KR102573629B1 - Composition for alleviate women's menopausal syndrome comprising Spartina alterniflora extracts as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for alleviating female menopausal syndrome containing an extract or a fraction thereof as an active ingredient.

Description

Composition for alleviating women's menopausal syndrome comprising Spartina alterniflora extracts as an active ingredient}

The present invention relates to a composition for alleviating female menopausal syndrome containing an extract or a fraction thereof as an active ingredient.

BACKGROUND OF THE INVENTION Due to modern industrialization, the economic level is improved and medical science is developed, so human lifespan is extended and the elderly population is increasing. Therefore, studies are being conducted to improve the health and quality of life of the elderly population.

In particular, women suffer from physical and mental difficulties due to menopausal symptoms that begin gradually after the age of 40. Menopause, experienced by about 20% of all women, is one of the aging phenomena and begins gradually from the age of 40. As the average lifespan is extended, there is a problem that accounts for more than 1/3 of a woman's life.

Female menopause is a kind of endocrine syndrome, and refers to a transitional period in which physiological and sexual functions are reduced or lost due to a decrease in female hormones, that is, estrogen, caused by overall and gradual aging of ovarian function. Estrogen exhibits essential regulatory effects such as catabolic and active actions in the uterus, vagina, skeletal muscle, and cardiovascular system of women (Couse and Korach, 1999; Korach et al, 1995). It is known to act on bone structure and cholesterol control, and is also applied to the evaluation of ovarian function.

Symptoms of menopause include symptoms due to vascular changes such as hot flashes, tachycardia, sweating or headaches, symptoms due to musculoskeletal changes such as muscle pain, arthralgia and back pain, symptoms due to urogenital changes such as frequent urination or incontinence, and atrophy of the uterus and vagina. , symptoms caused by changes in the brain nervous system such as memory loss, depression, loss of concentration and dizziness, general symptoms such as vision loss and changes in skin and hair are known. It is noteworthy that these important changes affecting women's life and health occur in 80% of women before and after menopause and progress over a long period of time, increasing the risk of developing diseases that are fatal to women's health, such as osteoporosis and cardiovascular disease. Yes (Turner et al, 1994; Versi et al, 2001)

Therefore, in order to improve the physical and mental health and quality of life of middle-aged women, the development of a treatment capable of improving menopausal symptoms is required. Examples of treatments that can improve menopausal symptoms include hormone replacement therapy or drug therapy using drugs such as non-steroidal agents. However, it is known that most of the drugs such as non-steroidal preparations have side effects such as headache and weight gain. In addition, in the case of replacement therapy using hormones such as estrogen, it is known that the risk of rejection and uterine bleeding, stroke, heart attack, breast cancer and uterine cancer may increase due to artificial hormone administration into the body (Swaran L, et al, Obstetrics & Gynecology, 91, 678-684, 1998)

Therefore, there is a demand for discovery of candidate substances similar to female hormones but having a low risk of side effects due to administration of female hormones.

On the other hand, Spartina alterniflora is a crested plant biotaxonomically. Cordgrass is used to make string. There are currently 17 known species in the genus Divine Grass, native to the Atlantic coast of North America from Newfoundland to Florida and along the Gulf of Mexico coast. Some species are native to Central and South America, Europe, and North Africa.

It has long, strong underground stems and many fine roots, forming a complex root system. It is erect, and the leaves are thick and flat. It is shaped like a rather wide long blade of 30 to 50 cm, with a sharp tip. The leaf width is 0.6 ~ 1.5 cm, and the height grows up to 0.5 ~ 3 m. They clump tightly together.

It forms a colony in the upper inlets of salt marshes or tidal flats in estuaries. It is well known as a primary invader to new territories, and expands radially once an individual has established itself. When they grow up, they merge with other groups around them, and at this time, the size and shape of the group changes according to changes in topography, such as the shape of the tidal channels. In addition, it is a perennial grass that turns from yellow-green to brown in autumn (originally it was raised to prevent coastal erosion), breeds by threatening natural halophytes, such as Chimyeoncho namunjae and Chaehong namul.

It is necessary to find a way to use these mud straps.

Domestic Patent Publication No. 2019-0092831 (2019.08.08)

Therefore, the inventors of the present invention completed the present invention by confirming that the extract or a fraction thereof has an effect of mitigating female menopausal syndrome as a result of research efforts using A.

Accordingly, an object of the present invention is to provide a composition for alleviating female menopausal syndrome containing an extract or a fraction thereof as an active ingredient.

In order to achieve the above object, the present invention, in one aspect, provides a composition for alleviating female menopausal syndrome, comprising an extract or a fraction thereof as an active ingredient.

The composition of the present invention contains, as an active ingredient, Spartina alterniflora extract or a fraction thereof.

In the present invention, the above composition may be applied to extraction by drying and crushing the above-ground and/or below-ground parts of the grass.

In the present invention, the term "above ground part" refers to the upper part of the surface of the ground around the surface of the plant. That is, it refers to the upper part of the surface of the earth excluding the root, which includes leaves, stems, stem barks, branches, branch barks, flowers, or both, except for the roots.

In the present invention, the term "subterranean part" refers to the part below the surface of the ground centering on the surface of the plant. That is, it refers to the root.

As used herein, the term "extract" refers to an extract obtained by extraction treatment, a diluent or concentrate of the extract, a dried product obtained by drying the extract, a crude or purified product of the extract, or a mixture thereof, and the extract itself and the extract. It includes extracts of all formulations that can be formed using The extract of the present invention may be extracted from natural, hybrid or mutant plants of each corresponding plant, and may also be extracted from plant tissue culture.

In the extraction of the present invention, the extraction method is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include solvent extraction, hot water extraction, ultrasonic extraction, filtration, reflux extraction, and the like, which may be performed alone or in combination of two or more methods.

In the present invention, drying of the extract may be performed by a known method within the range of not destroying useful components from the collected plant, and may be performed, for example, by natural drying in the shade. In addition, crushing is sufficient if the useful components of the plant can be sufficiently extracted in the subsequent extraction process, and it can be powdered. The drying and crushing processes may be performed in reverse order or repeatedly, if necessary.

In the present invention, the type of extraction solvent used for extracting chinensis is not particularly limited, and any solvent known in the art may be used. In the present invention, the extract may be obtained by extraction with water, C1 to C4 lower alcohol, or a mixed solvent thereof. In addition, non-limiting examples of the extraction solvent include water; C1 to C4 lower alcohols such as methanol, ethanol, propyl alcohol and butyl alcohol; polyhydric alcohols such as glycerin, butylene glycol, and propylene glycol; and hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; Or a mixture thereof may be used, and specifically, water, lower alcohol, 1,3-butylene glycol, and ethyl acetate may be used alone or in combination of two or more.

In the present invention, more specifically, ethanol, ethyl acetate, water, dichloromethane, butanol, hexane, and the like can be used as a solvent for extracting chinensis. A solvent extract can be prepared by extracting the grass grass using the solvent, and a dried extract obtained by distilling the solvent extract under reduced pressure and then freeze-drying or spray-drying can be prepared. In a specific embodiment of the present invention, ethyl acetate, water, dichloromethane, butanol, hexane, and ethanol were used as extracting solvents to prepare an extract of chinquapin, which was filtered and concentrated under reduced pressure.

The extraction is carried out at an extraction temperature of 10 to 80 ° C., specifically 15 to 50 ° C., using a solvent that is 1 to 10 times the weight, specifically 2 to 7 times the weight of the dry plant. Time to 30 days, specifically, a method of extracting for an extraction time of 12 hours to 18 days may be applied, and the process of obtaining a liquid crude extract by extracting 1 to 5 times in succession, including the drying and crushing can include

In the present invention, the extract is filtered to remove floating solid particles, for example, by filtering out particles using nylon or filter paper, or by filtering using freeze filtration, and then used as it is or freeze-dried, hot air dried, spray dried, etc. It can be dried and used.

The liquid crude extract may be separated from dried plant debris by vacuum filtration or the like, and then concentrated or dried. For example, the crude liquid extract may be a concentrated liquid concentrated under reduced pressure at 20 to 100 ° C., preferably 30 to 70 ° C., using a vacuum rotary concentrator, and the liquid extract may be dried to obtain a powdered extract. . The concentrated or powdered extract may be used by being soluble in water, alcohol, DMSO (dimethyl sulfoxide) or a mixed solvent thereof, if necessary.

As used herein, the term "fraction" refers to a result obtained by performing fractionation to separate a specific component or a specific component group from a mixture containing various components.

In the present invention, the fractionation method for obtaining the fraction is not particularly limited, and may be performed according to a method commonly used in the art. A non-limiting example of the fractionation method is a method of obtaining a fraction from the extract by treating a predetermined solvent to a complex extract obtained by extracting the extract.

In the present invention, the type of solvent used to obtain the fraction is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the fractionation solvent include polar solvents such as water and alcohol; and non-polar solvents such as hexane, ethyl acetate, chloroform, dichloromethane, and butanol. These may be used alone or in combination of two or more. When alcohol is used among the fractionation solvents, C1 to C4 alcohols may be specifically used. Preferably the water fraction is used.

In one embodiment of the present invention, the efficacy of relieving female menopausal syndrome was confirmed by measuring the estrogenic activity of the extract or a fraction thereof in MCF-7 cells and an animal model of cervical hypertrophy, and 50% ethanol was obtained by changing the solvent composition during extraction. It was confirmed that the root extract used showed the most excellent female climacteric syndrome alleviation effect. In addition, it was confirmed that the water fraction showed the best efficacy among the solvent fractions of the extract. In an animal model of cervical hypertrophy, when the water fraction of the 50% ethanol extract of Pygonia root was administered, an effect similar to that of estradiol-induced uterus enlargement was confirmed without significant change in body weight.

Therefore, the composition for alleviating female menopausal syndrome of the present invention can be used as a herbal medicine-based preventive or therapeutic agent for female menopausal symptoms with excellent effects, or as a health food.

A preventive or therapeutic agent for female menopausal syndrome containing the composition of the present invention as an active ingredient may further include appropriate carriers, excipients and diluents commonly used in pharmaceuticals. The female menopausal syndrome preventive or therapeutic agent may be formulated and used in a known dosage form such as powder, granule, tablet, capsule, suspension, emulsion, or syrup according to a conventional method. Among the preventive or therapeutic agents for female menopausal syndrome, the composition of the present invention is preferably included in an amount of 0.1 to 99.9% by weight.

Health food for preventing or improving female menopausal syndrome containing the composition of the present invention as an active ingredient may be provided in various forms. First, "health food" as defined in the present invention includes all health functional foods, health supplement foods, and functional foods manufactured and processed using raw materials or ingredients having useful functionalities for the human body, and is not limited to the definitions of relevant laws. It means to include all things that are consumed for the purpose of controlling consumer nutrients or obtaining beneficial effects on health.

The health food containing the composition of the present invention may further include suitable carriers, excipients, and diluents commonly used in pharmaceuticals or foods, and may be formulated into tablets, capsules, powders, granules, liquids, pills, etc. according to conventional methods. It can be provided in dosage form. In addition, the composition of the present invention may be provided in the form of being added to various foods or beverages. For example, it may be provided in the form of any one of beverages, powdered beverages, solids, chewing gum, tea, vitamin complexes, and food additives. Among the health foods, the composition of the present invention is preferably included in an amount of 0.1 to 99.9% by weight.

The preferred dosage of the composition of the present invention varies depending on the condition and weight of the patient, the severity of the disease, the type of drug, the route of administration and the period of administration, but the dosage adjustment according to these conditions can be appropriately selected by those skilled in the art. When used for prevention of female menopausal syndrome or as a health food, it is preferable to use 100 ~ 1,000mg per day based on an adult with a body weight of 60kg. it is desirable Administration may be administered once a day, or may be administered in several divided doses. The dosage is not intended to limit the scope of the present invention in any way.

In the present invention, it was confirmed that the extract or a fraction thereof exhibits an effect of inducing uterine hypertrophy similar to that of estradiol, a treatment for female menopausal syndrome, thereby relieving female menopausal syndrome containing an extract or a fraction thereof as an active ingredient can be used as a preventive or therapeutic agent for women's menopausal symptoms with excellent herbal medicine-based effects, and as a health food.

Figure 1 shows the estrogen-like activity of 50% ethanol extract and each fraction of the aerial part of P.
Figure 2 shows the estrogen-like activity of the 50% ethanol extract of the subterranean part of P. chinensis and each fraction.
Figure 3 shows the estrogen-like activity of extracts of the above-ground parts of P. japonica by extraction solvent.
Figure 4 shows the estrogen-like activity of extracts of the subterranean part of P. japonica for each extraction solvent.
Figure 5 shows the MCF-7 breast cancer cytotoxicity of the above-ground part extract of P.
Figure 6 shows the MCF-7 breast cancer cytotoxicity of the subterranean part extract of P.
Figure 7 shows the experimental progress schedule of the cervical hypertrophy animal model.
8 is a photograph confirming the improvement of uterine hypertrophy in the normal group, the control group, and the experimental group.
9 is a result of measuring the weight of the uterus to confirm the hypertrophy of the uterus in the normal group, the control group, and the experimental group.

Hereinafter, preferred examples and formulation examples are presented to aid understanding of the present invention. However, the following examples and preparation examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples or preparation examples.

[Example]

Preparation Example 1: Preparation of chinensis extract and fractions thereof

The roots and leaves of the sandalwood collected from the tidal flats near Dongmak Beach, Ganghwa Island were washed and dried in the shade. 30 mL of 0%, 30%, 50%, and 80% ethanol was used as an extraction solvent for 10 g of dried grass roots. Ultrasonic extraction was repeated twice for 1 hour at 25° C. to prepare an extract from the roots of Pygonia. Leaf extracts were prepared by ultrasonic extraction of 10 g of dried sage grass leaves using 100 mL of 0%, 30%, 50%, and 80% ethanol as extraction solvents at 25° C. for 1 hour each time.

[Table 1]

Preparation of extracts by solvent

After drying the extract of 1.1 g in 50% ethanol (EtOH) extract of the root of P. chinensis under reduced pressure, it was suspended in 250 mL of distilled water and solvent fractionation was performed by adding 250 mL of hexane. Solvent fractionation was performed a total of three times to obtain a hexane layer. Solvent fractionation was performed by adding 250 mL of dichloromethane to the remaining aqueous layer. A dichloromethane layer was obtained from a total of three solvent fractions. Solvent fractionation was performed by adding 250 mL of ethyl acetate to the remaining aqueous layer. Solvent fractionation was repeated a total of three times to obtain an ethyl acetate layer. 250 mL of saturated butanol was added to the remaining aqueous layer to obtain a solvent fraction, which was repeated three times to obtain a butanol layer. The hexane layer, the dichloromethane layer, the ethyl acetate layer, the butanol layer, and the remaining aqueous layer of the 50% ethanol extract of the grass root obtained through this process were dried under reduced pressure to obtain a hexane (Hex) fraction, a dichloromethane (MC) fraction, 37.8 mg, 36.9 mg, 25.2 mg, 241.2 mg, and 943.4 mg of the ethyl acetate (EA) fraction, the butanol (BuOH) fraction, and the water (H ₂ O) fraction, respectively, were obtained.

After drying the extract of 1.5 g in 50% ethanol extract of the chinensis leaf under reduced pressure, it was suspended in 250 mL of distilled water and solvent fractionation was performed by adding 250 mL of hexane. Solvent fractionation was performed a total of three times to obtain a hexane layer. Solvent fractionation was performed by adding 250 mL of dichloromethane to the remaining aqueous layer. A dichloromethane layer was obtained from a total of three solvent fractions. Solvent fractionation was performed by adding 250 mL of ethyl acetate to the remaining aqueous layer. Solvent fractionation was repeated a total of three times to obtain an ethyl acetate layer. 250 mL of saturated butanol was added to the remaining aqueous layer to obtain a solvent fraction, which was repeated three times to obtain a butanol layer. The resulting hexane layer, dichloromethane layer, ethyl acetate layer, butanol layer, and remaining aqueous layer were dried under reduced pressure to obtain the hexane (Hex) fraction, dichloromethane (MC) fraction, ethyl acetate (EA) fraction, butanol ( BuOH) fraction and water (H ₂ O) fraction were obtained at 102.2 mg, 91.8 mg, 111.4 mg, 531.1 mg, and 1015 mg, respectively.

[Table 2]

Preparation of solvent-specific fractions of 50% ethanol extract

Example 1: Identification of estrogenic activity of extracts and fractions of P.

MCF-7 human breast cancer cells (estrogen receptor positive) were cultured in 10% (v/v) fetal bovine serum (FBS, Gibco), 100 μg/ml penicillin G, and 100 μg/ml streptomycin sulfate. It was cultured in RPMI1640 medium (Gibco) and maintained at 37° C. temperature and humidified conditions of 5% CO ₂ . Cells used in the experiment were cultured in phenol red-free RPMI1640 medium (Gibco) containing 10% dextran-coated charcoal-stripped FBS.

Proliferation of MCF-7 cells was evaluated using an E-screen assay (Soto et al., Environ Health Perspect. 1995 Oct; 103(Suppl 7): 113-122). The E-screen assay uses the principle that ER-positive breast adenocarcinoma cells (MCF-7) overproliferate by activating ER signaling by a test substance. Usually, the relationship between the estrogenicity of the test substance and the degree of cell proliferation of MCF-7 is proportional, and the estrogenicity is confirmed by confirming the cell proliferation effect as an ER ligand. The cell culture medium used in this test was a phenol red-free medium containing charcoal stripped serum in order to thoroughly exclude external estrogenic effects.

MCF-7 cells (1×10 ⁴ cells/well) were cultured for 24 hours in a 48 well plate, and samples were treated at concentrations of 25, 50, and 100 μg/mL. In addition, the proliferation of MCF-7 cells was compared by treatment with ICI 182,780, an estrogen receptor antagonist. After sample treatment, incubation was performed for 144 hours, Ez-Cytox (DoGEN) reagent was added, incubation was performed for another 1 hour, and absorbance was measured at 450 nm. The cell proliferation increase effect for each sample was repeated three times and the average value was taken, and the ratio of the absorbance of the sample group to the absorbance of the control group to which no sample was added was expressed as a percentage and analyzed by comparison in a graph.

1.1. Estrogen-like activity test of extracts of the above-ground parts and each fraction

As shown in Figure 1, the above-ground part (leaf) extract and each fraction did not show a significant cell proliferation effect.

1.2. Estrogen-like activity test of subterranean extract and each fraction

As shown in FIG. 2 , the extract of the subterranean part (root) of P. chinensis showed a cell proliferation rate of 113.9, 119.5, and 118.4% at 25, 50, and 100 μg/mL, respectively, indicating a significant cell proliferation effect. The MCF-7 cell proliferation effect of the extract was suppressed when the extract of the subterranean part of the chinensis plant and the estrogen antagonist ICI 182,780 were simultaneously treated. The water fraction of the extract of the subterranean part of P. chinensis showed a significant cell proliferation effect by showing cell proliferation rates of 112.8, 117.9, and 116.6% at 25, 50, and 100 μg/mL, respectively. The MCF-7 cell proliferation effect of the extract was inhibited when the water fraction of the subterranean part extract and the estrogen antagonist (ICI 182,780) were simultaneously treated. Through this, the efficacy of the female menopausal syndrome improvement effect of the subterranean extract and the water fraction of P.

1.3. Estrogen-like activity test of extracts of the aerial parts of P.

As shown in Fig. 3, among the extracts of the aerial part of P. japonica, at a concentration of 25 μg/mL of the 30% ethanol extract, estrogen-induced cell proliferation and inhibition of cell proliferation by the antagonist were confirmed.

1.4. Estrogen-like activity test of extracts from the subterranean part of P. japonica by extraction solvent

As shown in FIG. 4 , estrogen-induced cell proliferation and inhibition of cell proliferation by the antagonist were observed at a concentration of 25 to 100 μg/mL of the 50% ethanol extract in the subterranean extract of P. As a result, among all the extracts of P. chinensis, the 50% ethanol extract of the subterranean part showed the most significant estrogen-like activity.

1.5. MCF-7 Breast Cancer Cytotoxic Effects of Extracts of the Aboveground Parts of Pygmy Flora

In order to evaluate the risk of breast cancer, a side effect of HRT, the cytotoxicity of breast cancer was confirmed. As shown in FIG. 5, all extracts of the above-ground parts of P. chinensis showed no effect on MCF-7 cancer cell proliferation. Therefore, it is judged that there is no risk of developing breast cancer.

1.6. Cytotoxic effect of MCF-7 breast cancer extracts from the subterranean part of P.

As shown in Fig. 6, all extracts of the subterranean part of Pygonia did not show the MCF-7 cancer cell proliferation effect. Therefore, it is judged that there is no risk of developing breast cancer.

Example 2: Confirmation of menopausal syndrome improvement efficacy using an animal model of uterine hypertrophy

2.1. Breeding of laboratory animals

14-day-old female SD rats were purchased from Daehan Biolink (Chungbuk, Korea) and adapted to the breeding environment for one week, and then 21-day-old females were used in the experiment. They were reared at a temperature of 22±2℃ and humidity of 50±10%, and the light/dark cycle was adjusted every 12 hours, and water and feed were freely fed. This experiment was conducted with the approval of the Institutional Animal Care and Use Committee (approval number: GIACUC-R2019035).

2.2. Uterine hypertrophy model production and sample administration

As shown in Table 3, six experimental animals were placed in each group, and the experimental groups were classified into a total of three groups. The group was divided into a normal group, a group treated with estradiol alone (control), and a sample administration group administered with the water fraction of 50% ethanol extract of 50% ethanol extract from estradiol treatment, dissolved in drinking water at a dose of 200 mg/kg/day and orally administered for 3 days. . As a positive control, estradiol was dissolved in corn oil at a concentration of 10 μL/4 mL/kg and injected subcutaneously to induce uterine hypertrophy.

[Table 3]

2.3. weight measurement

The body weight of the experimental animals was measured a total of 4 times, once each during the experiment period.

2.4. Uterine harvest and weighing

The uterus of the experimental animal was collected and weighed after removing fat and ovaries during dissection.

2.5. statistical analysis

Experimental results were expressed as mean ± standard deviation (SD). The significance of the difference between the means of each group was verified at the P < 0.05 level using one-way analysis of variance (ANOVA).

As shown in Table 4, compared to the normal group, no significant difference was found between the positive control group (86.88 g) and the normal group (88.45 g) treated with estradiol, an inducer of uterine hypertrophy. No significant difference was found between the groups (87.53 g) administered with the water fraction of the 50% ethanol extract of the radishes root, but the group administered with the water fraction of the 50% ethanol extract of the 50% ethanol extract of the chinensis root showed less weight loss than the control group.

[Table 4]

As shown in FIG. 8, the weight of the uterus was 50.33 mg in the control group compared to 41.50 mg in the normal group, and an increase in weight due to uterine hypertrophy induced by estradiol administration was confirmed. On the other hand, the group administered with the water fraction of the 50% ethanol extract of P. chinensis root showed a tendency to significantly increase compared to the Normal group with 49 mg. Based on the overall results, it was judged that the group administered with the water fraction of 50% ethanol extract of Pygonia root induced uterus hypertrophy. As such, intake of the water fraction of the 50% ethanol extract of the root of estradiol induces uterus hypertrophy similar to that of estradiol administration, and it is judged to be helpful in improving female menopausal symptoms with reduced estrogen.

Claims (9)

A composition for alleviating female menopausal syndrome comprising a 30% ethanol extract of the above-ground part, a 50% ethanol extract of the underground part, or a fraction thereof as an active ingredient.
delete delete According to claim 1,
The fraction is a composition fractionated with any one solvent selected from the group consisting of water, hexane, ethyl acetate, dichloromethane, alcohol having 1 to 4 carbon atoms, and combinations thereof of the extract.
delete delete According to claim 4,
Wherein the solvent is water.
A preventive or therapeutic agent for female menopausal syndrome comprising the composition of claim 1.
A health food for preventing or improving female menopausal syndrome comprising the composition of claim 1.