KR100525786B1 - Phytoestrogenic composition comprising an extract of chinese licorice root or isoliquiritin - Google Patents

Abstract

The present invention relates to a phytoestrogen composition comprising Glycyrrhiza uralensis Fisch extract or isoliquiritin as an active ingredient, which exhibits estrogen activity and proliferation inhibitory activity against cancer cells, and the licorice extract of the present invention. Or isoliquiritin shows estrogen but does not show androgens, inhibits the activity of endocrine-blocking bisphenol-A and inhibits the proliferation of cancer cells including breast cancer cells. It can be used for prevention and treatment.

Description

PHYTOESTROGENIC COMPOSITION COMPRISING AN EXTRACT OF CHINESE LICORICE ROOT OR ISOLIQUIRITIN}

The present invention relates to a phytoestrogen composition comprising Glycyrrhiza uralensis Fisch extract or isoliquiritin as an active ingredient exhibiting estrogen and anticancer activity.

Phytoestrogens, phytoestrogens, have been attracting attention as a substitute for synthetic estrogens that are at risk of developing cancer or cardiovascular disease in hormone replacement therapy (HRT), which is widely used as a method for treating menopausal symptoms. . Representative types of phytoestrogens are lignans and isoflavons, which are the main sources of vegetable oils, whole grains, vegetables, legumes, and fruits, and isoflavones are mainly found in soybean and legume products. The important function of phytoestrogens is to prevent breast cancer and prostate cancer by preventing the action of enzymes that proliferate cancer cells and promote the division of normal cells. It is also known to help prevent and treat osteoporosis, cardiovascular disease and menopausal symptoms.

Licorice is a legume and perennial herb, a medicinal plant found in Siberia, Mongolia and northern China. Licorice extract is used as a rectified component such as glycyrrhizic acid, glycyrrhetic acid, liquiritin, iso-liquiritin, and especially natural antibiotics. It acts as a good for sweat bands and acne, and also has anti-inflammatory effects to calm inflammation.

Accordingly, the present inventors have completed the present invention by confirming that the extract of licorice and isoliquiritin exhibits proliferation inhibitory effect on breast cancer cells and the like while showing an estrogen activity as a result of repeated studies to find a new phytoestrogen.

It is an object of the present invention to provide a new phytoestrogen and a composition containing the same, while exhibiting estrogen activity and having a proliferation inhibitory effect on cancer cells.

In accordance with the above object, the present invention provides a phytoestrogen composition containing an organic solvent extract of Glycyrrhiza uralensis Fisch or isoliquiritin as an active ingredient.

In addition, the present invention provides a phytoestrogen composition containing isoliquiritin as an active ingredient.

The licorice extract of the present invention can be produced by extracting licorice or its dried product with an organic solvent.

Specifically, 1 to 5 L of an organic solvent is added to 100 g of licorice or its dried product, and the extraction process at 60 to 90 ° C. is repeated 1 to 3 times. After that, the obtained extract is filtered, concentrated under reduced pressure, and dried to obtain licorice extract. You can get it.

Examples of the organic solvent that may be used in the extraction process in the present invention include C ₁ -C ₄ alcohol, C ₁ -C ₄ alcohol aqueous solution, ethyl acetate (EtOAc), trichloromethyl (CHCl ₃ ), hexane and the like. Preferred lower alcohols are methanol and ethanol.

In addition, isoliquirithin can be synthesized chemically and can be extracted from licorice. Specifically, 1 to 5 L of an organic solvent was added to 100 g of licorice or its dried product, extracted at 60 to 90 ° C., and then extracted with ethyl acetate (EtOAc) and trichloromethyl (CHCl ₃ ) to obtain an extract of the ethyl acetate layer. This can be obtained by silica gel column chromatography (eluent = CHCl ₃ : MeOH = 4: 1) and then reverse phase column chromatography (eluent = MeOH: H ₂ O: = 3: 2), and the chemical structure thereof is represented by the following Chemical Formula 1 As shown in.

Licorice extract and isoliquiritin of the present invention exhibits estrogen but no androgens, inhibits the activity of bisphenol-A, an endocrine barrier, and inhibits the proliferation of cancer cells including breast cancer cells. It can be used for alternative treatment and for the prevention and treatment of breast cancer. The licorice extracts of the present invention inhibited the proliferation of MCF-7 breast cancer cell lines at a concentration of 100 μg / ml and isoliquiritin at a concentration of 100 μg / ml, in particular hexane, CHCl ₃ and EtOAc extracts of licorice The low concentration of / ml showed a proliferation inhibitory effect.

Accordingly, the present invention provides a phytoestrogen composition comprising an organic solvent extract or isoliquiritin of licorice containing isoliquiritin as an active ingredient.

Licorice extract or isoliquiritin of the present invention may be mixed with a suitable pharmaceutically acceptable carrier or excipient or diluted with a diluent according to conventional methods to prepare a pharmaceutical composition having the above functions. Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like. Pharmaceutical compositions of the invention can be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders and the like.

The pharmaceutical compositions of the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular. Typical daily dosages of licorice extracts and fractions of the present invention range from 1 to 500 mg / kg body weight, preferably from 10 to 100 mg / kg body weight, and can be administered once or in several doses, isoli Typical daily doses of quiritine range from 0.1 to 50 mg / kg body weight, preferably 1 to 10 mg / kg body weight, and may be administered once or in several doses. However, it is to be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the route of administration, the age, sex and weight of the patient, and the severity of the disease, and therefore the dosage should in any way be regarded as the present invention. It does not limit the scope of.

In addition, licorice extract or isoliquiritin of the present invention can be added to food or beverages for the purpose of health promotion through phytoestrogens activity and cancer prevention. At this time, the amount of the extract in the food or beverage, in the case of healthy foods can generally be added to 0.1 to 50% by weight, preferably 0.1 to 1% by weight of the total food weight, based on 100 m1 for healthy drinks 0.1 to 50 g, preferably 0.1 to 1 g may be added. In addition, isoliquiritin can generally be added at 0.01 to 5% by weight, preferably 0.01 to 0.1% by weight of the total food weight for healthy foods, 0.01 to 5 g based on 100 m1 for healthy drinks, Preferably it can be added in the ratio of 0.01-0.1 g.

The health beverage composition of the present invention, except for containing the licorice extract or isoliquiritin as an essential ingredient, there is no particular limitation on the liquid component 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 (e.g., rebaudioside A, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. have.

In addition to the above, the beverage 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 the like. Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. These components can be used independently or in combination.

Hereinafter, the present invention will be described in more detail with reference to the following examples. only. The following examples are only for illustrating the present invention, but the scope of the present invention is not limited thereto.

Example 1: Preparation of Licorice Extract

After addition of 2 L of 80% EtOH per 100 g of licorice, the extract was first extracted at 70 to 80 ° C. for 3 hours using a reflux condenser, followed by cooling and filtering on a filter paper (Whatman No. 1 filter). If necessary, the residue on the filter paper was subjected to second extraction under the same conditions as the first, followed by filtration. The filtrate was concentrated to remove EtOH and lyophilized.

Meanwhile, after adding chloroform (CHCl ₃ ) to the prepared extract, shaking the sample and the solvent well mixed to separate the water layer and chloroform layer, the chloroform layer was removed, and then extracted by using 70% MeOH in the water layer, Ethyl acetate (EtOAc) was poured to separate the ethyl acetate layer, and water, 20, 40, 60% EtOH aqueous solution, and 100% EtOH were extracted and lyophilized using the ethyl acetate layer.

100 mg of each lyophilized extract was dissolved in 1 ml of 80% EtOH to prepare a 100 mg / ml sample stock solution, and diluted to the desired concentration using 80% ethanol as needed.

Example 2: Isolation of Isoliquiritin from Licorice Extract

The EtOH layer extract obtained in Example 1 was subjected to silica gel column chromatography (eluent = CHCl ₃ : MeOH = 4: 1), and then reversed phase column chromatography (eluent = MeOH: H ₂ O: = 3: 2) to give a yellow powder. Isoliquiritin was obtained.

Its ¹ H-NMR and ¹³ C-NMR data is shown in Table 1 below.

Test Example 1: Yeast Assay

In order to confirm whether the licorice extract and isoliquiritin prepared in Examples 1 and 2 exhibited hormonal activity, the extract was applied to recombinant yeast expressing human estrogen or androgen receptor to investigate the hormone production ability.

Saccharomyces cerevisiae ER + LYS 8127 (Duke University Medical Center) Donated from Dr. Donald P. McDonnell, USA), yeast nitrogen substrate (without amino acid, 67 mg / ml), 1% dextrose, L-lysine (36 μg / ml) and L-histidine (24 μg / ml) was added to selective growth medium (Sigma, USA) and then grown in shake incubator (200 rpm). Proliferated yeast was diluted to the appropriate concentration in the medium, 500 μM of CuSO ₄ (Sigma, USA) was added and the appropriate amount was dispensed into 50 ml tubes. In the yeast estrogen response (YER) test group and the yeast androgen response (YAR) test group, 100% EtOH licorice extract of 100 μg / ml and 1 mg / ml concentration prepared in Example 1 was used. %, And isoliquiritin at 10 μg / ml and 100 μg / ml concentrations isolated in Example 2. The control group was treated with dimethyl sulfoxide (Sigma, USA). In addition, as a positive control in the estrogen sex estradiol, the androgen resistance test test Nord - to the 10 ^-9 M, respectively were treated with testosterone was dissolved in DMSO.

The experimental group and the control group treated with each material were incubated for 18 hours in a shake incubator (200 rpm), and then the culture solution of each tube was diluted to the same concentration of 100 µg / ml and 1 mg / ml, and the 96-well microtiter plate. 100 μl was dispensed into each well of (Nunc, Netherland). Each well contains 2 mg / ml o-nitrophenyl-β-D-galactopyranoside (Sigma, USA), 0.1% lauryl sulfate (Sigma, USA), 50 mM β-mercaptoethanol (BDH chemical LTD, England) and Z buffer containing 200 U / ml oxalyticase (Enzogenetics, USA) were dispensed in 100 μl aliquots and after 20 minutes the color development was measured at 420 nm wavelength with a microplate reader.

All data are subjected to deonnet t test (Dunnett's t -test) and then analyzed using ANOVA statistical program SAS was analyzed at the significance level of P <0.05.

The estrogen reaction (ER) and androgen reaction (AR) of licorice extract and isoliquiritin measured by the above method are shown in FIGS. 1A, 1B and 2, respectively.

As can be seen in Figure 1a, licorice extract showed the highest estrogenity at 100 ㎍ / ml, concentration-dependent estrogen at 100 ㎍ / ml and 1 mg / ml, their estrogenity positive control group It was higher than phosphorus estradiol, and as can be seen in FIG. 1B, isoliquiritin showed the highest estrogenity at 10 μg / ml and concentration-dependent estrogen at 10 μg / ml and 100 μg / ml Their estrogenity was higher than that of estradiol, a positive control.

In addition, in the androgenicity search result of FIG. 2, the licorice extract showed less androgenicity than nor-testosterone used as a positive control at a concentration of 1 mg / ml. However, it was confirmed that androgenicity did not appear at the concentration of 100 μg / ml.

Test Example 2: Analysis of inhibition of proliferation and inhibition of bisphenol-A (BPA) activity of MCF-7 cell line

5 x 10 ⁴ cells / ml of MCF-7 cells (ATCC HTB-22) were placed in a 6-well culture plate and allowed to attach for 24 hours, followed by a mixture of 5% fetal bovine serum (FBS) and 3 ml / l PSN antibiotic (Gibco, USA) added phenol-red deficient D-medium (Gibco, USA) was incubated for 24 hours in an incubator maintained at 5% CO ₂ , 95% air, 100% humidity, 37 ℃.

After 24 hours, the medium was removed and the cultured cells were removed with a phenol-red deficient D-medium containing 5% DCC-FBS (dextran-coated charcoal-stripped FBS; Hyclone, USA) and 3 ml / L PSN antibiotic mixture. Test lipodium) or the isoliquiritin prepared in Example 2 to which the licorice extract prepared in Example 1 (10 μg / ml, 10 μg / ml or 1 mg / ml concentration) was added at 1 wt% The medium was incubated at 37 ° C. for 3 days in 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml or 500 μg / ml respectively. During this period, each test medium was changed once, and after 72 hours, cells were harvested by treatment with 1 ml of 0.1 N NaOH (Sigma Co.) and the DNA content was measured at OD 260 nm with a spectrophotometer.

On the other hand, to investigate whether licorice extract can inhibit the activity of cancer-inducing bisphenol-A (BPA) as an endocrine disruptor, in the experiments, BPA instead of licorice extract, or licorice extract and BPA It was treated at the same time. At this time, BPA was treated with 16 ㎍ / ml, the highest MCF-7 cell proliferation effect in the preliminary test, and licorice extract in the licorice extract and BPA simultaneous treatment group was used twice concentrated to the same amount as licorice alone treatment group. .

Figure 3 shows MCF-7 breast cancer cells and bisphenol A inhibitory effect of licorice extract extracted with various concentrations of EtOH, MCF cultured using the licorice extract extracted with the concentration of EtOH as an extraction solvent from 0 to 100% In -7 cells, the effect of inhibiting proliferation increased with increasing ethanol concentration. In addition, licorice EtOH 0% extract (water extract) did not show an inhibitory effect on MCF-7 breast cancer cells and bisphenol A.

Figure 4 shows the inhibitory effect of MCF-7 breast cancer cells of various concentrations of isoliquirithin, as shown here, in the isoliquiritin concentration of 100 ㎍ / ml did not show a significant proliferation of breast cancer cells, in particular Isoliquiritin at 300 ㎍ / ml concentration showed excellent breast cancer growth inhibitory effect.

5 shows the inhibitory effect of licorice extract according to the solvent MCF-7 breast cancer cells and bisphenol A. Licorice extract extracted with hexane, EtOAc, CHCl ₃ , 70% MeOH, respectively, 10 ³ times and 10 ⁴ times with 80% EtOH. Dilution resulted in 10 μg / ml or 100 μg / ml. The test compounds were diluted to show cytotoxicity tendency such as changing the shape of the cell at 100-fold and 1000-fold dilution for 10 ^four times.

As can be seen in FIG. 5, licorice extracts of 10 μg / ml to 100 μg / ml prepared using each solvent exhibited inhibitory effects on MCF-7 breast cancer cells and bisphenol A.

On the other hand, when the experiment was repeated using a licorice extract of 100 mg diluted 1 mg / ml, the concentration of the extract was high, it was impossible to measure the absorbance. Therefore, the anti-proliferative effect was measured by trypan blue staining, which counts and counts living cells by trypan blue after extract treatment. The results are shown in FIG.

6 is a result of observing the number of MCF-7 cells after 72 hours treatment of licorice extract extracted using hexane, EtOAc, CHCl ₃ , 70% MeOH as the extraction solvent at 1 mg / ml concentration, cancer cell death in all extracts The effect was observed. As can be seen from Figure 6, it can be seen that the number of cells also decreased significantly and died.

Test Example 3: Immature Uterotrophic Assay

Immature uterine proliferation assay (Kang, KS et al., Toxicology Letters , 118, 109-115, 2000) was performed using 80% EtOH extract of licorice, which had inhibitory effect in cell proliferation assay using MCF-7 cell line. .

Forty 18-day-old immature Sprague-Dawley rats were subjected to temperature 22 ± 3 ° C, relative humidity 50 ± 10%, ventilation times 10-12 times / hr, lighting time 12 hours, illuminance 150-. AIN-76A diet in a 200 lux breeding environment (0.6 g thiamine HCl, 0.6 g riboflavin 0.6 g, pyridoxine HCl 0.7 g, niacin 3.0 g, calcium pantothenate 1.6 g, folic acid 0.2 g, biotin 0.02 g, vitamin B12 (0.1%) ) 1.0 g, Vitamin A Palmitate (500,000 IU / g) 0.8 g, Vitamin D3 (400,000 IU / g) 0.25 g, Vitamin E Acetate (500 IU / g) 10.0 g, Menadione Sodium Bisulfite 0.08 g, Fine Powder Sucrose 981.15 g) was maintained while feeding. Each rat was suspended in corn oil with bisphenol A (40 mg / ml / 100 g body weight) and licorice EtOH extract (10 mg or 50 mg / ml / 100g body weight), respectively, and subcutaneously injected once daily for three days. 24 hours after the day of administration was cervical distal bone and lethal.

At necropsy, the liquid or fat in the uterus was removed, weighed, fixed in 10% formalin, and tissue samples were prepared by conventional methods and histopathological examinations were performed. The location of the uterine and vaginal incisions for histopathological examination is shown in FIG. 6. In particular, the height of the luminal epithelium of the uterine lumen was measured through a microscope, and vaginal epithelial cells were carefully observed.

One-way ANOVA was performed for statistical analysis of data on the body weight of test animals measured during the test, and the significance was tested at the p = 0.05 level. The statistical significance between and test group was tested (p <0.05).

As a result of measuring the body weight daily from the first injection to the lethality, as shown in FIG. 8, the weight gain rate was normal during the whole test period.

In addition, the absolute weight of the extracted uterus and vagina did not show an inhibitory effect as compared with the BPA-administered group, as shown in FIGS. 9 and 11. However, the relative weight obtained by dividing the absolute weight of the extracted uterus and vagina by the weight immediately before necropsy tends to have a concentration-dependent inhibitory effect on BPA on licorice extract, as shown in FIGS. 10 and 12. Seemed. If the absolute weight is only the data of the organs of each individual, the relative weight is more meaningful because it is the weight of each organ in the individual considering the weight of each individual.

As a result of histopathological examination, both the uterine epithelial cells and the vaginal epithelial cells were found to have an increased thickness of the epithelial cells in the BPA-administered group compared with the untreated control group. It was shown to inhibit epithelial cell proliferation by BPA in a concentration-dependent manner at / ml (Figs. 13 and 14).

Figure 13 is a histological picture of uterine epithelial cells, A-1 is a non-treated control group, A-2 is BPA administration group, A-3 is licorice 50 mg / ml / 100g + BPA administration group, A-4 is licorice 10 mg / ml / 100g + BPA administration group. In addition, Figure 13 is a tissue picture of vaginal epithelial cells, B-1 is a non-treated control group, B-2 is BPA administration group, B-3 is licorice 50 mg / ml / 100g + BPA administration group, B-4 is licorice 10 mg / ml / 100g + BPA administration group.

Formulation Example 1 Preparation of Pharmaceutical Formulation

Capsules were prepared by mixing the following ingredients to fill hard gelatine capsules:

                                              Volume (mg / capsule)

Active ingredients 20

(Licorice Extract or Isoliquiritin)

Dry starch 160

Magnesium Stearate 20

          Total 200 mg

Formulation Example 2: Food Containing Licorice Extract or Isoliquiritin

Foods containing licorice extract or isoliquiritin of the present invention prepared in Examples 1 and 2 were prepared as follows.

(1) Preparation of flour food

0.5% by weight licorice extract or 0.05% by weight of isoliquiritin was added to the flour, and bread, noodles, and the like were prepared using the mixture to obtain a food for health promotion.

(2) Manufacture of dairy products

To the milk was added 1% by weight licorice extract or 0.1% by weight isoliquiritin and the milk was used to prepare various dairy products such as butter and ice cream. However, licorice extract was added to coagulated milk protein in cheese production and coagulated milk protein obtained after fermentation in yogurt production.

(3) Preparation of Juice

1-5 g of licorice extract or 0.1-0.5 g of isoliquiritin was added to 1,000 ml of tomato or apple juice to obtain health promoting juice.

 Since the composition of the present invention containing licorice extract or isoliquiritin exhibits phytoestrogens activity and anticancer activity, it can be usefully used for hormone replacement therapy and prevention and treatment of cancer.

1 is an estrogen reaction (ER) of licorice extract (a) and isoliquiritin (b) measured by a yeast assay,

Figure 2 is an androgen reaction (AR) of licorice extract measured by the yeast assay,

Figure 3 shows the inhibitory effect on MCF-7 breast cancer cells and bisphenol A of licorice extract extracted with various concentrations of EtOH,

Figure 4 shows the inhibitory effect of various concentrations of isoliquitin on MCF-7 breast cancer cells,

5 shows the inhibitory effect on MCF-7 breast cancer cells and bisphenol A of licorice extract prepared by different extraction solvents,

Figure 6 shows the cytotoxicity of MCF-7 breast cancer cells of licorice extract prepared by different extraction solvents,

Figure 7 shows the location of the incision of the uterus and vagina for histopathological examination,

8 shows the weight change during the administration of licorice extract and BPA,

Figure 9 shows the absolute weight of the uterus after licorice extract treatment,

Figure 10 shows the relative weight of the uterus after licorice extract treatment,

11 shows the absolute weight of the vagina after licorice extract treatment,

12 shows the relative weight of the vagina after licorice extract treatment,

13 is a tissue picture of uterine epithelial cells,

14 is a histogram of vaginal epithelial cells.

Claims (16)

delete delete delete delete delete delete delete delete delete delete A composition for the prevention and treatment of breast cancer containing isoliquiritin as an active ingredient. delete delete delete delete delete