JP2002281995A - Flavonoid compound and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a flavonoid compound capable of enlarging the utilization range of naringin, and further to provide a method for producing the flavonoid compound. SOLUTION: The flavonoid compound has a structure represented by the formula (wherein, R1 is hydrogen and R2 is hydroxy group, or R1 is hydroxy group and R2 is hydrogen), and is 8-hydrcxynaringenin or 6-hydroxynaringenin having a hydroxy group at 8- or 6-position of naringenin. The flavonoid compound is obtained by subjecting the naringin to a fermentation treatment with microorganisms of Aspergillus saitoi. The fermentation treatment with the microorganisms comprises a mycelium-culturing step for subjecting a medium containing the Aspergillus and the naringin added thereto to a shaking culture to allow a vegetative mycelium of the Aspergillus to form the naringenin, and a sporulation step for forming the 8-hydroxynaringenin and the 6- hydroxynaringenin while progressing sporulation from the vegetative mycelium of the Aspergillus.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a flavonoid compound and a method for producing the same.

[0002]

2. Description of the Related Art Conventionally, naringin, which is a glycoside of naringenin, is a flavonoid abundantly contained in citrus fruits such as pomelo, summer tangerine, grapefruit and the like, particularly in the rind of the citrus fruits and in the capsular membrane. Since naringin has a strong bitter taste, it is mainly used to impart bitterness to beverages and the like. In addition, it has been known that naringin has an antitumor effect and an anti-inflammatory effect and also has an inhibitory effect on the generation of rat breast cancer by a carcinogen.

[0003]

As a part of the effective use of naturally occurring naringin, there is a high possibility that the range of use can be expected to be further expanded by converting naringin into a substance. In particular, since naringin has an extremely high absorbability in the body, it is expected that the substance conversion will enhance the value from a nutritional viewpoint.

[0004] The present invention was made as a result of earnest research aimed at further expanding the use of naringin.
An object of the present invention is to provide a flavonoid compound capable of expanding the range of use of naringin and a method for producing the same.

[0005]

In order to achieve the above object, the flavonoid compound according to the first aspect of the present invention comprises:
It has a structure represented by the following chemical formula 2, and is obtained by subjecting naringin to microbial fermentation treatment using Aspergillus saitoi.

[0006]

Embedded image However, R1 is hydrogen and R2 is a hydroxyl group, or R1 is a hydroxyl group and R2 is hydrogen.

[0007] A method for producing a flavonoid compound according to the present invention according to claim 2 is a method for producing a flavonoid compound according to claim 1, wherein naringin is converted to Aspergillus saii.
The flavonoid compound is produced by microbial conversion of the naringin by microbial fermentation treatment in itoi).

[0008] The method for producing a flavonoid compound according to the third aspect of the present invention is carried out in the second aspect of the present invention, wherein the microorganism fermentation treatment is performed by shaking culture of a medium containing naringin and Aspergillus cytoii. After performing a mycelium culture step of microbially converting naringin to naringenin into vegetative hyphae of Cytoii, a spore forming step of microbially converting flavonoid compounds from naringenin in the medium while performing spore formation from vegetative hyphae of Aspergillus cytoii. Is performed.

In the spore formation step after the mycelium culture step, either shaking culture or static culture may be used. However, in the case of stationary culture, the ratio of the surface area to the medium volume (specific surface area) is increased by reducing the depth of the medium to keep the entire medium under aerobic conditions, and the microorganisms produced by Aspergillus cytoii It is preferable to configure so as to increase the conversion efficiency.

[0010] The method for producing a flavonoid compound according to the invention described in claim 4 is carried out in the invention described in claim 3, and a preliminary culture step of shaking culture of a medium containing Aspergillus cytoii prior to the mycelium culture step. Is performed.

[0011]

Embodiments of the present invention will be described below in detail. The first flavonoid compound of the embodiment has a structure represented by the following chemical formula 3.

[0012]

Embedded image This first flavonoid compound has a chemical formula of C ₁₅ H ₁₂ O
₆ , a flavonoid compound having a molecular weight of about 289 (4 ′, 5,7,
8-tetrahydroxyflavanone or 2,3-dihydro-5,7,8-tri
hydroxy-2- (4-hydroxyphenyl) -4H-1-benzopyran-4-on
e). This flavonoid compound has a structure represented by the above-mentioned chemical formula (3), and naringenin (4 ′, 5,7-t)
An organic compound having a hydroxyl group at the 8-position of rihydroxyflavanone, so-called 8-hydroxynaringenin (8-hydroxyn)
aringenin).

The second flavonoid compound of the embodiment is:
It has a structure represented by the following chemical formula 4.

[0014]

Embedded image This second flavonoid compound has a chemical formula of C ₁₅ H ₁₂ O
₆ , a flavonoid compound having a molecular weight of about 289 (4 ′, 5,6,
7-tetrahydroxyflavanone or 2,3-dihydro-5,6,7-tri
hydroxy-2- (4-hydroxyphenyl) -4H-1-benzopyran-4-on
e). This flavonoid compound is an organic compound having a hydroxyl group at the 6-position of naringenin, that is, a so-called 6-hydroxynaringenin, based on the structure represented by the above formula (4).

These 8-hydroxynaringenin and 6-hydroxynaringenin
-Hydroxynaringenin (first and second flavonoid compounds) is soluble in methanol, ethanol and dimethylsulfoxide (DMSO), and slightly soluble, but soluble in water. Furthermore, while the above-mentioned naringenin has almost no antioxidant effect,
And the second flavonoid compound can exhibit an extremely high antioxidant effect. In particular, the 8-hydroxynaringenin can exert a much higher antioxidant effect than 6-hydroxynaringenin. And
Utilizing this high antioxidant activity, it can be added to, for example, foods and beverages and used for health foods and drinks having a health promoting activity. At this time, the first and second flavonoid compounds eliminate active oxygen in the living body to suppress the production of lipid peroxide, and cause lifestyle such as cancer, arteriosclerosis, and complications of diabetes caused by oxidative stress. Helps prevent disease.

The first and second flavonoid compounds include naringin as Aspergillus cytoi (As
pergillus saitoi). That is, these flavonoid compounds
Naringenin and neohesperidios
Aspergillus cytoii is cultured in a medium containing naringin, which is the glycoside of e), and the Aspergillus cytoii is microbial-converted into naringin, thereby producing the supernatant in the culture supernatant. The culture conditions at this time are 20 to 40 ° C. in order to favor the growth of Aspergillus cytoii and the aforementioned microbial conversion.
The culture temperature is preferably aerobic conditions.

As the medium, a medium for filamentous fungi such as a potato dextrose-containing medium or a Zapec medium, or various liquid mediums containing organic substances such as okara are preferably used. Furthermore, in order to inhibit fermentation other than the purpose of microbial conversion of the flavonoid compound from naringin, it is preferable that the minimum medium containing a minimum amount of nutrients is used.For example, monosaccharides and disaccharides are used to prevent alcohol fermentation. It is preferable not to include them inside. In addition, the above-mentioned medium was prepared at the start of the culture at Aspergillus.
In order to improve the growth of Cytoii, the pH is preferably in the range of 3 to 7.

Further, when naringin is added to the medium, it is preferable to contain a low-concentration organic solvent in order to enhance the solubility of naringin in the medium. Examples of the organic solvent include methanol, ethanol, DMSO
And the like. The content of the organic solvent in this medium is preferably 0.01 to 5% by volume, more preferably 0.01 to 0.1% by volume. When the content of the organic solvent in the medium is less than 0.01% by volume, a sufficient amount of naringin cannot be dissolved in the medium. On the other hand, when it exceeds 5% by volume, the growth of Aspergillus cytoii is inhibited.

On the other hand, the content of naringin added to the medium is preferably up to a saturation concentration as its solubility limit in order to efficiently obtain a large amount of flavonoid compounds. The saturation concentration is closely related to the content of the organic solvent, but is about 1% by weight or less. In addition, the concentration of Aspergillus cytoii added to the culture medium at the start of cultivation is 2 × 10 ⁶ cells / cell in order to efficiently obtain a large amount of flavonoid compounds in a short period of time.
mL (cfu / mL) or more.

Further, in order to enhance the efficiency of microbial conversion by Aspergillus cytoii, a hyphal culturing step of shaking and cultivating vegetative mycelia of Aspergillus cytoii in the above-mentioned medium is performed, and then spores which promote spore formation from the vegetative mycelia are obtained. It is preferable to perform the forming step.

Prior to the mycelium culture step, a medium containing only cells is preliminarily shake-cultured so that Aspergillus cytoii can form vegetative mycelia sufficiently (hereinafter referred to as a preculture step). To be described). This pre-culture step avoids the growth inhibition at the early stage of culture (early growth stage) of Aspergillus cytoii by an organic solvent added simultaneously to the medium to enhance the solubility of naringin,
This is performed to increase the efficiency of the conversion of naringin into microorganisms.

This pre-culture step is preferably performed until the vegetative mycelium of Aspergillus cyte occupies at least 1/3 of the culture surface, in order to avoid the growth inhibition in the initial stage of cultivation of Aspergillus cytoii, more preferably. It is configured to perform the process until it occupies about 1/3 to 2/3. In this preliminary culturing step, the period until the vegetative mycelium of Aspergillus cytoii occupies about 1/3 to 2/3 of the culture solution surface is generally 1 to 2 weeks. If the period of the preculture step is less than one week, the vegetative mycelium does not occupy one third of the liquid surface. On the other hand, when the period exceeds 2 weeks, the vegetative mycelium of Aspergillus cytoii grows in the whole container, and the nutrient components of the medium are deficient.
The decomposition of naringin progresses quickly due to the site, making the process control difficult.

The mycelium culturing step is a step after the addition of naringin to the medium, and the Aspergillus cytoii is cultured with shaking in the medium containing naringin while maintaining aerobic conditions. This is a step of microbial conversion of naringin into vegetative hyphae.
In this step, the vegetative mycelium of Aspergillus cytoii extremely efficiently performs a glycosidase reaction for generating naringenin by cleaving the bond between naringenin and neohesperidiose constituting naringin.

At this time, the content of naringin added to the medium may exceed the above-mentioned solubility limit. At this time, the naringin that has not been dissolved at the time of adding the naringin but settles at the bottom of the culture vessel can be appropriately dissolved in the culture medium by the stirring action by shaking and used for microbial fermentation. Further, when the medium contains the naringin exceeding the solubility limit, it is preferable to perform the shaking culture at about 50 rpm / min until the precipitate disappears in order to prevent the naringin precipitation at the bottom of the culture vessel. Preferably
As a result, more naringenin can be produced.

In the sporulation step, the vegetative mycelium of Aspergillus cytoii undergoes microbial conversion by culturing the vegetative hyphae of Aspergillus cytoii by culturing the vegetative hyphae of Aspergillus cytoii by culturing the vegetative mycelium of Aspergillus cytoii by culturing the culture medium after the hyphal culture step without changing the medium. It is a process. In the step of shifting from the mycelium culture step to the spore formation step, spore formation may start before the mycelium culture step is completed, so it is not possible to clearly distinguish it, but the spore formation has started. Can be visually confirmed once, and it can be grasped using the index as an index.

In this step, the vegetative mycelium of Aspergillus cytoii undergoes a hydroxylase reaction for adding a hydroxyl group to the 8- or 6-position of naringenin while promoting spore formation, thereby producing a flavonoid compound very efficiently. In addition, in this spore formation step, usually 8-
Hydroxynaringenin and 6-hydroxynaringenin are simultaneously produced in a predetermined ratio. The reaction for producing 8-hydroxynaringenin and 6-hydroxynaringenin is most efficiently performed from the early stage to the middle stage of the sporulation process in the culture vessel, and the yield increases. However, as the sporulation is completed from the middle stage, 8-hydroxynaringenin and 6-hydroxynaringenin are gradually decomposed and their contents decrease. For this reason, in order to obtain a larger amount of 8-hydroxynaringenin and 6-hydroxynaringenin, it is preferable that the culture is stopped after 3 to 10 days of culture after the addition of naringin. The flavonoid compounds may be extracted.

In addition, when static culture is performed in the spore formation step, the effect of suppressing spore formation due to physical stimulation at the time of shaking can be easily eliminated. -Hydroxynaringenin and 6-
The peak of hydroxynaringenin yield is also early. In addition, during this stationary culture, the entire medium is maintained under aerobic conditions by reducing the depth of the medium and increasing the ratio of the surface area to the volume of the medium (specific surface area), thereby increasing the activity of Aspergillus cytoii. It is preferable to increase the conversion efficiency of the microorganism.

On the other hand, when shaking culture is performed in the sporulation step, it is easy to maintain aerobic conditions even if the depth of the medium is appropriately increased, and furthermore, it is necessary to delay sporulation by physical stimulation. Therefore, the microbial conversion is carried out slowly, and the peaks of the yields of 8-hydroxynaringenin and 6-hydroxynaringenin are also slower than the stationary culture.

In the present embodiment, the shaking speed of the shaking culture is preferably in the range of 50 to 200 rpm / min. If the shaking speed is less than 50 rpm / min, the whole culture medium containing Aspergillus cytoii is not aerobic, so that hyphal growth cannot be sufficiently performed. On the other hand, when the shaking speed exceeds 200 rpm / min, the medium fluctuates violently and hyphal formation cannot be sufficiently performed.

Finally, the flavonoid compound is extracted and purified from the culture supernatant or the medium after the spore formation step. At this time, the medium was centrifuged (3000 rpm) to such an extent that the cell membrane of Aspergillus cytoii was not destroyed.
m) to obtain a supernatant fraction, and the supernatant fraction may be purified by reversed-phase liquid chromatography using a hydrophobic column. Since the precipitate fraction after the centrifugation also contains a relatively large amount of a flavonoid compound, the precipitate fraction was extracted with an organic solvent such as methanol or ethanol and sufficiently washed, followed by extraction. The extract may be configured to be purified by reversed-phase liquid chromatography.

Further, 8-hydroxynaringenin and 6-hydroxynaringenin
Since it has very similar properties to -hydroxynaringenin, it exhibits a remarkably similar elution pattern in the reverse phase liquid chromatography. For this reason, in order to separate both into separate fractions, it is more preferable to carry out reverse phase liquid chromatography or other separation and purification while slightly changing various conditions as necessary.

The effects exerted by the above embodiment will be described below. -The first flavonoid compound of the embodiment is obtained by subjecting naringin to microbial fermentation using Aspergillus cytoii, and is 8-hydroxynaringenin having a structure represented by Chemical Formula 3 above. Further, the second flavonoid compound of the embodiment is obtained by subjecting naringin to microbial fermentation using Aspergillus cytoii, and is 6-hydroxynaringenin having a structure represented by Chemical Formula 4 above. Since these flavonoid compounds are produced by converting naringin into microorganisms, it is possible to promote the effective use of naturally occurring naringin and to expand the range of its use.

Furthermore, since naringin as a raw material is contained in a large amount in the pericarp and membrane of pomelo, summer tangerine, grapefruit and the like, the residue (waste) after juice of juice is extremely effectively used. can do.
The residue has a very high concentration of naringin and is easily available in large quantities and at low cost. Therefore, these flavonoid compounds can be produced in extremely large amounts at low cost by using the residue as a raw material. On the other hand, regarding these first and second flavonoid compounds, literatures separated and purified from extracts of plants (for example, leaves of Scutellaria) and isomerization of plant extracts (for example, aglycones of safflower pigment) with acids. Are known. However, it is extremely difficult to obtain large amounts of flavonoid compounds at low cost by the methods according to these documents.

In addition, these flavonoid compounds
By having a structure in which a hydroxyl group is added to the 8th or 6th position of naringenin, it is possible to exhibit a significantly higher antioxidant effect than naringin and naringenin.
For this reason, active oxygen is eliminated in the living body to suppress the production of lipid peroxide, which can be used for prevention of lifestyle-related diseases such as cancer, arteriosclerosis, and complications of diabetes caused by oxidative stress.

The method for producing the first and second flavonoid compounds of the embodiment is characterized in that naringin is added to Aspergillus
It is obtained by microbial conversion of the naringin by microbial fermentation treatment at Cyto. Therefore, a flavonoid compound capable of expanding the range of use of naringin can be produced very easily. Furthermore, as well as using naringin which is a natural ingredient contained in citrus as a raw material, since Aspergillus cytoii used for brewing liquors such as shochu is used, there is almost no problem in ingestion to the human body. .

In addition, in the above-mentioned microbial fermentation treatment, a very simple operation process is performed by performing a mycelium culture step of shaking culture of a medium containing Aspergillus cytoii and naringin followed by a spore formation step. Thus, the first and second flavonoid compounds can be produced very efficiently. In addition, by performing a preliminary culture step prior to the mycelium culture step, growth inhibition of Aspergillus cytoii at an early stage of culture can be avoided, and the efficiency of microbial conversion of naringin can be easily increased.

[0037]

EXAMPLES Examples of the above embodiment will be described below. <Production of converted naringin product> Potato dextrose-broth medium (manufactured by DIFCO) was dispensed in 100 mL aliquots into a plurality of Erlenmeyer flasks (500 mL in volume) and sterilized in an autoclave (121 ° C, 15 minutes). After cooling, each flask was inoculated with 1.0 mL of a spore suspension of Aspergillus cytoii adjusted to a concentration of 2 × 10 ⁸ cells / mL or more into each flask. The vegetative mycelium was grown while shaking culture was performed at 100 rpm / min. The Aspergillus cytoi strain used was an Aspergillus cytoii strain (IAM No. 2210) obtained from the Applied Microbiology Research Promotion Association (aka IAM).

After growing the vegetative mycelium by shaking culture for 10 days, 5 mL of an ethanol-diluted 10% by weight naringin (manufactured by SIGMA) ethanol sterilized (105 ° C., 5 minutes) was added thereto, followed by the aerobic treatment. Shaking culture was performed under the conditions to further promote spore formation from the vegetative mycelium. In addition, when the state of spore formation at this time was monitored over time, spore formation started about 5 days after the injection of naringin, and spore formation was observed on the entire liquid surface of the medium three weeks later. I found out.

In this experiment, samples were taken at the time one week after the injection of naringin (the time when the spores began to appear on the liquid surface), that is, the period from the early stage to the middle stage of spore formation. The collected sample was centrifuged (3000 rpm, 15 minutes) to remove impurities by precipitation, and the supernatant was analyzed by high performance liquid chromatography (HPLC) (LC10A manufactured by Shimadzu Corporation, column manufactured by YMC). A303), and the change in flavonoid composition was examined. As a result, the ratio of the peak of the naringin conversion product to the entire flavonoid composition was about 3%.
It was found to be 0%.

Further, the remainder of the HPLC sample confirmed to contain the naringin-converted product was concentrated by an evaporator, followed by preparative HPLC (LC8A manufactured by Shimadzu Corporation, column: R353-151 manufactured by YMC).
A, SH343-5), and the naringin converted product was isolated and purified. As a result, 2
Peaks of various naringin conversion products were confirmed. The ratio of the peaks of these two naringin conversion products was about 4%.
7.5% (first naringin conversion) and 33.2%
(The second naringin conversion product).

<Structure Determination> The structures of the first and second naringin conversion products obtained in the above <Production of naringin conversion product> were determined. ¹ H NMR and ¹³ C NMR spectra were obtained by using a JEOL JNM-EX-400 NMR apparatus ( ¹ H NM) using tetramethylsilane (TMS) dissolved in DMSO-d6 as an internal standard.
R was analyzed at 400 MHz and ¹³ C NMR at 100 MHz). Mass spectrum (FAB-MS) is JEOL
It was measured by JMS-DX-705L. Each naringin conversion product is developed by thin-layer chromatography (TLC) (developing solvent is 1-butanol / acetic acid / water = 4/1/5).
Then, the Rf value (movement rate) was obtained. The results are shown in Tables 1 to 3.
Shown in

[0042]

[Table 1]

[0043]

[Table 2]

[0044]

[Table 3] As a result, the first naringin conversion product is 8-hydroxynaringenin having the structure represented by the above formula 3, and the second naringin conversion product is 6-hydroxynaringenin having the structure represented by the above formula 4. Was confirmed.

Further, the technical ideas that can be grasped from the above embodiment will be described below. The method for producing a flavonoid compound according to any one of claims 2 to 4, wherein the microbial fermentation treatment is performed in a medium containing 0.01 to 5% by volume of an organic solvent. With such a configuration, a relatively large amount of naringin can be dissolved in the medium while the growth inhibition of Aspergillus cytoii is reduced, and the microbial conversion efficiency can be easily increased.

The flavonoid compound according to any one of claims 2 to 4, wherein the culture supernatant obtained after the microorganism fermentation treatment is further purified by reversed-phase liquid chromatography using a hydrophobic column. Manufacturing method. With this configuration, the flavonoid compound can be isolated very easily.

[0047] The method for producing a flavonoid compound according to any one of claims 2 to 4, wherein the naringin is extracted from citrus peel or capsular membrane. With such a configuration, it is easy to produce flavonoid compounds in large quantities and at low cost.

A flavonoid compound having a structure represented by Chemical formula 3 and obtained by subjecting naringin to microbial fermentation using Aspergillus saitoi. With this configuration, the range of use of naringin can be expanded.

A flavonoid compound having a structure represented by Chemical Formula 4 and obtained by subjecting naringin to microbial fermentation using Aspergillus saitoi. With this configuration, the range of use of naringin can be expanded.

[0050]

As described above in detail, according to the present invention, the following effects can be obtained. According to the flavonoid compound of the first aspect of the invention, the range of use of naringin can be expanded.

According to the method for producing a flavonoid compound according to the second to fourth aspects of the present invention, a flavonoid compound capable of expanding the range of use of naringin can be easily produced.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court ゛ (Reference) C12R 1:66) (72) Inventor Yoshiaki Miyake 45-2, Kumanosho, Shishakatsu, Nishi-Kasugai-gun, Aichi Prefecture (72) Inventor Toshihiko Osawa, 2615-409, Tokugawacho, Higashi-ku, Nagoya, Aichi Prefecture (72) Inventor Kenichiro Minato 164-203, Ichikikimachi, Nishi-ku, Nagoya-shi, Aichi F-term (reference) 4B064 AE46 CA05 CB07 CB12 CC21 DA01 DA10 4C062 EE54 4C086 AA03 BA08 ZC37 ZC41

Claims (4)

[Claims] 1. A flavonoid compound having a structure represented by the following chemical formula 1 and obtained by subjecting naringin to microbial fermentation using Aspergillus saitoi. Embedded image However, R1 is hydrogen and R2 is a hydroxyl group, or R1 is a hydroxyl group and R2 is hydrogen. 2. A method for producing a flavonoid compound according to claim 1, wherein naringin is added to Aspergillus cytoii.
A method for producing a flavonoid compound, comprising subjecting said naringin to microbial conversion to produce a flavonoid compound by subjecting said naringin to microbial fermentation treatment in saitoi). 3. The microbial fermentation treatment includes the steps of: culturing a medium containing naringin and Aspergillus cytoii with shaking; performing a mycelial culturing step for microbial conversion of naringenin to naringenin from vegetative mycelium of Aspergillus cytoii; 3. The method for producing a flavonoid compound according to claim 2, wherein the spore formation step of microbial conversion of the flavonoid compound from naringenin in the medium is performed while spore formation is promoted from the vegetative hypha. 4. The method for producing a flavonoid compound according to claim 3, wherein a preliminary culture step of shaking culture of a medium containing Aspergillus cytoii is performed prior to the mycelium culture step.