Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Conjugate or complex
Reexamination Certificate
2001-06-18
2002-11-12
Barts, Samuel (Department: 1623)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Conjugate or complex
C424S756000, C536S124000, C536S127000, C536S128000, C536S004100, C536S008000, C435S200000, C514S456000
Reexamination Certificate
active
06479054
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The invention relates to a process for obtaining genistin from isoflavone inclusions.
BACKGROUND ART
In accordance with the change of lifestyle and the advance of the aging, the increase of lifestyle-related diseases has been a big problem. Prevention against malignant neoplasm, cardiac disease and cerebrovascular disease, the present three major cause of death, and bone fracture by osteoporosis which can be a cause of a bedridden state is strongly desired. For the prevention against them, a well balanced daily diet is important, though together with this it has been required to take actively a diet and food composition in which their prevention effects are recognized.
In such a situation, the attention to isoflavones, as a functional factor in foods is rising.
That is, isoflavones are recently shown to have a preventive and therapeutic effects for hyperlipidemia (J. Nutr. 126: 43-50 (1996); JP, 9-255570, A) and a preventive and therapeutic effects for osteoporosis (J. Nutr. 126: 161-167 (1996); JP, 8-231533, A).
Further, from the results of the epidemiological investigation and the animal experiments, it has been clarified that more intake of isoflavone results in higher bone density and lower cancer incidence (Shokuhin & Kaihatsu, vol. 31 No. 6: 44-47 (1996)).
Thus, it has been confirmed that isoflavones have a number of pharmacological effects such as an estrogen, antioxidant or anticancer actions.
Consequently, it is needed to provide isoflavones at lower costs.
Now, it is well known that in soybeans are contained daizein, genistein and its glycoside, daidzin, genistin, and malonate or acetate of each isoflavone glycoside, and conventionally isoflavones are obtained from soybeans.
As a conventional method to obtain isoflavones from soybeans, the followings are illustrated.
(1) JP, 4-21670, B
Extraction liquid of soybeans as it is or that after evaporation of solvent is contacted with a non-polar porous synthetic adsorption resin having a huge network structure to adsorb isoflavone derivatives, and then eluted from said synthetic resin using organic solvents or a mixed solvent of organic solvents and water, to obtain a solution containing isoflavone derivatives such as genistin.
(2) JP, 4-34526, B
From “broth”, “Yu”, “whey”, etc., physiologically active substances mainly consisting of saponins, flavonoids, isoflavonoids and glycosides thereof are obtained by adsorption-desorption with charcoal, active carbon or an active resin, wherein elution is carried out using an aqueous ethanol solution as the eluting solvent and the eluate is concentrated under reduced pressure to obtain a concentrate.
For example, from adsorbents of the active resin for “Yu”, a waste liquid in a tofu factory, are obtained respectively one consisting of 69% isoflavonoid type components with genistein as the main component and 8% saponin type components by elution with 60-95% ethanol, and one consisting of 81% isoflavonoid type components with genistein as the main component and 12% saponin type components by elution with 5-10N acetic acid.
(3) JP, 7-173148, A
Shed soybeans are immersed in warm water of 50° C. (adjusted to pH 9 by alkali), and the immersion liquid is added with synthetic resin (Diaion HP-20), stirred, and added with 1N-ammonia water. The resin adsorbent is eluted, and the elution liquid is concentrated under reduced pressure to give a syrup concentrate. The concentrate is extracted under reflux adding chloroform, and an extraction residue is recovered, added with 4% HCl methanolic solution, hydrolyzed at 100° C. under reflux for 6 hr. A hydrolyzate is concentrated under reduced pressure, washed thoroughly with distilled water, followed by drying the residue to give brown solid of an isoflavone mixture, with daizein and genistein as the main components.
(4) JP, 8-231533, A
This relates to a novel flavonoid glycoside, a therapeutic agent for osteoporosis comprising the flavonoid glycoside and an edible composition, and the flavonoid glycoside is obtained in the following way.
Defatted natto is extracted with methanol, concentrated to dryness by a rotary evaporator to give an extract. The methanolic extract is dissolved in distilled water, adsorbed to a column packed with a synthetic adsorbent (Diaion HP-20 (trade name); manufactured by Mitsubishi Kasei Co., Ltd.), eluted with 30% methanol and 70% methanol successively. The 70% methanol eluate is concentrated and chromatographed for separation using Sephadex LH-20 (manufactured by Pharmacia Co., Ltd.), making methanol as the eluting solvent to give a fraction containing various 6″-O-succinylisoflavones such as 6″-O-succinylgenistin.
(5) JP, 8-283283, A
Soybean is extracted with water, and the water extraction liquid is adsorbed to an adsorption resin to adsorb malonylisoflavone glycosides in the extraction liquid, followed by elution with an aqueous alcohol solution to obtain malonylisoflavone glycosides. The obtained malonylisoflavone glycoside solution is subjected to a heat treatment and/or an alkali treatment to lead to an isoflavone glycoside solution which is subjected to an acid treatment or an enzyme treatment to obtain isoflavone aglycons.
For example, water extraction liquid of 3 kg of shed soybeans is adsorbed and eluted with an aqueous methanol solution. The obtained eluate is analyzed by high-performance liquid chromatography to obtain each concentrate containing 1.61 g of malonyldaidzin and 1.76 g of malonylgenistin respectively.
(6) JP, 9-59166, A
This relates to an epidermal growth accelerating agent wherein malonylispoflavone glycos ides are effective components. The malonylisoflavone glycosides of effective components are obtained as follows.
20 L of warm water extract of shed soybeans is adjusted to pH 4.0 by hydrochloric acid, let stand for 2 hr, followed by addition of a filtration auxiliary agent and by suction filtration on Büchner funnel. The filtrate is passed through column packed with active carbon at the flow rate of 1.5 L/hr to adsorb malonylisoflavone glycosides, followed by wash of the column with 3 L of 1% ammonia water and subsequent elution with 5 L of 50% aqueous ethanol solution containing 1% ammonia. The obtained eluate is concentrated under reduced pressure at 50° C. to obtain 500 ml of concentrate containing 1.23 g of malonyldaidzin and 1.05 g of malonylgenistin.
(7) JP, 10-23878, A
This is a process for recovering isoflavones from an aqueous soy molasses feed stream, and comprises a step of heating the above feed stream to temperature for solubilization of genistin, a step of separating purified genistin products in a permeate of the above starting material by subjecting the above heated feed stream to ultrafiltration, a step of accelerating crystallization of genistin by cooling the above feed stream permeate treated by the above filtration, and a step of separating genistin by centrifugation or filtration of the above cooled permeate.
By inspecting the above conventional processes the followings can be mentioned.
In the invention (1), a solution containing daidzin, glycitin, genistin, daizein, genistein, etc., is obtained as an eluate, but a separation recovery of genistin from said eluate is not carried out.
In the invention (2), it is described that isoflavonoid type components consisting of 69-81% of genistein as the main component were obtained, but the obtainment of genistin is unclear.
In the invention (3), a chlorine containing organic solvent is used as the extracting solvent.
In the invention (4), the separation recovery of glycosides from an eluate is carried out by chromatography.
In the invention (5), the eluate is subjected to high-performance liquid chromatography as it is, and malonyldaidzin and malonylgenistin are separately obtained.
In the invention (6), the eluate is concentrated under reduced pressure to obtain a concentration liquid containing malonylgenistin.
In the invention (7), a precipitate of genistin is obtained from ultrafiltration liquid using the solubility difference due to temperature.
The above conventional processes a
Fujikawa Yoko
Tomita Tetsuji
Yatake Tsuneya
Yoshizawa Yasuko
Barts Samuel
Khare Devesh
Showa Sangyo Co. Ltd.
Wolf Greenfield & Sacks P.C.
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