KR100592482B1 - Methods for increasing liquiritigenin content in a licorice and its extracts and the method for the isolation and extraction of liquiritigenin from them - Google Patents

Abstract

The present invention is an active compound, such as anti-dementia, antibacterial, antioxidant, antiangiogenic, anti-skin cancer, elimination of cadmium-induced toxicity by using microorganisms having sugar hydrolysis activity or liquor coenzyme in licorice or licorice extract (liquiritigenin) content and a method for purifying the active compound (liquirigenin) from licorice, or licorice extract treated by the method.

Accordingly, the present invention is to enhance the content of liquiritigenin (active compound, such as anti-dementia, antibacterial, antioxidant, antiangiogenic, anti-skin cancer, cadmium-induced toxicity removal from licorice or licorice extract, active from this extract) By providing a method for purifying a compound (liquirithiinin), a health functional food composition having the functions of anti-dementia, antioxidant, anti-cancer, antibacterial, detoxification, pharmaceutical composition for treatment and prevention and anti-aging, sun protection, etc. Provides the effect of providing a functional cosmetics having the function of.

Licorice, Fermentation, Microorganism, Liquirigenin, Extraction, Purification

Description

 Method for increasing liquiritigenin content in a licorice and its extracts and the method for the isolation and extraction of liquiritigenin from them}

1 is a method and extract for enhancing the content of liquiritigenin, an active compound such as anti-dementia, antibacterial, antioxidant, antiangiogenic, anti-skin cancer, cadmium-induced toxicity removal from licorice or licorice extract according to the present invention Flow chart showing method for purifying active compound.

2 is a view showing the chemical structural formula of liquiritigenin (liquiritigenin) according to the present invention.

Figure 3 is a view showing the HPLC chromatogram comparing the change in the content of the liquid liquerigenin in the water extract after treatment with licorice or licorice extract according to the present invention microbial derived enzyme solution.

Figure 4 is a view showing the HPLC chromatogram comparing the change in the liquid content of liqueurinine in ethanol extract after treatment with the microorganism coenzyme solution from licorice or licorice extract according to the present invention.

Figure 5 is a view showing the result of measuring the content of the liquid liquerigenin produced after the enzyme treatment according to the present invention over time.

Figure 6 is a view showing the hydrogen nuclear magnetic resonance spectrum of the liquiditinein according to the present invention.

7 is a view showing the carbon nuclear magnetic resonance spectrum of the liquiditinein according to the present invention.

The present invention enhances the content of the extract of liquiritigenin, an active compound such as anti-dementia, antibacterial, antioxidant, antiangiogenic, anti-skin cancer, cadmium-induced toxicity removal from licorice or licorice extract, Liqueurinine).

More specifically, Glycyrrhiza uralensis belonging to the Rosacea legume and its related plant, Siberian licorice (G. globra var. Glandifera), Espanola licorice (G. globra) or the like, or their extracts, organic solvents Anti-dementia by treatment with Aspergillus kawachii, microorganisms having similar activity, or sugar hydrolase including glucosidase produced by the extract from traditional fermented foods, Increasing the content of liquiritigenin, which has antimicrobial, antioxidant, antiangiogenic, anti-skin cancer, and cadmium-induced toxicity, and extracting hot water, ethanol, soluble tea, silica gel And it relates to a method to separate and purify the active compound by Sephadex chromatography.

In general, licorice (Glycyrrhiza uralensis) is a perennial herbaceous perennial herbaceous reddish-brown, descending deep into the ground, with stalks growing straight up to 1-1.5m. Leaves are alternate, egg-shaped, pointed. Leaves are 2-5cm long, 1-3cm wide, without teeth. Flowers are similar to beans and bloom in male color in summer.

Liquiritigenin, which is known to be contained in small amounts in licorice, has been reported in the literature to exhibit various physiological activities such as anti-dementia, antibacterial, antioxidant, antiangiogenic, anti-skin cancer, elimination of cadmium-induced toxicity. have.

However, although the existing inventions related to the liquiriginine of licorice have international patents such as Japan and China, the method disclosed in the above patent is simply by using various solvents from licorice or other plants or through ion exchange resins. It is a method of purification, and it is known that there is no patent related to liquiriginine of licorice in Korea.

Accordingly, the present invention is intended to fundamentally solve the conventional problems as described above, and is an active compound, such as anti-dementia, antibacterial, antioxidant, antiangiogenic, anti-skin cancer, removal of cadmium-induced toxicity from licorice or licorice extract. By providing a method for enhancing the content of extract of liquiritigenin and purifying the active compound (liquirigenin) from the extract, the health functional food composition and treatment having the functions of anti-dementia, antioxidant, anticancer, antibacterial, detoxification, etc. And to provide a pharmaceutical composition for the prevention and functional cosmetics having the function of anti-aging, sun protection, and the like.

Hereinafter, an embodiment of a method of enhancing liquiritigenin content from licorice or licorice extract will be described with reference to the accompanying drawings.

First, the characteristics of the microorganism used in the present invention and the crude enzyme solution obtained therefrom are as follows. The microorganism used in the present invention was isolated from doenjang and is a fungus named aspergillus kawachii. The fungus was cultured in a medium mixed with bran and glucose solution and used for 5 to 15 days at 30 ° C, or 1 to 1000 mmol (preferably 100 mmol buffer) and sodium phosphate buffer (pH 7.0). 1 to 100 ml (preferably 30 ml) is added, and left to stand at 4 ° C. for 1 to 12 hours (preferably 5 hours), then filtered with gauze and the solution is 1 to 1 at 1000 to 20,000 rpm (preferably 12,000 rpm). Centrifuge for 60 minutes (preferably 15 minutes) and then use the obtained supernatant as the enzyme solution. The enzyme solution contains various glycolytic enzymes, and among them, β-glucosidase activity has the main characteristics.

First, about 1 kg of fine licorice is refluxed with water at 100 ° C. for microbial treatment of licorice or licorice extract and the extraction and purification of the resulting liquiriginine, followed by filtration and freeze drying of the filtrate.

Subsequently, 200 g of the lyophilisate was treated with a crude enzyme solution (26 g as a protein) obtained from Aspergyllus kawachii and left standing at 37 ° C. for 12 hours.

Then, about 500 ml of ethanol is added to the culture, and the ethanol soluble component and the insoluble component are divided, and the ethanol soluble component is concentrated under reduced pressure to obtain 15 g of the extract.

Subsequently, the extract was dispersed in 300 ml of distilled water, and then extracted three times with the same amount of dichloromethane. The combined portions soluble in dichloromethane were concentrated to obtain 200 mg.

In addition, silica gel column chromatography (Merck, Art No. 7734, column size: 1.5 × 30 cm, eluent: dichloromethane-methanol = 100-1 with respect to the dichloromethane soluble fraction given by 1-1 to give a slope Elute) to obtain 7 fractions.

Subsequently, the seventh of the seven fractions contained a large amount of liquirithiinin, and 71.2 mg of pure liquiritigenin was obtained by recrystallization in a mixed solvent of dichloromethane and methanol.

Herein, the liquirithiinin with an increased content from the licorice or licorice extract includes excipients, carriers, diluents, etc., which are acceptable as pharmaceutical, cosmetic, or food compositions, and thus, are well known using readily available ingredients. It can be formulated according to the method of.

In one example, the active ingredient may be mixed with the carrier, diluted with the carrier, or contained within the carrier in the form of a capsule, sachet, paper or other container. Here, when the carrier serves as a diluent, it may be a solid, semisolid, or liquid substance which acts as a vehicle, excipient or medium for the active ingredient. Thus, the preparation may be in the form of tablets, pills, dispersants, granules, elixirs, suspensions, emulsifiers, solutions, syrups, aerosols, soft and hard gelatin capsules, sterile injectable solutions, sterile packaged powders and ointments.

Examples of carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, Cellulose, polyvinylpyrrolidone, cellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil.

In addition, the licorithigenin extract of the licorice or licorice extract may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the components.

Accordingly, the liquiriginine extract of the licorice or licorice extract of the present invention can be formulated using known techniques in the art to rapidly, continuously or delayed release the active ingredient after administration to a patient, The cosmetic, cosmetic or food composition may be administered orally or parenterally.

For example, daily dosages of active compounds typically range from about 0.1 to 20 mg per kg of body weight and range from 100 mg to 10 g as water or ethanol extract when used as a functional food.

On the other hand, the active compound purification method in the extract enhanced the content of the liquirithigenin extract of licorice,

Microorganisms having a sugar hydrolytic activity or licorice treated with the microbial enzyme solution were extracted with hot water or ethanol, licorice was extracted with hot water or ethanol and concentrated to extract microorganisms having a sugar hydrolytic activity against the extract, or microbial enzyme liquid Processing to obtain a crude extract; Using the crude extract obtained by the step as it is or dispersing it in ethanol and dividing it into an ethanol soluble portion and an insoluble portion; Concentrating the ethanol soluble part separated by the above step, dispersing in water, partitioning extraction with dichloromethane, chloroform, ethyl acetate, and concentrating to obtain an organic solvent soluble fraction; Concentrating the organic solvent soluble fraction separated by the above step to purify the liqueuritinin having anti-dementia, antioxidant, anti-cancer, antibacterial, detoxification activity using silica gel, reversed phase silica gel, high-speed liquid, Sephadex chromatography It will proceed to step.

As a compound having anti-dementia, antioxidant, anti-cancer, antibacterial, and detoxifying activity purified by the above step, licorice or licoriginine with high content in licorice extract,

The literature reported that antidementia (Pan, Xuan; Kong, Ling-Dong; Zhang, Yong; Cheng, Christopher HK; Tan, Ren-Xiang.In vitro inhibition of rat monoamine oxidase by liquiritigenin and isoliquiritigenin isolated from Sinofranchetia chinensis. Acta Pharmacologica Sinica (2000), 21 (10), 949-953 and Hatano, Tsutomu; Fukuda, Toshiyuki; Liu, Yanze; Noro, Tadataka; Okuda, Takuo.Penolic constituents of licorice.IV.Correlation of phenolic constituents and licorice specimens from various sources, and inhibitory effects of licorice extracts on xanthine oxidase and monoamine oxidase.Yakugaku Zasshi (1991), 111 (6), 311-21), antibacterial (Hwang, Jae Ock; Ahn, Duk Kyun; Woo, Eun-Rhan ; Kim, Hyoung Ja; Seo, Seon Hee; Park, Hokoon.Studies on the antibacterial constituents of Baenongtang.Natural Product Sciences (1998), 4 (3), 130-135), antioxidant (Zou, Kun; Zhao, Yuying; Fu, Naiwu; Qiao, Liang; Zhang, Ruyi.Antioxidant constituents from Glycyrrhiza inflata Bat.root.Journal of Chinese P harmaceutical Sciences (1996), 5 (4), 182-185 and Zou, Kun; Zhang, Ru-Yi. Constituents from Glycyrrhiza inflata and antioxidant activities of phenols from the roots of Glycyrrhiza. Journal of Chinese Pharmaceutical Sciences (1994), 3 (1), 90), antiangiogenesis (Kobayashi, Shinjiro; Miyamoto, Takayuki; Kimura, Ikuko; Kimura, Masayasu.Inhibitory effect of isoliquiritin, a compound in licorice root, on angiogenesis in vivo and tube formation in vitro.Biological & Pharmaceutical Bulletin (1995), 18 (10), 1382-6), anti-skin cancer (Konoshima, Takao; Takasaki, Midori; Kozuka, Mutsuo; Inada, Akira; Nakanishi, Tsutomu; Tokuda , Harukuni; Matsumoto, Takeshi.Study on inhibitors of skin tumor promotion (V) .Inhibitory effects of flavonoids of Epstein-Barr virus activation.II.Soyakugaku Zasshi (1989), 43 (2), 135-41), cadmium-induced toxicity Kim, Sang Chan; Byun, Sung Hui; Yang, Chae Ha; Kim, Chul Young; Kim, Jin Woong; Kim, Sang Geon.Cytoprotective effects of Glycyrrhizae radix extract and its active component liquiritigenin against cadmium-induced toxicity (effects on bad translocation and cytochrome c-mediated PARP cleavage) .Toxicology (2004), 197 (3), 239-251), etc. It is known to exhibit various physiological activities.

Hereinafter, licorice, or liquiriginin, the content of which is increased in licorice extract, will be described with reference to FIGS. 1 and 2.

The active compound of the present invention is obtained by separating and purifying from a microorganism having a sugar hydrolysis activity or a licorice treated with the microbial enzyme solution, or a microorganism having a sugar hydrolysis activity to a licorice extract, or a mixture of the microorganism coenzyme solution. Treatment of microorganisms or enzyme solutions and extraction, separation and partial purification of liquerigenin are carried out as follows, for example.

As shown in Figures 1 and 2, the licorice treated with a microorganism having a hydrolytic activity or its microbial enzyme solution is extracted with hot water or ethanol, or licorice is extracted with hot water or ethanol and concentrated to extract sugar It is a step of obtaining a crude extract by treating a microorganism having hydrolytic activity, or a microbial enzyme solution thereof.

Here, the microorganisms used in the present invention were selected from 122 kinds of microorganisms isolated from doenjang by the present inventors, and the selection criteria were the highest in β-glucosidase activity. The microorganisms selected were identified as Aspergillus kawachii. The microorganism was inoculated in a sterilized medium by kneading 10 g of wheat bran and 10 ml of 1% glucose solution into a 100 ml Erlenmeyer flask, and autoclaving at 121 ° C. for 15 minutes. Cultivation was carried out for 25 days at 100 ° C, 100 rpm shaking, which was the most active condition for enzyme production, and the enzyme solution was placed in 30 ml of 100 mmol sodium phosphate buffer solution (pH 7.0) for 5 hours at 4 ° C and filtered with gauze. The supernatant was centrifuged at 12,000 rpm for 15 minutes.

At this time, the microorganism may be treated directly with licorice or licorice extract, or the microbial enzyme solution obtained by the above method may be treated with licorice or licorice extract. First, licorice is collected to remove foreign matter, and then finely chopped or crushed, and then hot water or ethanol (preferably 95% ethanol) for 2 to 24 hours, preferably 5 hours at room temperature to 100 ° C, preferably 100 ° C. After extraction, the extract is dried by lyophilization, distillation, distillation under reduced pressure, or spray drying.

For the dried product thus obtained, for example, the water extract of lyophilized licorice was dispersed in water at a concentration of 100 mg / ml, and then 1-100 ml of enzyme solution obtained from the microbial culture obtained by the above-mentioned method (preferably 1 mg of licorice extract). 1-10 mL of sugar coenzyme solution (about 100-100 μg as protein)] and the reaction solution at 20-50 ° C. (preferably 37 ° C.) for 10 minutes-36 hours (preferably 1 hour-12 hours). ) Incubate.

On the other hand, as a control, a treatment with an enzyme solution heated and inactivated and a licorice extract without any treatment are used. By this method, when the microbial enzyme solution is not treated as shown in FIGS. 3 and 4, or when the inactivated microbial enzyme solution is treated, a very small liquirithigenin peak can be observed by the treatment. have. As shown in FIG. 5 by the microbial enzyme solution treatment, the production of liquirithiinin increased with time, and the maximum content was reached in about 60 minutes of reaction.

Thereafter, 2-10 times (preferably 5 times) of ethanol is added to the reaction solution treated with the microorganism or microbial enzyme solution to separate the newly generated peaks, and the ethanol is divided into soluble and insoluble portions. Then, the ethanol soluble portion is collected, and after distillation, distillation under reduced pressure, or drying by spray drying, 3 times the amount of water is added to the dried product and dispersed therein, and an appropriate amount of dichloromethane, chloroform, ethyl acetate, ether, ethanol (preferably Partitioned dichloromethane).

At this time, the extract is distilled, distilled under reduced pressure, or dried by a spray dryer, and then separated by silica gel, reverse phase silica gel, Sephadex, preferably column chromatography using silica gel, etc. Obtained fractions. Pure liquiditinine can be obtained by recrystallization of this fraction from methanol, ethanol, water, dichloromethane, benzene, ethyl acetate, or a mixed solvent thereof.

As described above, the microorganisms having a sugar hydrolytic activity, or the compound of which licorice or licorice extract is greatly increased by the microbial enzyme solution treatment, are the result of the nuclear magnetic resonance spectrum shown in FIG. 5 and FIG. This compound was identified as liquirithigenin because it exactly coincided with the properties indicated.

As such, when the above-described method is used, about 0.5 g of liquirithiinine per kg of licorice extract treated with the microorganism having the maximum hydrolytic activity or its microbial enzyme solution can be obtained.

Liquirithiinin of the present invention has the spectroscopic properties as described above, and has a biological activity such as anti-dementia, antioxidant, anti-cancer, antibacterial, detoxification, etc., not only licorice but also by treating microorganisms of similar plants or microbial enzyme solution. In addition, since the same result can be obtained using Aspergyllus kawachii and microorganisms having similar functions in addition to the enzyme solution derived from this microorganism or the enzyme solution used in the present invention, the method of increasing the content of the compound of the present invention is particularly Licorice and Aspergillus kawachii or its enzyme solution is not limited only.

It is apparent to those skilled in the art that the present invention is not limited to the described embodiments, and that various modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, such modifications or variations will have to belong to the claims of the present invention.

As described above, the present invention enhances the content of liquiritigenin extract, which is an active compound such as anti-dementia, antibacterial, antioxidant, antiangiogenic, anti-skin cancer, and cadmium-induced toxicity removal from licorice or licorice extract. By providing a method for purifying the active compound (liquirithiinin) from the extract, a health functional food composition having the functions of anti-dementia, antioxidant, anti-cancer, antibacterial, detoxification, pharmaceutical compositions for treatment and prevention and anti-aging, It provides the effect of providing a functional cosmetics having a function such as UV protection.

Claims (4)

Finely slicing licorice and filtering the mixture by refluxing with water at 100 ° C. and then freezing and drying the filtrate; Treating the lyophilized dried product by the above step with a microorganism having a sugar hydrolytic activity, or a microbial enzyme solution thereof, and then leaving the sample to stand at 20 to 50 ° C. for 10 minutes to 36 hours; Adding 2 to 10 times the amount of ethanol to the culture cultured by the above step and dividing the ethanol soluble part and the insoluble part into ethanol soluble part to obtain an extract under reduced pressure; Dispersing the extract obtained by the above step in distilled water, extracting three times with dichloromethane, and concentrating the soluble fraction; Silica gel column chromatography on the dichloromethane soluble fraction concentrated by the above step to obtain seven fractions; Recrystallizing the seventh fraction fractionated by the above step in a mixed solvent of dichloromethane and methanol to obtain a pure liquirithigenin; a method of enhancing the content of the liquiritigenin extract of licorice . The method of claim 1, Liquritigenin (liquritigenin) content of the licorice, or licorice extract is enhanced by including a carrier, excipients, diluents, etc. characterized in that the production of anti-dementia, anti-cancer, anti-aging, antibacterial, antidote How to enhance the liquirithigenin extract content of licorice. Microorganisms having a sugar hydrolytic activity or licorice treated with the microbial enzyme solution were extracted with hot water or ethanol, licorice was extracted with hot water or ethanol and concentrated to extract microorganisms having a sugar hydrolytic activity against the extract, or microbial enzyme liquid Processing to obtain a crude extract; Using the crude extract obtained by the step as it is or dispersing it in ethanol and dividing it into an ethanol soluble portion and an insoluble portion; Concentrating the ethanol soluble part separated by the above step, dispersing in water, partitioning extraction with dichloromethane, chloroform, ethyl acetate, and concentrating to obtain an organic solvent soluble fraction; Concentrating the organic solvent soluble fraction separated by the above step to purify the liqueuritinin having anti-dementia, antioxidant, anti-cancer, antibacterial, detoxification activity using silica gel, reversed phase silica gel, high-speed liquid, Sephadex chromatography Purification method of the active compound in the liquirithynin extract of licorice, characterized in that proceeded to. The method of claim 3, wherein The liquiditinine is purified from the extract of liquiritinin from licorice using methanol, hexane, ethyl acetate, acetic acid, formic acid, chloroform, benzene, or various chromatographies using a mixed solvent thereof and a carrier such as silica gel and Sephadex. Purification method of the active compound in the liquirithigenin extract of licorice.

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