KR101963141B1 - Composition comprising extracts of Opuntia Ficus Indica, Momordica charantia and Passiflora incarnata for preventing or alleviating climacterium - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of menopausal symptoms, comprising an extract of Paeonia sp. The effect of the present invention on the specific activation of the estrogen receptor when mixed with the weight ratio of 4: 3: 1 was confirmed, Can be used as medicines and health functional foods useful for the prevention or treatment of diseases caused by < RTI ID = 0.0 >

Description

[TECHNICAL FIELD] The present invention relates to a composition for preventing or treating menopausal disorders, which comprises, as an active ingredient, an extract of Quercus mongolica,

The present invention relates to a pharmaceutical composition for the prevention or treatment of menopausal symptoms, comprising an extract of Paeonia sp.

With increasing interest in health and improved living standards, products and technologies related to health functional foods are diversifying and related markets are growing rapidly.

Health functional foods, including health supplements and special nutrition foods, have the characteristics of foods and medicines at the same time, but unlike medicines, they have a characteristic of being able to be taken on a daily basis with an emphasis on prevention rather than treatment of diseases.

In particular, when the ovary ages and becomes less functional as a woman becomes older, the production of ovulation and female hormones is no longer achieved, which is the result of menopause.

Usually, menopause is diagnosed when there is no menstrual period for one year. This change usually starts in the mid-40s and progresses gradually. From this time, until about one year after menopause, in which menstruation completely disappears, menopausal transition period, more commonly, It is called menopause and the average period is about 4-7 years.

On the other hand, the most common symptom in menopausal period is irregular menstruation. Symptoms of female hormone deficiency such as facial flushing and sweating appear. In severe cases, facial flushing is accompanied by fatigue, anxiety, depression, and memory impairment.

In order to control menopausal symptom, hormone therapy that replenishes female hormones deficient in menopause is being performed, but this hormone replacement therapy can increase the activity of cancer-induced gene and increase the risk of breast cancer or uterine cancer , Hypertension, or cardiovascular disease.

There are also reports that vegetal estrogen may help alleviate menopausal symptoms (Korean Patent Laid-Open Patent Application No. 2011-0017419), but the amount of food to be consumed to obtain the medicinal effect may be excessive, May cause nutritional imbalance, and there is still a problem that the stability against stimulation of the breast is not guaranteed.

The inventors of the present invention have been studying natural materials capable of improving menopausal disorders in that menopausal symptoms can be improved by activating the estrogen receptor beta (ER) Confirming the mechanism thereof, and completed the present invention.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating menopausal symptoms, which comprises an extract of Paeonia sp.

Another object of the present invention is to provide a health functional food composition for the improvement of menopausal disorders, which comprises an extract of Paeonia japonica, L. japonica extract and Clock flower extract as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to accomplish the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating menopausal symptoms, comprising an extract of Leptodendron sp.

In one embodiment of the present invention, the menopausal disorder is selected from the group consisting of osteoporosis, facial flushing, skin changes, poor circulation, arrhythmia, edema, sleeping disorder, sweating, muscle pain, urination, dysentery, urinary incontinence, vaginal dryness, Wherein the disorder is one or more symptoms selected from the group consisting of loss, concentration disorder, memory disorder, anxiety, and nervousness.

In another embodiment of the present invention, the composition is characterized by specifically activating estrogen receptor alpha or estrogen receptor beta.

In another embodiment of the present invention, the clock flower extract, Baicangbukcho extract and Yeoju extract are mixed at a weight ratio of 4: 3: 1.

In another embodiment of the present invention, the perennial herb extract is an extract of water.

In another embodiment of the present invention, the extract is a water-extracted extract.

In another embodiment of the present invention, the clock flower extract is an extract of ethanol.

In addition, the present invention provides a health functional food composition for improving menopausal symptoms, comprising an extract of Paeonia japonica, L. japonica extract, and a clock flower extract as an active ingredient.

According to the present invention, the perennial herb extract, Yeoju extract and clock flower extract can increase the transcriptional activity of the estrogenic gene or the estrogen responsive gene via the estrogen receptor alpha or beta, thereby improving the treatment, prevention and improvement effects of the menopausal period.

FIG. 1 is a graph showing the results of measurement of ERE-Luciferase activity according to an extraction method of Baekryujang extract.
FIG. 2 is a graph showing the results of HPLC analysis according to the extraction method of Baekryongkor extract.
FIG. 3 is a graph showing the results of measurement of ERE-Luciferase activity according to the extraction method of Yeast extract.
FIG. 4 is a graph showing the results of measurement of ERα and ERß-selective ERE-Luciferase activity according to the extraction method of Yeast extract.
FIG. 5 is a graph showing the results of HPLC analysis according to the extraction method of Yeast extract. FIG.
FIG. 6 is a graph showing the results of measuring the ERE-Luciferase activity according to the mixing ratio of Paeoniae japonica-Yaju-clock flower extract.
FIG. 7 is a graph showing the results of measurement of ERα and ERß-selective luciferase activity according to the mixing ratio of Paeoniae japonica-Yaju-clock flower extract.
FIG. 8 is a graph showing the results of evaluating cytotoxicity of MCF-7 cells according to the blending ratio of Paekcheoncho-Yeoju-clock flower extract.
FIG. 9 is a diagram showing an outline of the ER-ERE luciferase assay.
FIG. 10 is a graph showing the ERE-Luciferase activity of MCF7 cells in the extracts of Paekcheoncho-Yeoju-clock flower (4: 3: 1) and individual extracts (a: Paekcheoncho, b: Yeoju, c: (4: 3: 1).
FIG. 11 is a graph showing the results of evaluating the ERE-Luciferase activity of MCF7 cells in the extract of Baekjuncho-Yeoju-clock flower (4: 3: 1) and individual extracts.
FIG. 12 shows the results of confirming the ERE-luciferase activity at various concentrations of individual and perennial-Yeoju-clock flower extracts (4: 3: 1) in HEK293 cells expressing ERα protein (a: b: Yeoju, c: Clock flower, d: Paekcheoncho - Yeoju - clock flower extract (4: 3: 1)).
FIG. 13 is a graph showing the result of evaluating the enhancing effect of ERα and ERß-selective ER-ERE transcriptional activity of HEK293 cells against Paekcheoncho-Yeoju-clock flower extract (4: 3: 1) and individual extracts to be.
FIG. 14 is a graph showing the results of confirming ERE-luciferase activity at various concentrations of individual and perennial-Yeoju-clock flower extracts (4: 3: 1) in HEK293 cells expressing ERß protein specifically (a: , b: Yeoju, c: Clock flower, d: Baeknyecho-Yeoju-clock flower extract (4: 3: 1)).
15B is a MTT result of Yeoju, FIG. 15C is a MTT result of a clock flower, FIG. 15D is a result of MTT of a clock flower, (4: 3: 1), and FIG. 15E shows the result of synthesizing the above results.
16B is a result of MTT of Yeoju, FIG. 16C is a result of MTT of clock flower, FIG. 16D is a result of MTT of clock flower, FIG. (4: 3: 1) of Baeknyoncho-Yeoju-clock flower extract.
17 is a graph showing the results of confirming concentration-dependent ER-ERE transcription activity in MCF-7 cells.
18 is a graph showing the results of observing ERa-selective transcriptional activity of each extract in HEK cell line.
19 shows the results of observing ERß-selective transcriptional activity of each extract in an HEK cell line transfected with an ERß expression plasmid.

The present invention provides a pharmaceutical composition for preventing or treating menopausal symptoms comprising an extract of Paeonia japonica, L. japonica extract, and a clock flower extract as an active ingredient.

In addition, the present invention provides a health functional food composition for improving menopausal symptoms, comprising an extract of Paeonia japonica, L. japonica extract, and a clock flower extract as an active ingredient.

The composition comprises a pharmaceutical composition and a food composition.

Hereinafter, the present invention will be described in detail.

It was observed that the extract of Paeciliaceae according to the present invention, Yeoju extract and Clock flower extract, when mixed at a ratio of 4: 3: 1 best activates the estrogen receptor, thus confirming that the extract has the effect of improving the menopausal disorder. That is, the composition of the present invention can treat, prevent or ameliorate menopausal disorders by specifically activating estrogen receptor alpha and estrogen receptor beta.

Therefore, the extract of Paecilium japonica, Lycoris chejuensis and Clock Flower extract according to the present invention can be used for the prevention and treatment of osteoporosis, facial flushing, skin change, poor circulation, arrhythmia, edema, sleeping disorder, sweating, muscle pain, , Urinary frequency, urinary incontinence, vaginal dryness, dyspepsia, loss of libido, loss of motivation, concentration disorders, memory impairment, anxiety, and nervousness, and health functional foods for the improvement of menopausal disorders.

The Leucyrell extract of the present invention, Liliaceae extract and Timothy flower extract can be produced using conventional solvents known in the art, that is, under ordinary temperature and pressure conditions, using conventional solvents. Examples of the extraction solvent that can be used in the preparation of the extract of the present invention include water, C1 to C4 lower alcohols, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3- Butylene glycol, methylene chloride, and a mixed solvent thereof may be used alone or in combination. The solvent is preferably water, but is not limited thereto. The suitable amount of the extraction solvent is about 1 to 10 times of the dried weight of the flower of Baekjunju, Yeoju, and clock flower. As the extraction method, heat extraction, cold extraction, reflux cooling extraction and ultrasonic extraction may be used. It can be repeatedly used for extraction. The extracting temperature is not particularly limited as long as the extracting temperature is such a temperature as not to remove the active activity of the Baekjangju, Yaju, and clock flower components. Preferably, however, the extract is obtained by immersion at room temperature. , And the clock flower extract is preferably an ethanol extract.

The composition of the present invention may contain at least one known active ingredient having an effect of improving menopausal symptoms, together with a perennial herb extract, a lavender extract and a watch flower extract.

The composition of the present invention may further comprise at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, , And other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient according to a suitable method in the art.

The composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and the dose may be appropriately determined depending on the patient's weight, age, , Diet, administration time, method of administration, excretion rate, and severity of the disease. Preferably, the daily dose of the extract is from about 0.0001 to 500 mg / kg, preferably from about 0.001 to 300 mg / kg, and the daily dose is one to several times a day.

The composition of the present invention can be used alone or in combination with the methods of using surgery, hormone therapy, drug therapy and biological response modifiers for the improvement of menopausal disorders.

The composition of the present invention may be added to health functional foods for the purpose of improving menopausal disorders. When the perennial herb extract of the present invention is used as a food additive, it is possible to use the perennial herb extract, Yaju extract and clock flower extract as it is or to use it in combination with other food or food ingredients, Can be used. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, in the production of food or beverage, the perennial herb extract of the present invention, Yeast extract and watch flower extract are added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health functional foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, polysaccharides such as disaccharides such as maltose and sucrose, dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01-0.20 g, preferably about 0.04-0.10 g, per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the present invention provides a method for improving menopausal symptoms comprising administering to a subject an extract of Paeonia japonica, L. japonica extract, and a clock flower extract.

The subject is a mammal, including a human or non-human, and non-human mammals include, but are not limited to, mice, rats, dogs, cats, horses, cows, sheep, goats, pigs, rabbits and the like.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

[ Example ]

Example  1. Activity evaluation and composition analysis

1.1. Identification of ER / ERE-gene transcriptional activity for estrogen receptor activity

An ER-positive MCF7 cell line was used as a human breast cancer cell line (ATCC® HTB-22 ^™ ). Each cell was seeded into a 24-well plate at 5 × 10 ⁵ cells / well. At this time, charcoal-treated fetal bovine serum (FBS) was added and cultured using phenol-red-free DMEM medium (hereinafter referred to as estrogen-free medium).

Twenty-four hours after the start of the culture, plasmids (0.25 mg / well) of ERE3-Luciferase (three replicates of the ERE sequence) were transfected using Lipofectamine 2000 (Invitrogen). After culturing for 1 day, the cells were treated with estrogen-free media containing the appropriate drug concentration, and water-soluble cell extracts were extracted 24 hours later.

Luciferase activity in the extract was determined using a Luciferase assay system (Promega) reagent using a VICTOR 3 luminometer (Perkin Elmer).

Seven perennial plant extracts extracted by different extraction methods were used, and the extraction methods of the first to seventh perennial plant extracts are shown in Table 1 below.

Sample number Sample Characteristics One 1 2 Bacillus sp. Extract 2 3 100% hot water extract 60% 1 4 60% 2 hot water extract 5 70% 6 70% 7 80%

Experimental results showed that the best ERE-Luciferase activity was observed in sample No. 6 (80% hot water extraction) and the remaining six samples (except red bar) showed low ERE-Luciferase activity (Figure 1) .

1.2. Active and surface component profile analysis

100 mg / mL of Paeoniae Radix extract powder was dissolved in 70% methanol and ultrasonicated for 30 minutes and then centrifuged at 3,000 rpm for 10 minutes. The supernatant obtained by centrifugation was filtered with a 0.45 mm PTFE-syringe filter and then analyzed by Diode Array Detector-HPLC (Agilent 1260). HPLC analysis conditions are shown in Table 2 below.

Condition Contents Column Phenomenex Kinetex C-18 HPLC column
(4.6 x 150, 5 [mu] m) Column guard Phenomenex KrudKatcher Ultra HPLC in-LineFilter Column tempERαture 40 ℃ Mobile phase A Distilled water containing 0.1% formic acid Mobile phase B Acetonitrile (ACN) with 0.1% formic acid Mobile phase gradient 0 min: 20% B
20 min: 40% B
22 min: 20% B
25 min: 20% B Injection volume 20 mL Detection wavelength 360 nm Flow rate 1 mL / min

As can be seen in FIG. 2, there are large amounts of narcissin, glycoside of isorhamnetin, and four kinds of flavonol (quercitin, kaempferol, isorhamnetin) .

Example  2. Activity Evaluation and Composition Analysis of Yeast Extract

2.1. Identification of ER / ERE-gene transcriptional activity for estrogen receptor activity

Experimental methods were the same as those of Example 1, and five extracts of Y. japonica and Y. japonica extract, which were extracted by different extraction methods, were used. Table 3 shows the extracts of each of the used yeast extracts.

Sample number Sample Characteristics One Yeoju alcohol extract 1 2 Yeoju hot water extract 70% 1 3 Yeoju hot water extract 70% 2 4 Yeoju hot water extract 80% 5 Yeoju hot water extract 90% 6 Yeoju Chinese 7 Yeoju Indian

As a result, as shown in Fig. 3, ERE-Luciferase activity was remarkably observed in extracts 4 and 6 (red bars).

2.2. Evaluation of ERß-selective target gene transcription activity on estrogen receptor activity

Was dispensed an HEK293 cell line does not express the ER to ^{5 × 10 5 cells / well,} 24-well plate. At this time, charcoal-treated fetal bovine serum (FBS) was added and cultured using phenol-red-free DMEM medium (hereinafter referred to as estrogen-free medium).

The plasmids expressing ERα and ERβ (0.25 mg / well) and ERE3-Luciferase plasmid (0.25 mg / well) were co-transfected with Lipofectamine 2000 (Invitrogen) After 24 hours, water-soluble cell extracts were extracted using Passive lysis buffer (Promega).

Luciferase activity in the extract was determined using a Luciferase assay system (Promega) reagent using a VICTOR 3 luminometer (Perkin Elmer).

As a result, as shown in Fig. 4, when compared with the other six yeast samples, a high ER [alpha] and ERß-selective ERE-Luciferase activity was observed in the sixth yeast sample.

2.3. Active indicator material profile analysis

100 mg / mL of yujoo extract powder was dissolved in 70% methanol, sonicated for 30 minutes, and then centrifuged at 3,000 rpm for 10 minutes. The supernatant was filtered with 0.45 mm PTFE-syringe filter and analyzed by Diode Array Detector-HPLC (Agilent 1260). The HPLC analysis conditions were the same as in Table 2, except that the column temperature was 35 ° C.

As a result, as shown in FIG. 5, there was a significant unknown peak in the extracts of the Korean mulberry extracts 1 and 2 and the Chinese mulberry extract, and these substances were expected to be related to the estrogenic activity of the female mulberry.

Example  3. Baeknyoncho - Yeoju - Clock flower  Determine optimal blending ratio of extract

3.1. Identification of ER / ERE-gene transcriptional activity for estrogen receptor activity

The experimental method was the same as that described in Example 1. The extracts having the highest activity through ER / ERE gene transcription activity were selected in Examples 1 and 2, and the selected extracts are shown in Table 4 below.

100 years old OFI ) Yeoju (BM) Clock flower (PI) origin Domestic Domestic Italy Usage site Fruit Fruit Above ground Extraction method Hot water extraction 80% Hot water extraction 80% Alcohol extraction Appearance Brown, powder Brown, powder Brown, powder

The selected Paeoniae japonica, L. japonica and Clock flower extracts were mixed at various ratios.

Sample number OFI BM PI O: B: P  ratio μg / mL One 50 100 50 1: 2: 1 2 25 100 75 1: 4: 3 3 75 100 25 3: 4: 1 4 100 50 50 2: 1: 1 5 100 25 75 4: 1: 3 6 100 75 25 4: 3: 1 7 50 50 100 1: 1: 2 8 75 25 100 3: 1: 4 9 25 75 100 1: 3: 4

As a result, as shown in FIG. 6, the best ERE-Luciferase activity was observed at mixing ratios of 6 and 7, and it was found that the mixture weight ratio of Paeonia sp.

3.2. Evaluation of ERß-selective target gene transcription activity on estrogen receptor activity

The experimental method was the same as that described in Example 1.

As shown in FIG. 7, excellent ERα-selective luciferase activity was observed at the mixing ratio of 6-9, and excellent ERß-selective ERE-Luciferase activity was observed at the mixing ratios except for 3 and 8 times. ERα and ERß activity were influenced by the amount of fennel and flower blossom.

Selective rates of ERß / ERα above 1.5 were found in 1-3. However, these samples showed intrinsically weak ERE-Luciferase activity. Therefore, it was confirmed that the result of 6 (Baeknyoncho: Yejuju: clock flower = 4: 3: 1) is most appropriate considering the absolute ERE-Luciferase activity when selecting the mixture ratio of Paekcheoncho,

3.3. Cell-based  Toxicity assessment

MCF-7 cells were seeded on a 24-well plate at 5 × 10 ⁴ cells / well, treated with appropriate concentrations of test substance and control substance (Doxorubicin, 10 mM), cultured for 24 to 72 hours, Lt; / RTI >

100 mL of MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide; 400 mg / mL, PBS) was added and incubated in a cell incubator for about 2 hours. 100 mL of DMSO was added to the cell layer, and the mixture was stirred for about 3 minutes.

As shown in FIG. 8, when the metabolite of MTT released from DMSO was melted and measured, the extract of Paekcheon-Yeoju-clock flower showed no toxicity in MCF-7 cells as shown in FIG. 8, It was confirmed that the cell proliferation effect was not exhibited either.

Example 4: Perennial plant of the present invention, Yeoju, Clock flower blend extract (Paekcheoncho-Yeoju- clock flower Extract (4: 3: 1)) to determine the synergy effect

4.1. Test Methods

4.1.1. Test substance

The extracting method and properties of the extracts of Paeonia spp., Yeojoo, and Kwangju flowers provided by the Natural F & P Research Institute are as described in Table 4 of Example 4.1.

The stock solution of each sample was dissolved in dimethylsulfoxide (DMSO) (100 mg / ml), centrifuged at 13,000 rpm for 10 minutes, and the supernatant was filtered using a 0.45 mm syringe filter to remove the insoluble material . In cell experiments, the stock solution was diluted in the medium so that the amount of DMSO did not exceed 0.2% v / v, and the cells were treated.

17-estradiol (E2) was purchased from Sigma-Aldrich, and ICI 180,782 (ICI) was purchased from Tocris. Stock solutions of these drugs were also prepared with DMSO as the solvent.

4.1.2. Cell culture method

Human breast cancer cell MCF-7 cells, positive for estrogen receptor (ER), were cultured in RPMI 1680 medium supplemented with non-essential amino acids, antibiotics / antimycotics, sodium pyruvate and 10% fetal bovine serum (FBS) , And subculture was performed once a week on average. Human enbryonic kidney (HEK) cells, known to have little ER expression, were used to study ER-subtype-selective estrogenic signaling activity. Prior to 48-60 hours (2-3 days) of cell division, the medium was cultured in RPMI 1680 medium (hereinafter referred to as "estrogen-free medium") containing "stripped serum," which was usually treated with dextran- ). ≪ / RTI > The reason for using this medium is that phenol-red (a compound included in various kinds of media as a pH indicator) having ER agonist activity and ER (hormone) and growth factor Since the signal activity effect may interfere with the result of the test substance. Cell culture medium was purchased from Welgene (Daegu, Korea) and GBS was used for FBS and other media additives.

4.1.3. Setting the concentration of each test substance to consider the enhancement effect

The enhancing effect means that the effect exhibited by two or more components (or substances) is greater than the sum of the activities represented by the respective components. [Activity represented by 10 molecules of substance A + activity represented by 20 molecules of substance B] < [activity represented by a mixture of 10 molecules of substance A and 20 molecules of substance B].

The concentrations of the test substances were calculated as shown in Table 6 below in order to derive the single extract activity and the mixture extract activity in consideration of the mixing ratio of white: w: w = 4: 3: 1 according to the results of Example 4.1.

extract Centipede
(4 parts) Yeoju
(3 parts) Clock flower
(1 part) Baeknyoncho - Yeoju - Watch flower extract (4: 3: 1) density
(mg / ml) 50.0 37.5 12.5 100.0 250.0 187.5 62.5 500.0 500.0 375.0 125.0 1000.0 750.0 562.5 187.5 1500.0 1000.0 750.0 250.0 2000.0

4.1.4. ERE- luciferase  Transcriptional activity evaluation method ( Transactivation  assay)

A DNA plasmid (Figure 9) with an ERE (estrogen response element) sequence and a reporter gene (Luciferase) present in the promoter region of the gene whose expression is increased by 17β-estradiol (E2) After transfection into a positive breast cancer cell line (MCF-7), the cells were treated with a certain concentration of test substance (DMSO solution) and the luciferase activity was measured after 24 hours. On the other hand, in order to confirm ER-subtype selective ERE-transcription activity, a DNA plasmid capable of overexpressing each ER subtype (ie, ERα or ERβ) gene / protein in ER-negative HEK cell line was injected into the cell, We have implemented a model that can function as a subtype. FIG. 9 shows an outline of the ER-ERE luciferase assay. A plasmid having an estrogen response element, which is a DNA site in which a ligand-activated ER interacts with a promoter region of a firefly luciferase gene, is transfected into a cell, (The target gene whose ER-ERE activity is regulated by the ER-ERE activity). ER binds properly with the ligand (represented by the black dot in FIG. 9), forms a dimer, and has a suitable tertiary structure to bind to the ERE sequence. The ER-ERE binding binds to the coregulatory proteins necessary for transcription RNA polymerase is introduced to greatly increase the RNA synthesis of the target gene.

Cells were seeded (10 ⁵ cells / well) in a 24-well plate one day before transfection and ERE-Luciferase plasmid DNA (0.25 μg / well) was transfected using Lipofectamine 2000 transfection reagent (Invitrogen) . Expression plasmid DNA (0.25 μg / well) containing a gene encoding human ERα or ERβ was simultaneously transfected with Luciferase plasmid for transcriptional activity according to ER subtype expression. ER subtype selective transcriptional activity was performed in HEK293T cells.

E2 (ER agonist, 1 nM) and ICI 182,780 (ICI; Pure ER antagonist, 1 μM) were used as a positive control, and the experiment was conducted in the concentration range of 0.05 to 200 μg or more / ml of the referent. After transfection, 24 hours after transfection, medium was replaced with medium containing appropriate concentration of test substance. After incubation for 18-24 hours, Luciferase assay was performed.

Luciferase assay was performed by washing the cells twice with cold PBS, and lysate was extracted with 1 × passive lysis buffer (Promega). 50 μl of lysate was mixed with 50 μl of luciferase assay reagent. The generated luminescence intensity was measured using a SpectraMax M5 ^e (Molecular Device). The activity of the candidate substance was expressed as% value when the luciferase activity represented by E2 1 nM was taken as 100%.

4.1.5. Cytotoxicity evaluation method

MTT assay was performed to evaluate cytotoxicity. Absorbance was measured at a wavelength of 500 nm to 600 nm.

2 ml of MCF-7 cells (9 × 10 ⁴ cells / ml, 24-well plate) dissolved in estrogen-free RPIMI media were dispensed per well and cultured for 24 hours. , 10, 25, 50, 75, 100 μg / ml). DMSO was used as a vehicle control, and E2 (1 nM) and ICI (1 M) were used as a positive control.

After 24 hours, the culture medium was removed, and the cells were washed carefully with PBS to remove the cells. Then, MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide Stock solution dissolved in PBS) was diluted in the culture to a concentration of 1:10. Then, 500 μl of the diluted MTT medium solution was added to each well, followed by incubation for about 3 hours. After confirming that the MTT solution and the cells treated with the test substance met with reddish purple color, Gently so as not to fall off. DMSO (500 μl) was added to each well. The plate was gently shaken and waited for about 3 minutes. 100 μl / well of each cell extract was transferred to a 96-well plate and absorbance at 550 nm was measured (SpectraMax, Molecular Device).

4.2. result

4.2.1. Synergistic signaling activity by ER-ERE pathway versus individual extracts of Paekcheon-Yeoju- clock flower extract (4: 3: 1)

MCF7  In the cell Augmentative  Check the effect

As a result of examining ERE-Luciferase activity at various concentrations of individual and Paekcheon-Yeoju-clock flower extracts (4: 3: 1), individual extracts showed up to 22% (4: 3: 1) showed activity from 2000 μg / ml to 80% (FIG. 10d). The EC ₅₀ value was found to be 475 μg / ml when the maximum activity value of Paekcheoncho-Yeoju-clock flower extract (4: 3: 1) was 0.8 (relative to E2 1 nM).

The combined use of E2 + ICI (ER antagonist) was performed as an index showing that the activity value derived from this experimental system was mediated by ER. The activity of each extract at each concentration (Luciferase reading value) and concentration Table 7 shows the activity of the respective extracts of Hwaseong City.

Three
Total sample concentration (mg / ml) The sum of the activity at that concentration (column 1) Activity value at that concentration when treated for hundreds of days (column 2) Increment value = 2 columns - 1 column (3 columns) Simple sum ratio to enhancement value (3 columns ÷ 1 row) 100 466.49 364.37 (102.13) (0.22) 500 460.05 619.20 159.15 0.35 1000 423.32 871.13 447.81 1.06 1500 653.35 1043.81 390.46 0.60 2000 553.48 1199.42 645.94 1.17

Whether or not the treatment of the hundreds of treatments shows the enhancing effect is determined by the value of the third column (i.e., the enhancement value) in Table 3 as a positive value and the value thereof as the ratio of the last column. As shown in FIG. 11, the enhancement value was confirmed to be about 200% at the concentrations of 1000 and 2000 μg / ml. In conclusion, the extracts of Paekcheon-Yeoju-clock flower (4: 3: 1) showed clear enhancement effect on ER-ERE transcription activity in MCF7 cells.

ERα - Synergistic effect of selective ER-ERE activity

The ERE-luciferase activity at various concentrations of individual and perennial-Yeoju-clock flower extracts (4: 3: 1) was confirmed in HEK293 cells expressing ERα protein specifically, and the results are shown in FIG. As can be seen from Figs. 12a to 12c, the individual extracts exhibited up to 36% of activity (clock flower 250 μg / ml) as compared with the activity by E2, whereas, as shown in FIG. 12d, The maximum activity value of flower extract (4: 3: 1) was about 0.7 (E2 1 nM) compared to that of flower extract (4: 3: 1) , It was confirmed that the EC ₅₀ value was 125 μg / ml.

E2 + ICI (ER antagonist) was performed as an index showing that the activity value derived from this experimental system was mediated by ER. In the ERα-selective model, Paekcheoncho-Yeoju-clock flower extract (4: 3: 1) was also assayed in a manner similar to that in the MCF7 cell line. Table 8 below shows the results of the calculation of the enhancing effect of the activity value by the mixed extract of hundreds of times corresponding to the sum of the activity (luciferase reading value) and the concentration at each concentration of the individual extracts.

Three
Total sample concentration (mg / ml) The sum of the activity at that concentration (column 1) A hundred years  The activity values at that concentration (2 columns) Augmentation value  = 2 columns - 1 column (3 columns) Augmentation value  prepare Simple sum  Ratio (3 columns ÷ 1 row) 100 8282.3 5899.26 (2383.0) (0.29) 500 11035.7 10339.17 (696.5) (0.06) 1000 11760.8 11124.18 (636.6) (0.05) 1500 11941.7 14652.15 2710.4 0.23 2000 12770.3 14812.28 2041.9 0.16

As can be seen from FIG. 13, the enhancement value was found to be about 200% at the concentrations of 1500 and 2000 μg / ml during the treatment of 100 days. In conclusion, the extracts of Paekcheon-Yeoju-clock flower (4: 3: 1) exhibited the enhancing effect of ERα-selective ER-ERE transcription in high concentration treatment.

Synergistic effect of ERß-selective ER-ERE activity

The ERE-luciferase activity at various concentrations of individual and Paekcheo-Yeoju-clock flower extracts (4: 3: 1) was examined in HEK293 cells expressing the ERß protein specifically. The results are shown in FIG. As shown in FIGS. 14A to 14C, the individual extracts showed no significantly superior activity than the DMSO treatment with respect to the activity by E2. On the other hand, as shown in FIG. 14D, : 1) showed activity up to 50% at 2000 μg / ml. The EC ₅₀ value was found to be 474 μg / mL when the maximum activity value of Paekcheoncho-Yeoju-clock flower extract (4: 3: 1) was 0.5 (relative to E2 1 nM).

E2 + ICI (ER antagonist) was performed as an index showing that the activity value derived from this experimental system was mediated by ER. In the ERβ-selective model, Paekcheocho- 1) was also analyzed by a method similar to that in the MCF7 cell line. Table 9 below shows the results of calculation of the enhancing effect of the activity value by the mixed extract of hundreds of times corresponding to the sum of the activity (luciferase reading value) and the concentration at each concentration of the individual extracts. Similarly, at 2000 μg / ml concentration, the enhancement value was about 20% at 100 days treatment. In other words, it was once again confirmed that the extract of Paekcheon-Yeoju-clock flower (4: 3: 1) exhibits the enhancing effect of ERß-selective ER-ERE transcription in high concentration treatment.

Three
Total sample concentration (mg / ml) The sum of the activity at that concentration (column 1) A hundred years  The activity values at that concentration (2 columns) Augmentation value  = 2 columns - 1 column (3 columns) Augmentation value  prepare Simple sum  Ratio (3 columns ÷ 1 row) 100 203288.63 51086.12 (152202.5) (0.75) 500 223268.36 79405.76 (143862.6) (0.64) 1000 232314.13 82503.16 (149811.0) (0.64) 1500 234153.82 103264.12 (130889.7) (0.56) 2000 113728.17 135723.24 21995.1 0.19

4.2.2. A hundred years  Determination of cytotoxicity against individual extracts of the mixture

MCF7  Cytotoxicity of Extracts on Cell Lines

Cytotoxicity of the extracts on the MCF7 cell line was confirmed. As shown in FIGS. 15A to 15E, no significant toxicity was observed in all the concentration groups used in the experiment compared to the DMSO (solvent control group) treated groups.

ERß-selective HEK293  Cytotoxicity of extracts on cell lines

The results of confirming the cytotoxicity of the extract on the ERß-selective HEK293 cell line are shown in Fig. As shown in FIG. 16B, about 30% of the cytotoxicity was observed in the control group without tendency of the concentration-response relationship, suggesting the cell proliferation inhibitory activity consistently exhibited in MCF7 cells and ERα-HEK293 cells. As shown in FIGS. 16A and 16D, no treatment of cell proliferation or toxicity of 20% or more was observed in the treatment group of Paekcheon and Bacillus. However, as shown in FIG. 16C, about 30% or more of cytotoxicity was observed at low concentration in the clock flower.

4.2.3. Baeknyeoncho, gourd, clock flower extract 3 each species and baeknyeoncho-gourd-clock Flower Extract (4: 3: 1) to determine the concentration-dependent ER-ERE transcription activity of

In order to confirm the concentration-dependent ER-ERE transcriptional activity of each extract, the test substances were treated in MCF-7 and HEK (ERα and ERβ protein-specific system) cell lines. The concentration of the test substance was 10 ~ 1000 μg / mL, and the intracellular transcriptional activity was expressed as a fold value when the DMSO treatment group was set to 1.0.

MCF Confirmation of concentration-dependent ER-ERE transcriptional activity in -7 cells

The results of confirming the concentration-dependent ER-ERE transcription activity in MCF-7 cells are shown in Fig. As can be seen from FIG. 17, no transcription activity was observed in the total concentration group when Paekcheoncho and Yeoju were treated singly, and Paekcheoncho-Yeojoo-clock flower extract (4: 3: 1) (4: 3: 1) at 500 and 1000 μg / ml at the concentration of 1000 μg / ml, respectively. .

Of individual extracts ERα - selective concentration-dependent transcriptional activity

The results of observing ERa-selective transcriptional activity of each extract in HEK cell line transfected with the ERa expression plasmid are shown in FIG. As can be seen in FIG. 18, similar to the results in the MCF-7 cell line, no significant transcription activity was observed in the treatment with Paeonia japonica and Yeoju alone.

However, in the case of Baeknyoncho-Yeoju-clock flower extract (4: 3: 1), the transcription activity was 2.5 times or more at a concentration of 500 μg / ml or more and similar to the result in the MCF-7 cell line, The activity of extract (4: 3: 1) was confirmed to be superior to that of clock flower. Especially, at 1000 μg / mL, 4-fold increase in activity was observed at 4: 3: 1 of Paekchecho-Yaju-clock flower extract. Similarly, a synergistic effect was observed in the concentration of 500 μg / mL at 4: 3: 1.

ERß-selective concentration-dependent transcriptional activity of individual extracts

The results of observing the ERß-selective transcriptional activity of each extract in HEK cell line transfected with the ERß expression plasmid are shown in FIG.

As shown in FIG. 19, the clock flower exhibited a transcription activity of 1.5 times or more at a concentration of 500 μg / mL or more, and the other extract alone did not show significant activity. In the case of Yeoju, MCF-7 or ERα- But did not show activity.

In the case of Paekcheon-Yeoju-clock flower extract (4: 3: 1), the transcription activity was remarkable at 100 μg / ml or more, and no higher activity was observed at the higher concentration, and the maximum efficacy concentration 100 μg / mL. In other words, the synergistic effect of Baekjuncho-Yaju-clock flower extract (4: 3: 1) was observed at a concentration of 100 μg / ml.

4.2.4. conclusion

We have found an extract that regulates the ERα and ERβ mediated transcriptional activity in order to develop a plant extract containing a substance that regulates the molecular target of ER expression in women's menopausal symptoms as a functional food that can help menopausal women .

Each extract showed transcriptional activity in a concentration dependent manner, but no transcription activity was observed in the case of Paekcheoncho and Yeoju in the case of single treatment, and in case of clock flower, transcription activity was remarkable at 100, 500 and 1000 μg / ml. On the other hand, the transcription activity of Paekcheoncho-Yeoju-clock flower extract (4: 3: 1) was significantly higher than 100 μg / ml and both the MCF-7 and HEK cell line conditions showed the same concentration of single extract Showed superior transcriptional activity in comparison.

Therefore, it was found that the three extracts could activate ERα and ERβ-ERE transcripts at a certain level, but when mixed with the extracts, they could express excellent activity at low concentrations. To develop the mixed extract material, 3, and 1, respectively, were found to have an enhancing effect on the selective activity of ERα and ERβ. Especially at high concentration (2000 μg / ml), the enhancement effect was clearly shown in all active models.

Namely, the mixed extract of Paekcheoncho-Yeoju-clock flower extract (4: 3: 1) from the above example exhibited superior activity than that of the single extract individually, and thus the ER is targeted even at a low concentration It can be applied.

Examples of formulations for the composition of the present invention are illustrated below.

Formulation example  1: Preparation of pharmaceutical preparations

1. Manufacturing of powder

Bacillus thuringiensis extract 200mg

Lactose 100 mg

The above components were mixed and packed in airtight bags to prepare powders.

2. Preparation of tablets

Bacillus thuringiensis extract 200mg

100 mg of corn starch

Lactose 100 mg

Magnesium stearate 2 mg

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

3. Preparation of capsules

Bacillus thuringiensis extract 200mg

100 mg of corn starch

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

4. Preparation of injections

Bacillus thuringiensis extract 200mg

100 mg mannitol

Na ₂ HPO _{4 12} H ₂ O 2 mg

Sterile sterilized water for injection

Injection was prepared by mixing the above components per ampoule (2 mL) according to the usual injection preparation method.

Formulation example  2: Manufacturing of food

Foods containing the perennial herb extract, Yeoju extract and watch flower extract of the present invention were prepared as follows.

1. Preparation of cooking seasoning

Healthy cooking sauce was prepared with 20-95 wt.

2. Manufacture of tomato ketchup and sauce

0.2-1.0% by weight of Bacillus subtilis extract was added to tomato ketchup or sauce to prepare healthy tomato ketchup or sauce.

3. Manufacture of flour food

0.5 to 5.0% by weight of Bacillus thuringiensis extract was added to wheat flour, and breads, cakes, cookies, crackers and noodles were prepared by using this mixture to prepare foods for health promotion.

4. Manufacture of soups and gravies

0.1-5.0% by weight of Hwaseong City Extract was added to soup and juice to produce health promotion meat product, noodle soup and juice.

5. Manufacture of ground beef

A ground beef for health promotion was prepared by adding 10% by weight of Hwaseong city extract to ground beef.

6. Manufacture of dairy products

5-10% by weight of Bacillus thuringiensis extract was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation example  3: Manufacturing of beverages

*One. Manufacture of carbonated drinks

Add the following ingredients: 100% extract, 10-15% of sugar, 5-10% of sugar, 0.05-0.3% of citric acid, 0.005-0.02% of caramel and 0.1-1% of vitamin C, and add 75-80% of purified water to the syrup made. The syrup was sterilized at 85-98 ° C for 20-180 seconds, mixed with cooling water at a ratio of 1: 4, then 0.5-0.82% of carbon dioxide gas was injected, and a carbonated drink containing the extract of Paeonia japonica, .

2. Manufacture of health drinks

(70%, 0.12%), Vitamin C (0.02%), Calcium pantothenate (100%), Water extract, (0.02%) and licorice extract (solid content 65%, 0.01%) were uniformly blended and sterilized instantaneously and then packaged in glass bottles, plastic bottles and other plastic bottles.

3. Manufacture of vegetable juice

Healthy vegetable juice was prepared by adding 0.5g of Baekjangcho extract, Yeojoo extract and Clock flower extract to 1,000ml of tomato or carrot juice.

4. Manufacture of fruit juice

Healthy fruit juice was prepared by adding 0.1g of Baekjangcho extract, Yeoju extract and Clock flower extract to 1,000ml of apple or grape juice.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (14)

A pharmaceutical composition for preventing or treating menopausal symptoms , comprising an extract of Opuntia Ficus Indica , a extract of Momordica charantia and a flower of Passiflora incarnata ,
Wherein the clock flower extract, Baicangbukcho extract, and Yeoju extract are mixed at a weight ratio of 0.5: 4: 0.5 to 3: 1.
The method according to claim 1,
The menopausal disorder may be selected from the group consisting of osteoporosis, facial flushing, skin changes, hypotonia, arrhythmia, edema, sleeping disorder, sweating, muscle soreness, urination, dysuria, urinary frequency, urinary incontinence, vaginal dryness, dyspareunia, Anxiety, anxiety, and neuropsychiatric disorder in a mammal, including a human, a human,
The method according to claim 1,
Wherein the composition specifically activates an estrogen receptor alpha or an estrogen receptor beta. &Lt; Desc / Clms Page number 18 &gt;
The method according to claim 1,
The pharmaceutical composition for preventing or treating menopausal disorder according to claim 1, wherein the composition comprises a clock flower extract, a banyan seedlings extract, and a fennel extract at a concentration of 500 to 2000 μg / ml.
The method according to claim 1,
Wherein the perennial herb extract is an extract from water.
The method according to claim 1,
Wherein the extract is a water-soluble pharmaceutical composition for preventing or treating menopausal symptoms.
The method according to claim 1,
Wherein the clock flower extract is an extract from ethanol.
A health functional food composition for the improvement of menopausal disorders, comprising an extract of Opuntia Ficus Indica , a extract of Momordica charantia and a flower of Passiflora incarnata ,
Wherein the clock flower extract, Baicangbukcho extract, and Yeoju extract are mixed at a weight ratio of 0.5: 4: 0.5 to 3: 1.
9. The method of claim 8,
The menopausal disorder may be selected from the group consisting of osteoporosis, facial flushing, skin changes, hypotonia, arrhythmia, edema, sleeping disorder, sweating, muscle soreness, urination, dysuria, urinary frequency, urinary incontinence, vaginal dryness, dyspareunia, Anorexia nervosa, anxiety disorder, and nervous irritation, wherein the health functional food composition for improving menopausal symptoms is one or more symptoms selected from the group consisting of anxiety, anxiety, and nervousness.
9. The method of claim 8,
Wherein said composition specifically activates estrogen receptor alpha or estrogen receptor beta. &Lt; RTI ID = 0.0 &gt; 18. &lt; / RTI &gt;
9. The method of claim 8,
The composition of claim 1, wherein the composition comprises a clock flower extract, a banyan seedlings extract, and a fennel extract at a concentration of 500 to 2000 μg / ml.
9. The method of claim 8,
Wherein the perennial herb extract is an extract from water.
9. The method of claim 8,
Wherein said extract is a water-soluble extract.
9. The method of claim 8,
Wherein the clock flower extract is an extract of ethanol.

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