JP2009155293A - Fermented licorice extract and production method thereof, and external medicine for skin and food and drink for beautification containing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide fermented licorice extract excellent in various physiologically active functions including an antioxidant function and an anti-aging function, inexpensively producible, high in safety, not badly influencing the quality of an object to be added concerning taste, odor, feeling after use, and the like, and widely usable for an external medicine for skin and a food and drink for beautification, and further an external medicine for skin and a food and drink for beautification blended with the fermented licorice extract as an active constituent for various physiologically active functions including an antioxidant function and an anti-aging function. <P>SOLUTION: The fermented licorice extract and a production method thereof are characterized by obtaining by fermenting at least one of licorice powder and licorice extract using a fungus of the genus Aspergillus. The external medicine for skin and the food and drink for beautification containing the fermented licorice extract are also provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a fermented licorice extract, a method for producing the same, and a skin external preparation and a cosmetic food and drink containing the fermented licorice extract.

  Licorice is a root and rhizome of plant licorice (Glycyrrhiza glabra, G. uralensis, G. inflata, etc.) belonging to the genus Glycyrrhiza, which has antiallergic action, anti-inflammatory action, detoxification action, etc. Since it has a physiological activity, it has been used as a herbal medicine and herb for a long time. As an active ingredient contained in licorice, glycyrrhizin is a typical example, but flavonoid components such as liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin and the like are also included.

The flavonoid is a general term for compounds having a basic skeleton structure of C6-C3-C6 (C: carbon), and has various physiological activities such as an antioxidant action, a carcinogenesis-inhibiting action, an anti-inflammatory action, and an arteriosclerosis-inhibiting action. It is known to have an effect. Flavonoids are widely present in plants and can be ingested in large amounts from vegetables and fruits in daily life. In recent years, it has been reported that the presence or absence of a bioactive action and the strength are strong or weak depending on the type of the basic skeleton structure of the flavonoid or the difference in the functional group to be bound (for example, see Non-Patent Documents 1 to 3).
For example, 8-hydroxy liquiritigenin and 3′-hydroxy liquiritigenin, which are flavonoids, have been reported to exist in acacia, spinach and the like and have a strong antioxidant action (for example, Non-Patent Document 4). , 5).

In addition, it has been reported that the physiological activity may increase as a result of changes in the basic skeletal structure of flavonoids contained in plants or the functional groups to be bound by fermentation.
For example, in fermented soybean foods, isoflavone compounds, which are flavonoids contained in soybeans, have been reported to be converted to isoflavone compounds having an orthodihydroxy structure during the fermentation process and exert a strong antioxidant effect (for example, Patent Document 1).
For example, citrus fruits such as orange and lemon are used as filamentous fungi. It has been reported that when fermented using saitoi, hesperidin, which is a flavonoid, is converted to 8-hydroxyhesperetin and exhibits a strong antioxidant effect (see, for example, Patent Document 2).
However, there is no report that the 8-hydroxy liquiritigenin and the 3′-hydroxy liquiritigenin were produced by fermentation.

  Regarding the fermentation of licorice, it has been reported that an aqueous extract of licorice is subjected to lactic acid fermentation, and the fermented product is used as a functional food composition, an antiandrogen agent, a peripheral vasodilator, and an arteriosclerosis inhibitor. (For example, see Patent Documents 3 to 5). However, in the lactic acid fermentation techniques described in Patent Documents 3 to 5, the basic skeleton structure of flavonoids contained in licorice or the functional group to be bonded does not change.

  Therefore, up to now, for example, by changing the basic skeleton structure of flavonoids or the functional group to which it is bonded, it is excellent in various physiological activities such as antioxidant and anti-aging effects, and can be manufactured at low cost, and safety The fermented licorice extract that is high and does not adversely affect the quality of the added object in terms of taste, smell, feeling of use, etc., and can be widely used for skin external preparations and beauty foods and drinks, has not yet been provided, At present, there is a strong demand for prompt provision.

JP 2002-308868 A JP 2002-275175 A Patent No. 2801964 JP-A-10-236967 JP 2003-26592 A Plant Flavonoids in Biology and Medicine (1986), Alan R. et al. Liss Inc. (NY) Plant Flavonoids in Biology and Medicine II (1988), Alan R. et al. Liss Inc. (NY) The Flavonoids (1994), Chapman & Hall (London) Biochemical Journal (1966), 98 (2), 493-500 Journal of Agricultural and Food Chemistry (2001), 49 (6), 2767-2773.

An object of the present invention is to solve the conventional problems and achieve the following objects. That is, the present invention is firstly excellent in various physiologically active effects such as an antioxidant action and an anti-aging action and can be produced at low cost by changing the basic skeleton structure of flavonoid or the functional group to be bonded. Providing fermented licorice extract that is highly safe and that can be widely used for external preparations for skin and beauty foods and drinks without adversely affecting the quality of the additive in terms of taste, smell, feeling of use, etc. The purpose is to do.
The present invention also provides, secondly, a skin external preparation and a cosmetic food and drink comprising the fermented licorice extract of the present invention as an active ingredient for various physiologically active actions including an antioxidant action and an anti-aging action. The purpose is to provide.

  In order to solve the above-mentioned problems, the present inventors have made extensive studies and as a result, obtained the following findings. That is, as a result of fermenting licorice water extract containing liquiritin and isolicritin using Aspergillus fungi, the liquiritin is microbially converted to 8-hydroxyliquiritigenin through microbial conversion to liquiritigenin. And the isolicritin was microbially converted to 3′-hydroxyisoquiritigenin via microbial conversion to isoliqueritigenin; the fermented licorice extract obtained by the fermentation treatment, The superoxide removal action, radical scavenging action and MMP-1 activity inhibitory action are remarkably increased as compared with those before fermentation treatment, and are useful as antioxidants, anti-aging agents, skin external products and cosmetic foods and drinks. ;

The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> A fermented licorice extract obtained by fermenting at least one of licorice powder and licorice extract using Aspergillus genus filamentous fungi.
<2> Aspergillus fungi are oryzae, A.M. Kawachi, A.K. awamori, A.M. sojae, A.M. saitoi and A.I. The fermented licorice extract according to <1>, which is at least one selected from niger.
<3> The fermented licorice extract according to any one of <1> to <2>, which contains at least one of 8-hydroxyliquiritigenin and 3′-hydroxyisoquiritigenin.
<4> An external skin product containing the fermented licorice extract according to any one of <1> to <3>.
<5> A cosmetic food or drink comprising the fermented licorice extract according to any one of <1> to <3>.
<6> A method for producing a fermented licorice extract, comprising a step of fermenting at least one of licorice powder and licorice extract using Aspergillus genus filamentous fungi.
<7> Aspergillus filamentous fungi oryzae, A.M. Kawachi, A.K. awamori, A.M. sojae, A.M. saitoi and A.I. <1> The method for producing a fermented licorice extract according to <6>, wherein at least one filamentous fungus selected from niger is used.
<8> One or more selected from ammonium sulfate, ammonium nitrate, urea, amino acids, yeast extract, peptone, protein and meat extract are used as a medium for fermentation. <6> to <7> It is a manufacturing method of fermented licorice extract.
<9> In the step of fermentation treatment, liquiritin contained in at least one of licorice powder and licorice extract is microbially converted to 8-hydroxyliquiritigenin via microbial conversion to liquiritigenin; And isolicritin contained in at least one of licorice powder and licorice extract is microbially converted into 3′-hydroxyisoquiritigenin via microbial conversion into isoliqueritigenin. The method for producing a fermented licorice extract according to any one of <6> to <8>, wherein at least one of them is performed.

According to the present invention, the conventional problems can be solved and the object can be achieved. First, for example, the basic skeletal structure of flavonoid or the functional group to be bonded is changed, so that the antioxidant action and the anti-oxidation are achieved. It is excellent in various physiological activities including aging, can be manufactured at low cost, has high safety, does not adversely affect the quality of the additive in terms of taste, smell, feeling of use, etc. It is possible to provide a fermented licorice extract that can be widely used for food and drink for food use and a method for producing the same.
In addition, according to the present invention, secondly, the external preparation for skin and cosmetics, wherein the fermented licorice extract of the present invention is blended as an active ingredient for various physiologically active effects including antioxidant and anti-aging effects. Food and drink can be provided.

(Fermented licorice extract)
The fermented licorice extract of the present invention is obtained by fermenting at least one of licorice powder and licorice extract using Aspergillus fungi.

-Aspergillus fungi-
The Aspergillus filamentous fungus is not particularly limited and may be appropriately selected depending on the intended purpose. However, it is preferably isolated from koji used in fermented foods such as sake, miso, and soy sauce.
There is no restriction | limiting in particular as a seed | species of the said Aspergillus genus filamentous fungi, According to the objective, it can select suitably, for example, A. oryzae, A.M. Kawachi, A.K. awamori, A.M. sojae, A.M. Saitoi, A.I. niger and the like.

-Licorice powder, licorice extract-
The licorice is not particularly limited as long as it is a root and rhizome of a plant licorice belonging to the genus Glycyrrhiza, which can be appropriately selected according to the purpose. Examples of the plant liquorice include Glycyrrhiza glabra, G. et al. uralensis, G. et al. inflata and the like. The licorice is rich in flavonoid components such as liquiritin, isoliquiritin, liquiritigenin and isoliquiritigenin.

  The licorice powder can be easily obtained by a method generally used for crushing plants. For example, the licorice powder can be obtained by drying the licorice and then crushing it using a crusher.

  The licorice extract can be easily obtained by a method generally used for plant extraction. The licorice extract includes a licorice extract, a dried product obtained by drying a diluted solution of the extract, or a crude product or a purified product thereof.

  The licorice, which is the raw material for extraction, can be obtained by drying or pulverizing it as it is or using a crusher and subjecting it to solvent extraction. Drying may be performed in the sun or using a commonly used dryer. The licorice may be used as a raw material for extraction after pretreatment such as degreasing with a nonpolar solvent such as hexane or benzene. In addition, the extraction process by the polar solvent of licorice can be performed efficiently by performing pretreatments, such as degreasing.

As the solvent used for the extraction, it is preferable to use water, a hydrophilic organic solvent, or a mixed solvent thereof at a temperature from room temperature to the boiling point of the solvent.
Examples of water that can be used as the extraction solvent include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, as well as water that has been subjected to various treatments. Examples of the treatment applied to water include purification, heating, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering, and the like. The water that can be used as the extraction solvent includes purified water, hot water, ion exchange water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.

Examples of the hydrophilic organic solvent include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene glycol, propylene glycol, Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycerin, and a mixed solvent of these hydrophilic organic solvents and water can be used.
In addition, when using the mixed solvent of the said water and a hydrophilic organic solvent, in the case of a lower alcohol, 1 mass part-90 mass parts with respect to 10 mass parts of water, and in the case of a lower aliphatic ketone, 10 masses of water. It is preferable to add 1 to 40 parts by mass with respect to parts. In the case of polyhydric alcohol, it is preferable to add 1 part by mass to 90 parts by mass with respect to 10 parts by mass of water.

  In the present invention, it is not necessary to employ a special extraction method for extracting a licorice extract from licorice, which is an extraction raw material, and extraction can be performed using any extraction device at room temperature or under reflux heating.

  Specifically, in a treatment tank filled with an extraction solvent, licorice as an extraction raw material is added, and while being occasionally stirred, if necessary, it is allowed to stand for 30 minutes to 2 hours to elute soluble components, The solid matter is removed by filtration, and the extraction solvent is distilled off from the obtained extract, and the extract is obtained by drying. The amount of the extraction solvent is usually 5 to 15 times (mass ratio) of the extraction raw material. The extraction conditions are usually about 1 to 4 hours at 50 to 95 ° C. when water is used as the extraction solvent. Moreover, when the mixed solvent of water and ethanol is used as an extraction solvent, it is normally 30 minutes-about 4 hours at 40 to 80 degreeC.

  The resulting licorice extract is diluted, concentrated, dried, purified, etc. according to a conventional method to obtain a diluted or concentrated solution of the extract, a dried product of the extract, or a crudely purified product or purified product thereof. Processing may be performed.

-Fermentation treatment-
The fermentation treatment method is not particularly limited as long as Aspergillus filamentous fungi are used, and can be appropriately selected from known fermentation methods according to the purpose. As the fermentation treatment, for example, there is a method of inoculating precultured Aspergillus filamentous fungi in a medium adjusted to an appropriate temperature after sterilizing the medium together with an aeration and stirring type fermenter, and performing aeration and stirring while controlling the culture temperature. Can be mentioned.

There is no restriction | limiting in particular as said aeration stirring type fermenter, It can select suitably according to the objective from the normally used aeration stirring type fermenter, For example, for bacteria, for yeast, for filamentous fungi etc. Can be mentioned. There is no restriction | limiting in particular as the method of the said preculture, For example, it can carry out by the liquid culture by a small fermenter, a flask, a test tube etc.
The components of the medium are not particularly limited as long as the cells used for fermentation can grow, and can be appropriately selected from the components of the medium normally used for fermentation according to the purpose. The nitrogen source in the medium is not particularly limited, but it is possible to select one or more selected from ammonium sulfate, ammonium nitrate, urea, amino acids, yeast extract, peptone, protein, and meat extract in terms of high fermentation efficiency. preferable.
Although there is no restriction | limiting in particular as pH of the said culture medium in a fermentation process, It is preferable that it is 3.5-8.0 at a point with the high efficiency of fermentation. Although there is no restriction | limiting in particular as the temperature of the said culture medium in a fermentation process, It is preferable that it is 20-37 degreeC at the point with the high efficiency of fermentation.

In the course of the fermentation treatment, liquiritin contained in at least one of licorice powder and licorice extract is microbially converted to 8-hydroxyliquiritigenin through microbial conversion to liquiritigenin. Further, isolicritin contained in at least one of licorice powder and licorice extract is microbially converted to 3′-hydroxyisoquiritigenin through microbial conversion to isoliqueritigenin. Liquiritin is a glycoside of liquiritigenin, and isoliquiritin is a glycoside of isoquiritigenin.
The “microorganism conversion” means that the conversion of the molecular structure of each flavonoid is performed using aspergillus filamentous fungi-derived components (for example, enzymes) as a catalyst.

  The fermented licorice extract obtained by the fermentation treatment can be used as it is as a composition having various physiological activities such as an antioxidant action and an anti-aging action. It is easier to use the one that has been. In obtaining a dried fermented licorice extract, carriers such as dextrin and cyclodextrin may be added to improve hygroscopicity. In addition, since the fermented licorice extract has a unique odor, it can be purified for the purpose of decolorization, deodorization, etc. within a range that does not cause a decrease in its physiological activity. Since it is not used in a large amount when it is added to an agent or cosmetic food or drink, there is no practical problem even if it is not purified. The purification can be performed, for example, by activated carbon treatment, adsorption resin treatment, ion exchange resin treatment, or the like.

-8-hydroxyliquiritigenin, 3'-hydroxyisoquiritigenin-
As described above, the fermented licorice extract of the present invention is obtained as a result of microbial conversion in the fermentation process, and is represented by the following 8-hydroxy liquiritigenin and 3′-hydroxy represented by the following structural formulas (1) and (2), respectively. Contains isoliquiritigenin.

８−ヒドロキシリクイリチゲニン（８−ＯＨＬ）

３’−ヒドロキシイソリクイリチゲニン（３’−ＯＨＩ）

8-Hydroxyliquiritigenin (8-OHL)

3'-hydroxyisoquiritigenin (3'-OHI)

There is no restriction | limiting in particular as content of the said 8-hydroxy liquiritigenin and the said 3'-hydroxyiso liquiritigenin in the said fermented licorice extract, or fermentation conditions are changed by concentrating a fermentation liquid. The content can be adjusted to a desired value.
The content of 8-hydroxyliquiritigenin and 3′-hydroxyisoquiritigenin can be measured by a known method. For example, the fermented licorice extract is separated using HPLC, and the corresponding peak is obtained. It can be calculated based on the area.

The fermented licorice extract of the present invention has various physiologically active actions including an antioxidant action and an anti-aging action, and based on these actions, for example, used as an antioxidant, an anti-aging agent, etc. can do.
The antioxidant effect of the fermented licorice extract of the present invention is exhibited based on at least one of a superoxide scavenging action and a radical scavenging action.
The anti-aging effect of the fermented licorice extract of the present invention is exhibited based on the MMP-1 activity inhibitory action.
The reason for exerting the superoxide scavenging action, the radical scavenging action and the MMP-1 activity inhibiting action is that 8-hydroxyliquiritigenin and 3′-hydroxyisoquiritigenin contained in the fermented licorice extract It is thought to be due to the action of
Moreover, if the fermented licorice extract of this invention is a bioactivity action which 8-hydroxy liquiritigenin and 3'-hydroxyisoquiritigenin have, other than the said radical scavenging action and the said MMP-1 activity inhibitory action Naturally, it can also exert its physiological activity.

The fermented licorice extract of the present invention has various excellent physiologically active effects including an antioxidant effect and an anti-aging effect, and is excellent in usability and safety when applied to the skin. It is suitable for blending into the skin external preparation of the present invention described later.
In addition, the fermented licorice extract of the present invention has various excellent physiological activities including antioxidation and anti-aging, and is not digested in the digestive tract. Therefore, it is particularly suitable for blending into the beauty food or drink of the present invention described later.

(Method for producing fermented licorice extract)
The method for producing a fermented licorice extract of the present invention includes a step of fermenting at least one of licorice powder and licorice extract using Aspergillus genus fungi, and further, for example, fermentation It includes other steps such as a step of drying the treated product, a step of purifying the fermented product, and a step of concentrating the fermented product.
Aspergillus fungi, licorice powder, licorice extract, fermentation treatment and the like in the method for producing the fermented licorice extract are as described above in each item of the fermented licorice extract of the present invention, and thus the description thereof is omitted. To do.

(Skin external preparation)
The external preparation for skin of the present invention contains the fermented licorice extract of the present invention as an active ingredient, and further contains other components appropriately selected as necessary.

  Here, the use of the external preparation for skin is not particularly limited and can be appropriately selected from various uses. For example, an ointment, cream, emulsion, lotion, pack, jelly, lip balm, lipstick, bath preparation, astringent , Hair tonic, hair cream, hair liquid, shampoo, pomade, rinse, and the like.

  There is no restriction | limiting in particular as a compounding quantity of the fermented licorice extract of this invention in the said whole skin external preparation, Although it can adjust suitably with the kind of skin external preparation, 0.0001 with respect to the said whole skin external preparation. % By mass to 10% by mass is preferable, and 0.001% by mass to 1% by mass is more preferable.

The external preparation for skin can further contain various main ingredients and auxiliaries usually used in the production of cosmetics and other components as long as they do not impair the object and effects of the present invention.
The other components are not particularly limited and include components appropriately selected according to the purpose. For example, astringents, bactericides, antibacterial agents, ultraviolet absorbers, humectants, cell activators, fats and oils, waxes , Hydrocarbons, fatty acids, alcohols, esters, surfactants, fragrances, and the like. When used in combination with the fermented licorice extract, these components may act synergistically with various physiological activities of the fermented licorice extract, resulting in superior effects that are normally expected. .

  The external preparation for skin of the present invention has high safety when used on the skin, and effectively exhibits various excellent bioactive actions including antioxidative action and anti-aging action. It is useful for anti-oxidation and anti-aging.

(Beauty food and drink)
The cosmetic food and drink of the present invention contains the fermented licorice extract of the present invention as an active ingredient, and further contains other components appropriately selected as necessary.
Here, the above-mentioned beauty foods and drinks are those that are less likely to harm human health and are taken orally or by digestive tract administration in normal social life. It is not limited to categories such as quasi-drugs, and includes, for example, foods that include a wide range of foods that are taken orally, such as general foods, health foods, health functional foods, quasi-drugs, and pharmaceuticals.

  The cosmetic food and drink of the present invention may be prepared by blending the fermented licorice extract with any food or drink so as not to impede its activity, or a nutritional supplement mainly composed of the fermented licorice extract. It may be food.

  There is no restriction | limiting in particular as said beauty food-drinks, Although it can select suitably according to the objective, For example, drinks, such as a soft drink, a carbonated drink, a nutrition drink, a fruit drink, a lactic acid drink; Ice cream, ice sherbet , Shaved ice and other frozen desserts; noodles such as buckwheat, udon, harusame, gyoza skin, sushi mai, Chinese noodles, instant noodles; rice cakes, candy, gum, chocolate, tablet confectionery, snacks, biscuits, jelly, jam, cream, Baked goods such as baked goods, bread; crab, salmon, clams, tuna, sardines, shrimp, skipjack, mackerel, whale, oyster, saury, squid, red sea bream, scallop, abalone, sea urchin, sea bream, tocobushi, etc .; Processed fishery and livestock products such as ham and sausage; Dairy products such as processed milk and fermented milk; salad oil, tempura oil, margarine, mayonnaise, Oil and fat processed foods such as yotoning, whipped cream, dressing; seasonings such as sauce and sauce; curry, stew, parent and child rice bowl, rice bowl, miscellaneous cooking, Chinese rice bowl, bowl, tempura, eel rice, hayashi rice, oden, marbodorf, Retort pouch foods such as beef bowl, meat sauce, egg soup, omelet rice, dumplings, shumai, hamburger, meatballs; various forms of health foods and nutritional supplements; pharmaceuticals such as tablets, capsules, drinks, troches, etc. Examples include foreign products.

As said other component, the auxiliary | assistant raw material or additive normally used in manufacturing the said cosmetics food / beverage products is mentioned.
The raw material or additive is not particularly limited and may be appropriately selected depending on the intended purpose.For example, glucose, fructose, sucrose, maltose, sorbitol, stevioside, rubusoside, corn syrup, lactose, citric acid, Tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dl-α-tocopherol, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, gum arabic , Carrageenan, casein, gelatin, pectin, agar, vitamin Bs, nicotinamide, calcium pantothenate, amino acids, calcium salts, pigments, fragrances, preservatives, and the like.

  The addition amount of the fermented licorice extract of the present invention in the beauty food or drink varies depending on the type of the beauty food or drink to be targeted and cannot be unconditionally defined, but the original taste of the beauty food or drink is impaired. What is necessary is just to add in the range which is not, 0.001 mass%-50 mass% are preferable with respect to various object cosmetics food / beverage products, and 0.01 mass%-20 mass% are more preferable. Further, in the case of a cosmetic food or drink in the form of granules, tablets or capsules, 0.01% by mass to 100% by mass is preferable, and 5% by mass to 100% by mass is more preferable.

  The cosmetic food and drink of the present invention can be taken orally on a daily basis, and has various physiological activities including antioxidant and anti-aging effects by the action of fermented licorice extract, which is an active ingredient. It can be exhibited extremely effectively.

  Note that the fermented licorice extract, external preparation for skin, and food and drink for cosmetics of the present invention are preferably applied to humans, but as long as the respective effects are exerted, animals other than humans can be used. Can also be applied.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

(Production Example 1)
-Production of fermented licorice extract-
As a raw material for extraction, 100 g of pulverized licorice root was put into 1,000 ml of water, kept at 80 ° C. for 2 hours with gentle stirring, and then filtered. The filtrate was concentrated under reduced pressure at 40 ° C. and further dried with a vacuum dryer to obtain an extract (powder).
Aspergillus sojae (A. sojae) was sterilized (121 ° C, 20 minutes) and cooled in a 500 mL Erlenmeyer flask with baffle to which 120 mL of a medium containing 3% by weight bread crumbs and water (pH 5.0) was added. The preculture was obtained by shaking culture at 28 ° C. for 2 days. In the main culture, 6 L (pH 5.0) of a medium containing 5% by mass licorice extract, 0.2% by mass yeast extract and water is introduced into a 10 L jar fermenter, sterilized (121 ° C., 20 minutes) and cooled. Thereafter, a preculture solution (120 mL) was inoculated, and cultured for 18 days under the culture conditions of a culture temperature of 28 ° C., a stirring speed of 150 rpm, and an aeration rate of 1 vvm to obtain the fermented licorice extract of Production Example 1.

(Comparative Example 1)
-Production of licorice extract-
As a raw material for extraction, 100 g of pulverized licorice root was put into 1,000 ml of water, kept at 80 ° C. for 2 hours with gentle stirring, and then filtered. The filtrate was concentrated under reduced pressure at 40 ° C. and further dried with a vacuum dryer to obtain an extract (powder).

Example 1
-Component composition analysis of fermented licorice extract-
Using the fermented licorice extract of Production Example 1 and the licorice extract of Comparative Example 1 as samples, HPLC (liquid chromatography) analysis was performed under the following conditions.

[HPLC conditions]
Mobile phase: (A) 0.1% TFA aqueous solution, (B) 0.1% TFA acetonitrile solution
0-5 minutes A: B = 80: 20
5-35 minutes A: B = 80: 20 to 10:90 linear gradient,
35-40 minutes A: B = 10: 90
Flow rate: 0.4 mL / min Detection: UV254, 330 nm
Column temperature: 40 ° C
Injection volume: 10 μL
Column: Zorbax Eclipse XDB-C18

前記表１中、「＋」はピークが検出されたことを示す。 Based on the area of the peak obtained by HPLC analysis, the content (mass%) of various flavonoids relative to the entire sample was quantified. The results are shown in Table 1.

In Table 1, “+” indicates that a peak was detected.

  As shown in Table 1, liquiritin and isoliquiritin detected in the licorice extract of Comparative Example 1 are not detected in the fermented licorice extract of Production Example 1, but in the fermented licorice extract of Production Example 1, instead. New peaks RT3.8 and RT10.0 were detected.

(Example 2)
-Structural analysis of RT3.8 and RT10.0-
RT3.8 and RT10.0 were purified under the following conditions, and structural analysis was performed under the following conditions using the purified RT3.8 and RT10.0 as a sample.

40 g of the fermented licorice extract obtained in Production Example 1 was subjected to ODS column chromatography, silica gel column chromatography, and recycle HPLC (LC-918, manufactured by Nihon Analytical Industry, JAIGEL GS-310, manufactured by Nihon Analytical Industry). Fractionation and purification of RT3.8 and RT10.0 were performed.
The structure was determined for purified new peaks RT3.8 and RT10.0. ¹ H NMR and ¹³ C NMR spectra were obtained using a tetramethylsilane (TMS) dissolved in methanol-d4 as an internal standard, and a JEOL AL-400 NMR apparatus ( ¹ H NMR was 400 MHz, ¹³ C NMR was 100 MHz. ). The results are shown below.

<RT3.8>
1H-NMR chemical shift δ (assigned hydrogen): 2.71 (1H, dd, J = 2.9, 16.8 Hz), 3.08 (1H, dd, J = 12.7, 16.8 Hz) (3 -H2), 5.41 (1H, dd, J = 2.9, 12.7 Hz, 2-H), 6.52 (1H, d, J = 8.8 Hz, 6-H), 6.81 ( 2H, d, J = 8.6 Hz, 3 ′, 5′−H), 7.29 (1H, d, J = 8.8 Hz, 5-H), 7.36 (2H, d, J = 8. 6Hz, 2 ', 6'-H)
13C-NMR chemical shift δ (assigned carbon): 45.0 (3-C), 81.4 (2-C), 110.8 (6-C), 115.6 (10-C), 116.2 (3 ', 5'-C), 119.3 (5-C), 129.2 (2', 6'-C), 131.2 (1'-C), 133.9 (8-C) , 152.6 (7-C), 153.9 (9-C), 158.8 (4'-C), 193.8 (4-C)

<RT10.0>
1H-NMR chemical shift δ (assigned hydrogen): 6.36 (1H, d, J = 8.9 Hz, 5′-H), 6.74 (2H, d, J = 8.6 Hz, 3,5-H ), 7.44 (1H, d, J = 8.9 Hz, 6′-H), 7.48 (1H, d, J = 15.4 Hz, α-H), 7.50 (2H, d, J = 8.6 Hz, 2, 6-H), 7.68 (1 H, d, J = 15.4 Hz, β-H)
13C-NMR chemical shift δ (assigned carbon): 108.4 (5′-C), 115.0 (1′-C), 116.8 (3, 5-C), 118.3 (α-C) , 123.2 (6′-C), 127.7 (1-C), 131.7 (2, 6-C), 133.6 (3′-C), 145.4 (β-C), 153.1 (4′-C), 154.2 (2′-C), 161.3 (4-C), 193.9 (C═O)

  From the NMR measurement results, the new peak RT3.8 is a compound in which a hydroxyl group is bonded to the 8-position of liquiritigenin, so-called 8-hydroxyliquiritigenin, and RT10.0 is a hydroxyl group at the 3′-position of isoliqueritigenin. It was confirmed to be a compound to which is bound, so-called 3′-hydroxyisoquiritigenin.

(Example 2)
-Radical scavenging test for DPPH-
Using each fermented licorice extract of Production Example 1 and licorice extract of Comparative Example 1 as samples, 1,1-diphenyl-2-picrylhydradyl radical (DPPH), which is a very stable radical, was used according to the following test method. The radical scavenging action was tested.

3 mL of each sample solution was added to 3 mL of a 1.5 × 10 ⁻⁴ mol / L DPPH ethanol solution, and the container was immediately sealed, shaken and allowed to stand for 30 minutes, and then the absorbance at a wavelength of 520 nm was measured.
As a control, the same operation was performed using a solvent in which the sample solution was dissolved instead of the sample solution, and the absorbance at a wavelength of 520 nm was measured. Further, as a blank, after adding 3 mL of the sample solution to ethanol, the absorbance at a wavelength of 520 nm was measured immediately.
And the radical elimination rate (%) was computed from the measurement result by following (1) Formula.
Radical scavenging rate (%) = {1- (BC) / A} × 100 (1)
[However, in the above formula (1),
A: Absorbance of control,
B: Absorbance when the sample solution is added,
C: Absorbance of the blank, respectively. ]

Next, the sample concentration was decreased stepwise to measure the radical scavenging rate, and the sample concentration (μg / mL) at which the DPPH radical scavenging rate was 50% was determined by interpolation. The results are shown in Table 2.
Further, as a reference example, radical scavenging test for DPPH was conducted in the same manner as described above using 8-hydroxyliquiritigenin, 3′-hydroxyisoquiritigenin, liquiritigenin and isoliquiritigenin as samples. The rate (%) was calculated. The results are shown in Table 2.

表２の結果から、製造例１の発酵甘草抽出物が、高いＤＰＰＨに対するラジカル消去作用を有することが認められた。

From the results in Table 2, it was confirmed that the fermented licorice extract of Production Example 1 has a high radical scavenging action on DPPH.

(Example 3)
-Superoxide elimination test (NBT method)-
Using each fermented licorice extract of Production Example 1 and licorice extract of Comparative Example 1 as samples, the superoxide scavenging action was tested by the following test method.

3 mmol / L xanthine, 3 mmol / L EDTA, 1.5 mg / mL bovine serum albumin (BSA) solution, 0.75 mmol / L nitroblue tetrazolium (NBT) 0.1 mL, and 0.05 mol / L Na 2.4 mL of ₂ CO ₃ buffer (pH 10.2) was placed in a test tube, 0.1 mL of each sample solution was added thereto, and the mixture was allowed to stand at 25 ° C. for 10 minutes. Subsequently, the xanthine oxidase solution was added, it stirred rapidly, and it left still at 25 degreeC for 20 minutes. Thereafter, 0.1 mL of 6 mmol / L copper chloride was added to stop the reaction, and the absorbance at a wavelength of 560 nm was measured. The absorbance measured at this time was defined as “absorbance upon addition of sample solution and enzyme solution”.
The same operation and measurement of absorbance were performed without adding the enzyme solution. The absorbance measured at this time was defined as “absorbance when the sample solution was added and no enzyme solution was added”.
The same measurement was performed when distilled water was added without adding the sample solution. The absorbance measured at this time was defined as “absorbance when no sample solution was added and enzyme solution was added”.
Moreover, the same measurement was performed when distilled water was added without adding the enzyme solution and without adding the sample solution. The absorbance measured at this time was defined as “absorbance when no sample solution was added and no enzyme solution was added”.
And the superoxide elimination rate was calculated | required from the measurement result by following (2) Formula.
Superoxide erasure rate (%) = {1- (AB) / (CD)} × 100 (2)
[However, in the above formula (2),
A: Sample solution addition, absorbance at the time of enzyme solution addition,
B: Absorbance when sample solution is added and enzyme solution is not added,
C: No sample solution added, absorbance at the time of enzyme solution addition,
D: Absorbance when no sample solution is added and when no enzyme solution is added. ]

Next, the sample concentration was decreased stepwise to measure the superoxide erasure rate, and the sample concentration (μg / mL) at which the superoxide erasure rate was 50% was determined by interpolation. The results are shown in Table 3.
In addition, as a reference example, 8-hydroxyliquiritigenin, 3′-hydroxyisoquiritigenin, liquiritigenin, and isoliquiritigenin were used as samples to perform a superoxide elimination test in the same manner as described above. The rate (%) was calculated. The results are shown in Table 3.

表３の結果から、製造例１の発酵甘草抽出物が、高いスーパーオキサイド消去作用を有することが認められた。

From the results in Table 3, it was confirmed that the fermented licorice extract of Production Example 1 had a high superoxide scavenging action.

Example 4
-Matrix metalloproteinase-1 (MMP-1) activity inhibition test-
Using each fermented licorice extract of Production Example 1 and licorice extract of Comparative Example 1 as samples, MMP-1 activity inhibitory action was tested by the following test method. This test method is a partial modification of the Wunsch and Heidrich method.

In a test tube with a lid, 50 μL of a test sample dissolved in 0.1 mol / L Tris-HCl buffer (pH 7.1) containing 20 mmol / mL calcium chloride, MMP-1 (COLLAGENASE Type IV from Clostridium histolyticum (manufactured by Sigma) ) 50 μL of the solution and Pz-peptide (Pz-peptide: Pz-Pro-Leu-Gly-Pro-D-Arg-OH (manufactured by BACHEM Feinchemikaline AG)) solution 400 μL were mixed and reacted at 37 ° C. for 30 minutes. The reaction was stopped by adding 1 mL of a 25 mmol / L citric acid solution. Thereafter, 5 mL of ethyl acetate was added and shaken vigorously. This was centrifuged (1,600 × g, 10 minutes), and the absorbance of the ethyl acetate layer at a wavelength of 320 nm was measured. A blank test was performed and corrected in the same manner. From the obtained measurement results, the MMP-1 activity inhibition rate was determined by the following formula (3).
MMP-1 activity inhibition rate (%) = {1- (C−D) / (A−B)} × 100 (3)
[However, in the formula (3),
A: Absorbance at a wavelength of 320 nm with no test sample added and enzyme added,
B: Absorbance at a wavelength of 320 nm with no test sample added and no enzyme added,
C: Absorbance at a wavelength of 320 nm with addition of test sample and enzyme,
D: Absorbance at 320 nm with addition of test sample and without addition of enzyme. ]

The inhibition rate was measured by gradually reducing the sample concentration, and the sample concentration (μg / mL) at which the inhibition rate was 50% was determined by interpolation. The results are shown in Table 4.
Further, as a reference example, 8-hydroxyliquiritigenin, 3′-hydroxyisoquiritigenin, liquiritigenin, and isoquiritigenin are used as samples, and matrix metalloproteinase-1 (MMP-1) in the same manner as described above. An activity inhibitory action test was performed, and the MMP-1 activity inhibition rate (%) was calculated. The results are shown in Table 4.

表４の結果から、製造例１の発酵甘草抽出物が、高いマトリックスメタロプロテアーゼ−１（ＭＭＰ−１）活性阻害作用を有することが認められた。

From the results of Table 4, it was confirmed that the fermented licorice extract of Production Example 1 has a high matrix metalloproteinase-1 (MMP-1) activity inhibitory action.

(Formulation example 1)
-Emulsion-
An emulsion was prepared from the following composition by a conventional method.
-Fermented licorice extract of Production Example 1 ... 0.10 g
・ Jojoba oil: 4.00 g
・ 1,3-Butylene glycol ・ ・ ・ 3.00g
・ Polyoxyethylene cetyl ether (20E.O.) ... 2.50 g
・ Olive oil ... 2.00g
・ Squalane ... 2.00g
・ Cetanol ... 2.00g
・ Glyceryl monostearate ... 2.00g
・ Oleic acid polyoxyethylene sorbitan (20E.O.) ... 2.00g
・ Methyl paraoxybenzoate 0.15 g
-Jaundice extract ... 0.10g
・ Dipotassium glycyrrhizinate ... 0.10g
・ Ginkgo biloba extract ... 0.10g
・ Conchiolin ... 0.10g
・ Owaku extract ... 0.10g
・ Chicken extract ... 0.10g
・ Fragrance ... 0.05g
・ Purified water: remainder (total 100.00 g)

(Formulation example 2)
-Lotion-
A lotion was produced from the following composition by a conventional method.
-Fermented licorice extract of Production Example 1 ... 0.10 g
・ Glycerin ... 3.00g
・ 1,3-Butylene glycol ・ ・ ・ 3.00g
・ Oleic acid polyoxyethylene sorbitan (20E.O.) ... 2.00g
・ Methyl paraoxybenzoate 0.15 g
・ Citric acid ... 0.10g
・ Sodium citrate: 0.10 g
・ Oil-soluble licorice extract ... 0.10g
・ Seaweed extract ... 0.10g
・ Cudin extract ... 0.10g
・ Xylobiose Mixture ... 0.05g
・ Fragrance ... 0.05g
・ Purified water: remainder (total: 100.00 g)

(Formulation example 3)
-Cream-
A cream was produced from the following composition by a conventional method.
-Fermented licorice extract of Production Example 1 ... 0.10 g
・ Squalane ... 10.00g
・ 1,3-butylene glycol ... 6.00g
・ Liquid paraffin ・ ・ ・ 5.00g
・ Salah beeswax 4.00 g
・ Cetanol ... 3.00g
・ Glyceryl monostearate ... 3.00 g
・ Lanoline ... 2.00g
・ Oleic acid polyoxyethylene sorbitan (20E.O.) ... 1.50 g
・ Methyl paraoxybenzoate ... 1.50g
・ Stearic acid: 1.00 g
・ Yeast extract ... 0.10g
・ Perilla extract ... 0.10g
-Linden extract ... 0.10g
・ Jiuyu extract ... 0.10g
・ Fragrance ... 0.10g
・ Purified water: remainder (total: 100.00 g)

(Formulation example 4)
−Pack−
A pack was produced from the following composition by a conventional method.
-Fermented licorice extract of Production Example 1 0.20 g
・ Polyvinyl alcohol: 15.00g
・ Ethanol ... 10.00g
・ Propylene glycol: 7.00 g
・ Polyethylene glycol ... 3.00g
・ Sage extract ... 0.10g
・ Toki extract ... 0.10g
・ Carrot extract ... 0.10g
・ Ethyl paraoxybenzoate 0.05g
・ Fragrance ... 0.05g
・ Purified water: remainder (total: 100.00 g)

(Formulation example 5)
-Tablet dietary supplements-
The following mixture was tableted to produce a tablet-shaped dietary supplement.
-Fermented licorice extract of Production Example 1 ... 30 g
・ Powdered sugar (sucrose) 178g
・ Sorbit ・ ・ ・ 10g
・ Glycerin fatty acid ester: 12g

(Formulation example 6)
-Granular dietary supplement-
The following mixture was formed into granules to produce a dietary supplement.
-Fermented licorice extract of Production Example 1 20g
・ Beat oligosaccharide ... 1000g
・ Vitamin C ... 167g
・ Stevia extract ... 10g

(Formulation example 7)
-Granular dietary supplement-
The following mixture was formed into granules to produce a dietary supplement.
-Fermented licorice extract of Production Example 1 20g
・ Beat oligosaccharide ... 1000g
・ Vitamin C ... 167g
・ Stevia extract ... 10g

The fermented licorice extract of the present invention has various excellent physiologically active actions including antioxidative action and anti-aging action, and is effective in preventing or improving in vivo oxidation prevention and skin aging, for example. Therefore, it can be suitably used as an antioxidant, an anti-aging agent and the like. Furthermore, the fermented licorice extract of the present invention has various excellent physiological activities including antioxidant and anti-aging effects, and is effective in preventing and improving skin wrinkles and skin elasticity reduction. For example, it is widely used in ointments, creams, emulsions, lotions, packs, jellies, lip balms, lipsticks, bath preparations, astringents, hair tonics, hair creams, hair liquids, shampoos, pomades, rinses and the like.
In addition, cosmetic foods and drinks to which the fermented licorice extract of the present invention is added have various excellent physiological activities including antioxidant and anti-aging effects even when taken orally, and are also excellent in safety. For example, it is widely used for health foods, dietary supplements and the like.

Claims (9)

  A fermented licorice extract obtained by fermenting at least one of licorice powder and licorice extract using Aspergillus spp.   Aspergillus filamentous fungi are oryzae, A.M. Kawachi, A.K. awamori, A.M. sojae, A.M. saitoi and A.I. The fermented licorice extract according to claim 1, wherein the extract is one or more selected from niger.   The fermented licorice extract according to any one of claims 1 to 2, containing at least one of 8-hydroxyliquiritigenin and 3'-hydroxyisoquiritigenin.   An external skin product comprising the fermented licorice extract according to any one of claims 1 to 3.   A cosmetic food or drink comprising the fermented licorice extract according to any one of claims 1 to 3.   A method for producing a fermented licorice extract, comprising a step of fermenting at least one of licorice powder and licorice extract using an Aspergillus genus fungus.   Aspergillus filamentous fungi are oryzae, A.M. Kawachi, A.K. awamori, A.M. sojae, A.M. saitoi and A.I. The method for producing a fermented licorice extract according to claim 6, wherein at least one filamentous fungus selected from niger is used.   The fermented licorice extract according to any one of claims 6 to 7, wherein at least one selected from ammonium sulfate, ammonium nitrate, urea, amino acid, yeast extract, peptone, protein and meat extract is used as a medium used for fermentation. Production method.   In the step of fermentation treatment, liquiritin contained in at least one of licorice powder and licorice extract is microbially converted to 8-hydroxyliquiritigenin via microbial conversion to liquiritigenin, and licorice At least one of the fact that isolicritin contained in at least one of the powder and licorice extract is microbially converted to 3'-hydroxyisoquiritigenin via microbial conversion to isoliqueritigenin The method for producing a fermented licorice extract according to any one of claims 6 to 8, wherein one is performed.