KR100637358B1 - Composition comprising the extract of Glycyrrhizae radix for preventing the toxicity of Endocrine Disruptors - Google Patents

Abstract

The present invention relates to a composition for defense of toxicity by environmental hormone containing licorice extract as an active ingredient, licorice extract of the present invention is a cell abnormality caused by the toxicity of bisphenol A in MCF-7 cells, breast cancer cell line Inhibition of proliferation and inhibition of abnormal growth of uterine and vaginal epithelial cells, which are toxic to bisphenol A in vivo, resulted in a decrease in BPA toxicity, so the composition containing licorice extract was toxic to bisphenol A. It can be usefully used as a composition for the treatment and prophylaxis of medicines or dietary supplements.

Licorice, Extract, Environmental Hormone, Bisphenol A, Toxicity, Cell, Proliferation, Composition, Medicine, Health Food.

Description

Composition comprising the extract of Glycyrrhizae radix for preventing the toxicity of Endocrine Disruptors}             

Figure 1a is a measure of the effect of inhibiting the BPA activity of ethanol extract at each concentration of licorice, Figure 1b is a measure of the effect of inhibiting the BPA activity of fractions for each solvent licorice,

Figure 2a to 2g relates to the results of the endocrine system disorder test of licorice 100% ethanol extract, Figure 2a is a measure of weight change during the licorice extract administration period, Figure 2b is the absolute uterine weight after licorice extract treatment Figure 2c is a measure of relative uterine weight after licorice extract treatment, Figure 2d is a measure of absolute vaginal weight after licorice extract treatment, Figure 2e is a measure of relative vaginal weight after licorice extract treatment Figure 2f is a diagram showing the histological picture of uterine epithelial cells after licorice extract treatment, Figure 2g is a diagram showing the histology of vaginal epithelial cells after licorice extract treatment.

The present invention relates to a toxic defense composition by environmental hormone containing licorice extract.

Endocrine disruptors are chemicals that enter the living organisms and disrupt or disrupt the normal functioning of the endocrine system, commonly known as Endocrine Disruptors. Environmental hormone is a substance that acts on the endocrine system of the human body and acts like a female hormone to block hormone secretion, excess and under-secretion, leading to reproductive dysfunction such as infertility, sexual dysfunction, and reduced sperm count. In addition, exposure to this can lead to weight loss, growth inhibition, hepatotoxicity, testicular cancer, breast cancer, diabetes, decreased immune function, hyperlipidemia, lower urethral fever, latent and birth defects, and wild animal genital malformation, sex ratio imbalance, and mating reduction. It is known that phenomena such as mating, same sex, decrease in the number of pups and femaleization of males occur. Environmental hormones, unlike biological hormones, are not easily degraded and remain in the environment and body for many years, and have been reported to be concentrated in fats and tissues of organisms such as the human body (Golden, RJ, et al., Crit. Rev. Toxicol). , 28 , pp 109-227, 1998; Soto, AM, et al., Environ.Health Perspect , 92 , pp167-173, 1991).

After the issue of environmental hormone was raised for the first time in the 70s, the seriousness of the environmental hormones continued to be debated until the mid-90s. Since then, research on environmental hormones has been carried out systematically. As of 1998, 69 species were classified as environmental hormones in the United States and 67 species in the EU and Japan.In Korea, 41 ingredients are included in pesticides. A total of 67 species are estimated to be environmental hormones.

Chemical substances known as environmental hormones include dioxin (TCDD) generated when burning garbage, PCB, DDT, alkylphenols as synthetic detergent raw materials, and bisphenol A as plastic raw materials. Environmental hormones include household cleaners, insecticides, herbicides, It is detected in all areas related to daily life, such as automobile fumes, disposable tableware made of polystyrene (cup noodle containers, etc.), can beer, canned beverages, even baby bottles.

Bisphenol A (hereinafter referred to as BPA), which has been a problem in large quantities in the Nakdong River in 1999, is an exogenous estrogen that binds to the estrogen receptor and causes a similar effect. That is, similar hormones are made to reduce testicles and females, as well as females to cause abnormal uterine epithelial tissue growth, and toxicities in pregnant women are passed on to the fetus. BPA is the main component of plastics, which is contained in synthetic resins and polycarbonates or dental care sealants, which are widely used in household goods such as bottle caps, beverage cans, internal coatings, food packaging materials, and food containers. have.

Gaido, KW, et al., Toxicol. Appl. Pharmacol. , 143 , pp205-212, 1997. Since it is 15,000 times lower than phosphorus 17 β-estradiol, it has been reported that the ability of BPA to exogenous estrogen is very low. However, Milligan et al. (Milligan, SR, et al., Gen. Com. Endocrinol ., 112 , 89-95, 1998) show that BPA is 10,000 times lower than estradiol when subcutaneously injected into mice undergoing ovarian surgery. In addition, BPA has a weak estrogen effect in vitro, but in vitro, it acts as an estrogen receptor antagonist or agonist. It is known to increase the weight of the prostate gland in males of procrastination.

As described above, despite the results of many biotoxicity test results on environmental hormones such as BPA, BPA has been widely used as a packaging agent for special containers such as heat-sensitive devices and pressure-sensitive papers in the dental and industry.

Therefore, it is urgent to develop toxic defense substances against environmental hormones such as BPA. In Korea, Korean ginseng saponin has been reported to have an active substance that can protect against toxicity caused by TCDD (dioxin), a powerful environmental hormone, especially reproductive toxicity, and improve the quality of sperm. Patent No. 293764 discloses a dioxin toxic antidote based on ginseng. Red ginseng is also known to reduce the toxicity of BPA by activating the metabolism of foreign substances, detoxifying BPA and then exhaling it in vitro.

However, in addition to the defensive action of ginseng, research on toxic defense substances of environmental hormones is insufficient worldwide. Therefore, if you find a breakthrough ingredient from crops grown only in Korea or find an active ingredient among Korea's own plants that are not native to foreign countries, and develop new drugs that defend the toxicity of environmental hormones, new drug candidates or materials Compared with other foreign companies, it will be able to take advantage of competitiveness. Moreover, if it is based on a Chinese medicine that has been prescribed for a long time without major side effects, it is expected that a new drug that can be recognized in the world market can be obtained at a relatively low cost and in a short time.

In light of these trends, we selected a variety of crops to protect against environmental hormone toxicity among the 132 crops based on the regional characteristics of Korea. .

One of them, Glycyrrhizae radix , is a generally safe herbal medicine frequently applied in many combination prescriptions in oriental pharmacology (Kamei, J., et al., Eur. J. Pharmacol. , 469 , pp159-163, 2003). Licorice is a perennial herb, root diameter is circumferential, main root is very long, rough and large, and the outer shell is reddish brown to dark brown. Root is sweet and is used as sweetener and herbal medicine. It is distributed in northeastern China, Siberia and Mongolia.

Glycyrrhizae radix extract is liquiritin, liquiritigenin, liquiritin apioside, isoliquiritin, isoliquiritin apioside ) And flavonoids, including aglycones (Kamei, J., et al., Eur. J. Pharmacol. , 469 , pp159-63, 2003).

Recent studies have shown that liqueurine apioxide has a pharmacological effect of inhibiting cough in a dose-dependent manner. In addition, it has been suggested that estradiol E2-related endometrial carcinogenicity was suppressed by licorice in animal models. In addition, licorice acts as a natural antibiotic, which is effective against sweat bands and acne, and has anti-inflammatory effects to calm inflammation.

In the previous study, we found that licorice extract has an estrogen receptor, which has anti-cancer activity in MCF-7 cells, a human breast cancer cell line used in endocrine disruptors. It confirmed that there was. It was also confirmed that BPA induced cell proliferation up to 1.5-2.0 fold when exposed to MCF-7 cells at a concentration of 16 ng / ml for 72 hours.

Therefore, based on these findings, the present inventors have focused on the fact that licorice has a specific growth inhibitory activity in breast cancer cells of humans sensitive to estrogen, and tested the toxic defense effect of BPA on licorice. Confirmed to complete the present invention.

An object of the present invention is to inhibit aberrant proliferation of cells due to BPA toxicity in MCF-7 cells, a breast cancer cell line, and to inhibit aberrant proliferation of uterine and vaginal epithelial cells, which are toxic by BPA in vivo. By providing a composition for toxic defense by BPA containing licorice extract exhibiting a BPA toxicity reduction effect.

In order to achieve the above object, the present invention provides a composition for the treatment and prevention of toxicity by environmental hormone containing Glycyrrhizae radix crude extract and non-polar solvent soluble extract as an active ingredient.

The environmental hormones include BPA, TCDD, PCB, DDT, Ilkyphenol, preferably BPA.

The crude licorice extract includes water and C ₁ to C ₄ lower alcohols such as methanol, ethanol, butanol and the like, and a mixture thereof, preferably an extract available in methanol and ethanol.

The ethanol may be 100%, 80%, 60%, 40%, 20% ethanol, preferably 100% ethanol by concentration.

The licorice nonpolar solvent soluble extract includes an extract soluble in a nonpolar solvent such as hexane, chloroform, methylene chloride or ethyl acetate, preferably hexane, ethyl acetate.

Hereinafter, the present invention will be described in detail.

The licorice crude extract and nonpolar solvent soluble extract of the present invention can be obtained as follows.

After the licorice of the present invention is purchased or collected, dried and pulverized with a crusher, a volume of water up to about 2 to 40 times, preferably about 15 to 25 times the weight of the licorice sample and C such as methanol, ethanol, butanol, and the like Lower alcohols of ₁ to C ₄ or mixed solvents having a mixing ratio of about 1: 0.1 to 1:10, preferably about 60 to 100 ° C., preferably about 80 to 1 hour 48 hours with methanol, preferably After extraction using hot water extraction, cold needle extraction, reflux cooling extraction or ultrasonic extraction, preferably hot water extraction method for about 3 hours, the supernatant is recovered by vacuum filtration to obtain a primary filtrate, and the residue is The extraction was carried out in the same manner as above to obtain a secondary filtrate, and then the above-described procedure of combining the primary filtrate and the secondary filtrate was repeated 1 to 5 times, preferably 1 to 3 times, and the supernatant was Collect and persimmon Concentrated, it is possible to obtain the licorice crude extract.

In addition, the licorice non-polar solvent soluble extract of the present invention, after suspending the licorice crude extract in distilled water or methanol, and about 1 to 100 times, preferably about 1 to 5 times the volume of hexane, chloroform, ethyl acetate The same nonpolar solvent can be added to obtain a non-polar solvent soluble layer 1 to 10 times, preferably 1 to 5 times, to extract and separate. It is also possible to further carry out conventional fractionation processes (Harborne JB Phytochemical methods: A guide to modern techniques of plant analysis, 3rd Ed. , Pp 6-7, 1998).

More preferably, the crude licorice extract obtained by the above process, preferably licorice methanol extract, water and organic solvents such as butanol, hexane, chloroform, ethyl acetate, etc. in order from low polarity solvent to high polarity, preferably Solvent fractions and concentrated under reduced pressure in the order of hexane, chloroform, ethyl acetate, butanol, water in order to obtain a licorice hexane, chloroform, ethyl acetate, butanol fraction.

The present invention provides a pharmaceutical composition for the treatment and prevention of toxicity by bisphenol A containing crude licorice extract and nonpolar solvent soluble extract obtained by the above-mentioned preparation as an active ingredient.

In addition, licorice has been used as a herbal medicine for a long time, the extracts of the present invention extracted therefrom also have no problems such as toxicity and side effects.

As a result of searching for the effect of inhibiting abnormal growth of cells by BPA of licorice methanol extract and each solvent fraction obtained by the above method, it was confirmed that the growth inhibition effect in all fractions, of which the growth inhibition effect of 100% ethanol extract was the most Excellence could be confirmed.

The pharmaceutical composition for treating and preventing toxicity by bisphenol A of the present invention comprises 0.1 to 90% by weight of the extract based on the total weight of the composition.

Pharmaceutical compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

The pharmaceutical dosage forms of the extracts of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

Pharmaceutical compositions comprising extracts according to the invention, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Carriers, excipients and diluents that may be included in the composition comprising the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations may include at least one excipient such as starch, calcium carbonate and sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the extracts of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The extract of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

In addition, the present invention provides a dietary supplement for the improvement and prevention of toxicity by bisphenol A comprising the crude licorice extract or a non-polar solvent soluble extract and a food acceptable additive. Examples of the food to which the extract can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

It may also be added to foods or beverages for the purpose of preventing and improving toxicity by bisphenol A. At this time, the amount of the extract in the food or beverage may be added in 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1g based on 100 ml. have.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for having the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the extract of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the extracts of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited by the following Experimental Examples.

Example 1 Preparation of Licorice Methanol Extract

Licorice used in the present invention was purchased on the Chinese medicine building materials and used to accurately discriminate. After drying, 2000 ml of 100% methanol was added to 100 g of crushed licorice, followed by hot water extraction for 3 hours while heating to 80 ° C. The filtrate was filtered through two filters to obtain a primary filtrate. The residue was added with 500 ml of 100% methanol and extracted at 80 ° C. for 3 hours to obtain Whatman No. The filtrate was filtered through two filter papers to obtain a second filtrate. The first filtrate and the second filtrate were combined and concentrated under reduced pressure using a rotary evaporator to obtain about 32 g of a licorice methanol extract.

Example 2. Preparation of Licorice Ethanol Extract

Licorice used in the present invention was purchased on a Chinese medicine building material and accurately discriminated and used. 100 ml, 80%, 60%, 40%, 20% ethanol and 0% ethanol (distilled water) were added to 100 g of the ground licorice, which was pulverized, and extracted for 3 hours while heating to 80 ° C. . The filtrate was filtered through two filters to obtain a primary filtrate, and the residue was further added with 500 ml of 100%, 80%, 60%, 40%, 20% ethanol and 0% ethanol (distilled water), respectively, at 80 ° C. for 3 hours. While extracting Whatman No. 2 filtered through a filter paper to obtain a secondary filtrate, and then the primary and secondary filtrates were combined and concentrated under reduced pressure using a rotary evaporator, liquor 100% ethanol extract 9-10 g, licorice 80% ethanol extract 28- 30 g, licorice 60% ethanol extract 26-28 g, 40% ethanol extract 27-29 g, 20% ethanol extract 28-30 g, 0% ethanol extract 29-31 g were obtained, respectively.

Example 3. Preparation of Licorice Hexane Soluble Fraction

After diluting by adding 100 ml of 10% methanol to about 32 g of the licorice methanol extract obtained in Example 1, adding 100 ml of hexane and mixing the mixture, the mixture was fractionated 3-4 times to obtain a water-soluble fraction and a licorice hexane-soluble fraction. The hexane soluble fraction was concentrated under reduced pressure using a rotary evaporator to obtain 0.75-0.95 g of licorice hexane soluble fraction.

Example 4 Preparation of Licorice Chloroform Soluble Fraction

100 ml of chloroform was added to the licorice water-soluble fraction obtained in Example 3, mixed, and then fractionated three to four times to obtain a water-soluble fraction and a licorice chloroform-soluble fraction. The chloroform-soluble fraction was concentrated under reduced pressure using a rotary evaporator. 0.6-0.7 g of licorice chloroform soluble fraction was obtained.

Example 5 Preparation of Licorice Ethyl Acetate Soluble Fraction

100 ml of ethyl acetate was added to the licorice water-soluble fraction obtained in Example 4, mixed, and then fractionated three to four times to obtain a water-soluble fraction and a licorice ethyl acetate-soluble fraction. The ethyl acetate-soluble fraction was subjected to a rotary evaporator. Concentration under reduced pressure afforded 0.8-0.9 g of licorice ethyl acetate soluble fraction.

Example 6 Preparation of Licorice Butanol Soluble Fraction

100 ml of butanol was added to the licorice water-soluble fraction obtained in Example 5, mixed, and then fractionated three to four times to obtain a water-soluble fraction and a licorice-butanol-soluble fraction, and the butanol-soluble fraction was concentrated under reduced pressure using a rotary evaporator. 5.0-6.0 g of licorice butanol soluble fraction was obtained.

Experimental Example 1. E-Screen Assay

E-screen assays were developed by Soto et al. (Soto, AM, et al., Environ. Health. Perspect., 103 (Suppl . 7) , pp113-122, 1995) to examine the estrogen properties of current chemicals. It is widely used as an in vitro method. The E-Screen Assay is based on two principles: That is, (1) certain molecules in the serum used for cell culture media inhibit the growth of estrogen-sensitive human cells, and (2) estrogens offset these inhibitory effects and cause cell proliferation.

MCF-7 cell line, a human breast cancer cell, reacts with intracellular estrogen receptors and causes cell proliferation when exposed to estrogen-like or estrogen-like environmental hormones. Using the E-screen assay, which is a test method developed by designing the characteristics of MCF-7 cells, that is, the binding reaction with estrogen receptor, BPA, which is an environmental hormone, and various organic solvent extracts of licorice, which are expected to be toxic defense substances, were extracted. Simultaneous exposure to MCF-7 cells was attempted.

First, phenol red-free D-media with 5% fatal bovine serum (FBS) and 3 ml / l PSN antibiotic mixture (GIBCO, USA) added. ) MCF-7 cells (ATCC, USA) were incubated in a 5% CO ₂ , 95% air (air), 100% humidity (SANYO, JAPAN) maintained at 37 ℃. Cells used for testing (5 × 10 ⁴ / ml) were plated in 6-well culture plates and attached for 24 hours before phenol red-glass D-medium. Incubated for 24 hours, after 24 hours to remove the medium (medium), the mixture of BPA and each licorice extract, that is, the licorice methanol extract of Example 1, the licorice ethanol extract of Example 2, the extract The mixture of licorice hexane soluble fraction of Example 3, licorice chloroform soluble fraction of Example 4 and licorice ethyl acetate soluble fraction of Example 5 was mixed with 5% DCC_FBS (dextran-coated charcoal-stripped FBS, Hyclone, USA) The mixture was diluted in PSN antibiotic mixture (test media) / 1 L medium and incubated at 37 ° C. for 3 days. During that time, the test medium was replaced once and the harvested cells were measured for DNA content at OD 260 nm with a spectrophotometer (BD).

As a result, BPA multiplies human breast cancer cells by about 1.5 times, whereas licorice extract, that is, licorice methanol extract of Example 1, licorice hexane soluble fraction of Example 3, licorice chloroform soluble fraction of Example 4, In the case of cells exposed simultaneously with the licorice ethyl acetate soluble fraction of Example 5, proliferation of cells by virtue of BPA toxicity in MCF-7 cells, which are breast cancer cell lines, at high and low concentrations (100 ug / ml, 10 ug / ml). In all groups except licorice chloroform soluble fraction of Example 4, the growth of cells similar to or less than that of the control group was observed when exposed to BPA at the concentration of 100 μg / ml. In addition, the licorice ethanol extract of Example 2, except for licorice 0% ethanol (DW) extract, inhibited the activity of BPA in human breast cancer cells concentration-dependently at a high concentration (100 ug / ㎖), Example 2 Licorice 100% of the ethanol extract (hereinafter referred to as G) was shown to be the most toxic protective effect (see Fig. 1a and 1b).

Experimental Example 2. Immature Uterotrophic assay

In the licorice 100% ethanol extract of Example 2 which was shown to have the strongest inhibitory activity against BPA activity in the E-screen assay, a cell proliferation assay using the MCF-7 cell line of Experimental Example 1 We tested the effects of BPA toxic defenses in vivo by conducting an endocrine disorder test (Immature Uterotrophic assay).

2-1. Laboratory animals

As experimental animals, Sprague-Dawley rats were used as 18-day-old immature rats (immature rats, purchased from Hallim experimental animals), and the diet was AIN-76A (http: //www.bio-serv.com/newcatalog/diets/saltmix.html) was administered directly.

2-2. Statistical method

One-way ANOVA was performed for statistical analysis of data on the body weight of the test animals measured during the test, and the significance was tested at the p = 0.05 level. Dunnett's t-test) was used to test the statistical significance between the control and test groups (p <0.05).

2-3. Experiment method

After 18 days of age, immature rats were treated with BPA and licorice 100% ethanol extract of Example 2 in a subcutaneous route simultaneously for 3 days, and then weighed 24 hours after the last dose. After the cervical spine At necropsy, the liquid or fat in the uterus was isolated, weighed, fixed in 10% formalin, histologic specimens prepared, and histopathologically examined. In particular, the height of the luminal epithelium was measured under a microscope (BH-8, Olympus, JAPAN), and carefully observed for vaginal epithelial cells.

The licorice 100% ethanol extract of Example 2 was subcutaneously administered to immature female rats by concentration for 3 days, and the weight gain rate was normal during the whole test period. The amount of uterine embolism in rats was increased by 155% compared to normal in BPA alone group, and increased by 85% in vagina, whereas BPA combined with 100% ethanol extract of licorice of Example 2 were 103% in uterus and vaginal Only 42% increased and a tendency to decrease in weight was observed. In addition, the relative weight of the vaginal and uterine organs showed a tendency to inhibit the BPA concentration-dependently when the 100% ethanol extract of Example 2 was administered and showed a statistically significant change. As such, licorice extract was found to reduce BPA toxicity by preventing abnormal growth of uterine and vaginal epithelial cells due to toxicity by BPA without specific toxicity in rats (see FIGS. 2A to 2E).

On the other hand, histopathological results showed that the thickness of epithelial cells was increased in the BPA-administered group compared with the untreated group when uterine epithelial and vaginal epithelial cells were observed. When licorice 100% ethanol extract was simultaneously exposed, 10 mg / It was shown that both uterine and vaginal suppression of epithelial cell proliferation by BPA at concentration and dose-dependently at ml and 50 mg / ml (see FIGS. 2F and 2G).

Experimental Example 3. Acute Toxicity Test

3-1. Oral administration

       ICR-based mice and Sprague dooli rats were divided into four groups of 10 rats each, and the licorice 100% ethanol extract of Example 2 of the present invention was orally administered at a dose of 100, 250, 500 and 1000 mg / kg, respectively. As a result of daytime toxicity, none of the four groups died and no symptoms were apparent from the control group.

3-2. Intraperitoneal administration

       The licorice 100% ethanol extract of Example 2 of the present invention was divided into 4 groups of 10 ICR mice (weight 25 ± 5 g) and Sprague dooli rats, respectively, at 25, 50, 100 and 200 mg / kg, respectively. After 24 hours of intraperitoneal administration, no toxicities were observed in all four groups and no symptoms were apparent in the control group.

       As a result, it was confirmed that the licorice extract of the present invention has little acute toxicity.

Comparative Example 1. Search for anti-proliferative properties of herbal medicines

Fractions with anti-proliferative properties were screened using MCF-7 proliferation assay among crop fractions based on 132 Chinese herbal medicines. Among them, licorice ( Glycrrhiza uralensis Fisch) was confirmed to have the most effective results in inhibiting the proliferation of MCF-7 cells and the activity of the endocrine disruptor BPA (see Table 1 below).

Kinds BPA (16 ng / ml) * Test substance (100 ug / ml) Brown rice 1.6338 barley 2.4516 corn 3.0849 buckwheat 3.1181 Black rice 3.1418 Big head 1.7283 sweet potato 1.8115 licorice 0.5441 Donkey 1.3765 Schisandra 1.1691 Cloves 0.6213 Bamboo leaf 2.5212 Ogapi 1.2335 Earth 1.2937 Safflower seed 1.1714 Unit:% (of untreated)

Hereinafter, an example of the preparation of a pharmaceutical composition including the extract of the present invention will be described, but the present invention is not intended to limit the present invention but is only specifically intended to be described.

Formulation Example 1 Preparation of Powder

Licorice Extract Powder 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2 Preparation of Tablet

Licorice Extract Powder 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation Example 3 Preparation of Capsule

Licorice Extract Powder 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

Licorice Extract Powder 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO _4, 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

Licorice Extract Powder 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method for preparing a liquid, each component is added and dissolved in purified water, lemon flavor is added to the mixture, and then the above ingredients are mixed, purified water is added, the total is adjusted to 100 ml by adding purified water, and then filled into a brown bottle. Sterilized to prepare a liquid.

Formulation Example 6 Preparation of Healthy Drink

Licorice Extract Powder 100mg

15 g of vitamin C

100 g of vitamin E (powder)

Iron lactate 19.75 g

3.5 g of zinc oxide

Nicotinamide 3.5 g

0.2 g of vitamin A

0.25 g of vitamin B ₁

0.3 g of vitamin B ₂

Water quantification

After mixing the above components in accordance with the conventional healthy beverage manufacturing method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and refrigerated Used to prepare the healthy beverage composition of the invention.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

As described above, the licorice crude extract and the non-polar solvent soluble extract of the present invention inhibit the abnormal growth of BPA toxic cells in MCF-7 cells, which are breast cancer cell lines, and are toxic by BPA in vivo. Since inhibiting aberrant proliferation of the uterine and vaginal epithelial cells exhibits a BPA toxicity reduction effect, the composition comprising licorice extract is useful as a pharmaceutical or health functional food for the treatment and prevention of toxicity by environmental hormones, preferably BPA Can be used.

Claims (9)

A pharmaceutical composition for the treatment and prevention of toxicity by bisphenol A containing crude extract of Glycyrrhizae radix as an active ingredient. The pharmaceutical composition of claim 1, wherein the crude extract is an extract available in water, C ₁ to C ₄ lower alcohols, or a mixed solvent thereof. The pharmaceutical composition of claim 2, wherein the crude extract is an extract available in methanol or ethanol. Glycyrrhizae radix A pharmaceutical composition for the treatment and prevention of toxicity by bisphenol A containing a non-polar solvent soluble extract as an active ingredient. The pharmaceutical composition according to claim 4, wherein the nonpolar solvent soluble extract is an extract soluble in hexane, chloroform or ethyl acetate. delete The pharmaceutical composition according to claim 1 or 4, comprising 0.02 to 90% by weight of the extract relative to the total weight of the composition. A dietary supplement for the improvement and prevention of toxicity by bisphenol A comprising the crude licorice extract of claim 1 or the licorice nonpolar solvent soluble extract of claim 4 and a food acceptable additive. The health functional food according to claim 8, which is a health beverage.

Images