determined based on the results of liquid chromatography and thin layer chromatography.

Then, physicochemical analysis of each isoflavone derivative was carried out.

Regarding analysis means, NMR analysis is described in J
NM-GSX 270 (made by JEOL Ltd.), mass spectrometry (FAB-M
S) used JMS-SX 102 (manufactured by JEOL Ltd.), and infrared absorption analysis (IR) used IR-700 (“KBr tablet method”: manufactured by JASCO Corporation).

The description regarding the NMR data is based on a comparison between the NMR spectrum data of soybean isoflavone glycoside (genistin) (Table 1 below) and the NMR spectrum data of each isoflavone derivative (Tables 2 to 4). .

[0024]

[Table 1]

7- (6-succinyl-β-glucopyracino
) -3- (4-Hydroxyphenyl) -4H-1-benzopyran-4
-On

[0026]

[Table 2]

From the results in Table 2 above, the NMR of genistin is shown.
The differences from the spectral data were as follows.

¹ H-NMR: (CD ₃ OD, 270 MHz) ppm'δ 'value.

8.11 (1H, d, J = 8.9Hz, 5-H), 7.16 (1H, dd, J
= 8.9 and 2.4Hz, 6-H) new signal appears.

¹³ C-NMR: (CD ₃ OD, 270 MHz) ppm'δ 'value.

128.3 (C-5), 120.2 (C-10), 117.1 (C-6)
The emergence of a new signal.

Regarding the melting point, mass spectrometry and infrared absorption analysis (maximum absorption wavelength) of this compound, the following numerical values were obtained.

Melting point: 231 ° C FAB mass (m / z): 517 (C ₂₅ H ₂₄ O ₁₂ + H) ⁺ IR ν (cm ^-1 ): 3370, 2360, 2318, 1730, 1621, 152
4, 1445, 1247, 1070,885, 830. 7- (6-succinyl-β-glucopyrazinol) -5-hydro
Xy-3- (4-hydroxyphenyl) -4H-1-benzopyran-4
-On

[0034]

[Table 3]

From the results in Table 3 above, the NMR of genistin is shown.
The differences from the spectral data were as follows.

¹ H-NMR: (CD ₃ OD, 270 MHz) ppm'δ 'value.

The appearance of a new signal at 6.48 (1H, d, J = 1.2Hz, 6-H).

¹³ C-NMR: (CD ₃ OD, 270 MHz) ppm'δ 'value.

163.3 (C-5), 107.9 (C-10), 101.0 (C-6)
The emergence of a new signal.

Regarding the melting point, mass spectrometry and infrared absorption analysis (maximum absorption wavelength) of this compound, the following numerical values were obtained.

Melting point: 228 ° C. FAB mass (m / z): 533 (C ₂₅ H ₂₄ O ₁₃ + H) ⁺ IR ν (cm ^-1 ): 3432, 1714, 1648, 1612, 1515, 144
1, 1251, 1175, 107 4,837. 7- (6-succinyl-β-glucopyracinol) -3- (4-hi
Droxyphenyl) -6-methoxy-4H-1-benzopyran-4-
on

[0042]

[Table 4]

From the results in Table 4 above, the NMR of genistin is shown.
The differences from the spectral data were as follows.

¹ H-NMR: (CD ₃ OD, 270 MHz) ppm'δ 'value.

The appearance of a new signal at 3.86 (3H, s, OMe-H).

[0046]

INDUSTRIAL APPLICABILITY The present invention provides a novel isoflavone derivative obtained from fermented soybeans (natto), so that not only the known physiological action exhibited by conventional isoflavone derivatives but also a completely novel physiology. In addition, it can be expected to be useful for elucidating the mechanism of action of isoflavone derivatives in the soybean fermentation process.
It has various effects such as being able to contribute academically.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Takenori Okudaira 4-6-8 Souyama-cho, Kita-ku, Kobe-shi, Hyogo Prefecture (72) Inventor Hitoshi Ishida 3107-2 Sena, Shizuoka-shi, Shizuoka Prefecture (72) Kunio Tsuji 1375-11 Ikeda, Shizuoka City, Shizuoka Prefecture

Claims (6)

[Claims] 1. A compound of the following structural formula: An isoflavone derivative in which R ₁ in the above formula is hydrogen or a methoxyl group and R ₂ is hydrogen or a hydroxyl group. 2. The isoflavone derivative is 7- (6-succinyl-β-glucopyracinol) -3- (4-hydroxyphenyl) -4H-1-benzopyran-4-one; 7- (6-succinyl-β- Glucopyracinol) -5-hydroxy-3- (4-
Hydroxyphenyl) -4H-1-benzopyran-4-one; or 7- (6-succinyl-β-glucopyracinor) -3- (4-
(Hydroxyphenyl) -6-methoxy-4H-1-benzopyran-
The isoflavone derivative according to claim 1, which is 4-one. 3. A method for producing an isoflavone derivative, which comprises the following steps: (a) soybean is fermented with Bacillus subtilis, and (b) soybean fermented with Bacillus natto, Alcohol extraction, (c) the step (b)
The alcohol extract obtained in step (1) is purified by adsorption column chromatography, and (d) the purified product obtained in step (c) is purified by gel column chromatography. Process for producing isoflavone derivative. 4. A method for producing an isoflavone derivative, which comprises the following steps: (a) a concentrated hot water extract of soybean or a medium in which soy isoflavone glycoside is added to the hot water extract. , Culturing Bacillus subtilis, (b) removing the cells from the culture solution obtained in the step (a), (c) the culture solution obtained in the step (b), adsorption column Chromatographically purifying, and (d) the step
A method for producing an isoflavone derivative, which comprises a step of purifying the purified product obtained in (c) by gel column chromatography. 5. The production method further comprises the step of (e) purifying the purified product obtained by the gel column chromatography by reverse phase column chromatography.
The method for producing an isoflavone derivative according to claim 3 or 4, characterized in that 6. The isoflavone derivative is 7- (6-succinyl-β-glucopyrazinol) -3- (4-hydroxyphenyl) -4H-1-benzopyran-4-one; 7- (6-succinyl-β- Glucopyracinol) -5-hydroxy-3- (4-
Hydroxyphenyl) -4H-1-benzopyran-4-one; or 7- (6-succinyl-β-glucopyracinor) -3- (4-
(Hydroxyphenyl) -6-methoxy-4H-1-benzopyran-
The method for producing an isoflavone derivative according to any one of claims 3 to 5, which is 4-one.