KR102380208B1 - Composition for prevention and alleviating of menopausal symptom comprising Gastrodia elata extract - Google Patents

Abstract

The present invention relates to a composition for relieving, preventing, or treating female menopause comprising a Chunma extract as an active ingredient. As can be seen through the present invention, Chunma extract has a proliferative effect on MCF-7 cells, and estrogen receptor It was also found that the TFF1 gene expression level, a product of the pathway, has increased cell proliferation via the estrogen receptor. Therefore, Chunma extract can be used as a phytoestrogen-like substance to alleviate, prevent, or treat female menopausal diseases.

Description

Composition for prevention and alleviating of menopausal symptom comprising Gastrodia elata extract

The present invention relates to a composition for alleviating and preventing female menopausal symptoms comprising a cheonma extract as an active ingredient.

Due to modern industrialization, the economic level is improved and the human lifespan is extended due to the development of medicine, and the elderly population is increasing. Therefore, studies are being conducted to improve the health and quality of life of the elderly.

In particular, women are experiencing physical and mental difficulties due to the symptoms of menopause that begin gradually after the age of 40. Menopause, which about 20% of all women experience, is one of the aging phenomena, and it starts slowly from the age of 40, but there is a problem that it takes up more than 1/3 of a woman's life as the average lifespan is extended.

Female menopause is a type of endocrine syndrome, and refers to a transition period in which physiological and sexual functions decrease or disappear due to a decrease in female hormones, that is, estrogen due to general and gradual aging of ovarian function. Estrogen exerts essential regulatory effects such as catabolic and active action in the uterus, vagina, skeletal muscle, and cardiovascular system in women (Couse and Korach, 1999; Korach et al, 1995). It is known to act on bone structure and cholesterol regulation, and it is also applied to the evaluation of ovarian function.

Symptoms of menopause include hot flashes, tachycardia, symptoms due to vascular changes such as sweating or headache, symptoms due to musculoskeletal changes such as muscle pain, joint pain and back pain, frequent urination or incontinence, and urogenital changes such as atrophy of the uterus and vagina Common symptoms such as symptoms caused by changes in the brain nervous system, such as memory loss, depression, loss of concentration and dizziness, loss of vision, 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 diseases 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, there is a need to develop a treatment method that can improve menopausal symptoms.

Examples of treatments that can improve menopausal symptoms include drug therapy using drugs such as hormone replacement therapy or nonsteroidal agents. However, it is known that most of the drugs such as nonsteroidal drugs 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 in the body (Swaran L, et al, Obstetrics & Gynecology, 91, 678-684, 1998)

Therefore, there is a need to discover candidate substances that are similar to female hormones and have a low risk of side effects due to the administration of female hormones.

[Prior Patent Literature]

Republic of Korea Patent Publication 10-2019-0092831

The present invention has been devised in response to the above needs, and an object of the present invention is to provide a novel composition for preventing or alleviating female menopause.

In order to achieve the above object, the present invention provides a food composition for relieving women's menopause comprising a cheonma extract as an active ingredient.

In one embodiment of the present invention, the Chunma extract is preferably extracted using water, C2-C4 alcohol, or a mixed solvent thereof, but is not limited thereto.

In a more preferred embodiment of the present invention, the Cheonma extract is preferably an ethanol extract, but is not limited thereto.

In addition, the present invention provides a pharmaceutical composition for preventing or treating female menopause comprising an extract of Chunma as an active ingredient.

In one embodiment of the present invention, the Chunma extract is preferably extracted using water, C1-C4 alcohol or a mixed solvent thereof, but is not limited thereto.

In a more preferred embodiment of the present invention, the Cheonma extract is preferably an ethanol extract, but is not limited thereto.

In addition, the present invention provides a phytoestrogens composition comprising a cheonma extract as an active ingredient.

In one embodiment of the present invention, the Chunma extract is preferably extracted using water, C1-C4 alcohol or a mixed solvent thereof, but is not limited thereto.

In a more preferred embodiment of the present invention, the Cheonma extract is preferably an ethanol extract, but is not limited thereto.

In addition, the present invention provides a composition for increasing gene expression of trefoil factor (Trefoil factor 1; TFF1) comprising a cheonma extract as an active ingredient.

In addition, the present invention provides a method of increasing the expression of the trefoil factor (Trefoil factor 1; TFF1) gene in the cells by treating the cellulosic extract in vitro.

In one embodiment of the present invention, the cell is preferably an MCF-7 cell, but is not limited thereto.

Chunma extract according to the present invention can be made by extracting Chunma or dried products thereof, and Chunma can be used without limitation, such as cultivated or commercially available ones.

Chunma extract according to the present invention can be extracted using a conventional extraction method in the art, such as solvent extraction method, ultrasonic extraction method, filtration method and reflux extraction method. As a preferred preparation example, the Chunma extract can be obtained by immersing the dried Chunma root in water, alcohol, or a mixed solvent thereof to obtain an extract, then filtering the extract, freeze-drying or spray-drying it again, and concentrating under reduced pressure, but is not limited thereto. does not

In this case, as the extraction solvent, water, C1-C4 alcohol, or a mixture thereof may be used, and the C1-C4 alcohol may preferably be ethanol, but is not limited thereto.

The preventive and therapeutic agent for menopausal symptoms containing the composition of the present invention as an active ingredient may further include appropriate carriers, excipients and diluents commonly used in pharmaceuticals.

In addition, the pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods. .

In the case of formulation, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants are usually used. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, and the like. The solid preparation may include at least one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, and gelatin in addition to the active ingredient.

In addition to simple excipients, lubricants such as magnesium stearate talc may also be included. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents, may be included. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, laurin, cacao butter, glycerogelatin, etc. may be used.

The dosage of the extract disclosed herein may vary depending on the patient's condition and weight, the degree of disease, drug form, administration route and period, and may be selected from a range commonly used in the art. In one aspect, the daily dose of the active ingredient may be 0.0001 to 0.5 g/kg based on dry weight, and in one aspect, 0.001 to 0.1 g/kg may be administered.

In one aspect of the present invention, the composition for improving women's menopausal symptoms may be a health functional food composition. For example, there are various foods, beverages, gum, tea, vitamin complexes, health supplements, etc., and may be used in the form of powders, granules, tablets, capsules or beverages. In addition to the active ingredient, the food composition of each dosage form can be appropriately selected and formulated by those skilled in the art without difficulty depending on the dosage form or purpose of use in addition to the active ingredient, and a synergistic effect may occur when applied simultaneously with other raw materials.

First, "health food" as defined in the present invention includes all health functional foods, health supplements, and functional foods manufactured and processed using raw materials or ingredients useful for the human body, and is not limited to the definition of related laws. It is meant to include all intakes for the purpose of regulating consumer nutrients or obtaining useful effects on health.

In this case, the amount of the active ingredient contained in the food or beverage may include 0.001 to 90% by weight of the total food weight in the case of a general food composition, but is not limited thereto. In addition, in the case of a health food composition, it may include 10 to 90% by weight of the total weight of the food, but is not limited thereto. In one aspect, when ingested as a food composition, the daily dose of the active ingredient may be 0.0001 to 0.1 g/kg based on dry weight, but is not limited thereto.

In one embodiment, the composition may contain other ingredients capable of giving a synergistic effect to the main effect within a range that does not impair the main effect of the present invention. For example, in order to improve physical properties, additives such as fragrances, pigments, bactericides, antioxidants, preservatives, humectants, thickeners, inorganic salts, emulsifiers, and synthetic polymers may be further included.

In addition, auxiliary components such as water-soluble vitamins, oil-soluble vitamins, high molecular peptides, high molecular weight polysaccharides and seaweed extract may be further included. According to the formulation or purpose of use, a person skilled in the art can appropriately select and mix the ingredients without difficulty, and the amount added can be selected within a range that does not impair the purpose and effect of the present invention. For example, the amount of the components added may be in the range of 001-5 wt%, more specifically 0.01-3 wt%, based on the total weight of the composition.

The dosage form of the composition according to the present invention may be in various forms such as solutions, emulsions, viscous mixtures, tablets, powders, tablets, calps, liquids, granules, pills, pieces, pastes, syrups, gels, jellies, bars or edible pills. may be, and it may be administered by various methods such as simple drinking, injection administration, spray method, or squeeze method.

Hereinafter, the features of the present invention will be described.

The present invention intends to proceed with a patent for a phytoestrogens composition for alleviating and preventing female menopausal symptoms using a cheonma extract, which exhibits the same efficacy as estrogen.

As can be seen through the present invention, the present invention investigated the proliferation effect of MCF-7 cell of Chunma 70% EtOH extract, and through qRT-PCR, the estrogen receptor was stimulated by increasing the expression of TFF1 gene, the product of the estrogen receptor pathway. It was also found to have cell proliferation ability through the

Furthermore, if we examine the tendency of protein expression, we can confirm which pathway of the estrogen receptor pathway is activated and the molecular mechanism of Chunma. (E2 is 17β-estradiol, EC is an estrogenic compound, and it is a substance with estrogen-like activity in the cell.)

1 is a figure showing the experimental results to find estrogen-like substances,
Figure 2 is a figure showing the concentration-dependent effect of Chunma extract on MCF-7 cells,
Figure 3 is a figure showing the TFF1 gene expression level confirmation (qRT-PCR),
Figure 4 shows the activation pathway of the estrogen receptor pathway.

Hereinafter, the present invention will be described in more detail by way of preparation examples and examples.

However, the following Preparation Examples and Examples are only provided to explain the present invention in detail, and the scope of the present invention is not to be construed as being limited by these Preparation Examples and Examples.

<Preparation Example 1> Preparation of Chunma extract

After shredding the dried Chunma root, put 850 g in an extraction container, immerse 12 ℓ of 70% ethanol, shake at room temperature for 24 hours to extract, and then filter (Whatman No2) to obtain the primary extract, and the same as the primary extract. The residue was extracted by this method to obtain secondary and tertiary extracts.

After mixing all of the first and second extracts, the mixed pressure extract was concentrated and dried with a rotary vacuum concentrator to obtain a Chunma extract (126 g).

Example 1; E-screen assay

The theory that will be the core of the experiment to find estrogen-like substances is MCF-7 cells, which are human female breast cancer cells.

What is noteworthy about MCF-7 cells is that they are estrogen (especially E₂, 17-β-estradiol)-induced growth cells and are estrogen receptor (ER) α-positive cells. That is, estrogen stimulates MCF-7 through ERα, and the proliferation of MCF-7 cells proceeds.

In the present invention, it was confirmed that the number of cells increased together with the estrogen-treated group in the MCF-7 cell-treated group with the Chunma extract (FIG. 1). The extraction of each medicinal material was obtained through the same process as the preparation of the Chunma extract.

SRB assay setting conditions and experimental procedures are as follows.

- Cell : MCF-7

- Culture media : RPMI1640 + 10% FBS + 1% PS, 37℃, 5% CO₂

Positive control: E₂ (17-β-estradiol) (10^-10M, in EtOH),

negative control: EtOH

① Cell seeding

To provide sufficient space for MCF-7 cells to attach, seeding was performed in a 24-well plate, and the number of cells was 1*10⁴ cells per well.

The medium was carried out from seeding to phenol-red free DMEM (10% CD-FBS, 1% PS).

After seeding, it was incubated for 24 hours in an incubator (37℃, 5% CO₂).

② Sample processing

The positive control used in this experiment was 17β-estradiol (E2, in 100% EtOH, final concentration 10^-10M), and the vehicle control was automatically 100% EtOH.

In the case of EtOH, the sample was prepared by diluting it to an appropriate concentration so that it became 1%, which is the concentration that does not affect the cells when treating the medium.

For the sample, 10 kinds of natural products existing in the natural product library in the laboratory were selected and 100mg/ml of 70% EtOH extract (4℃, 12hr extraction) was processed so as to have a final concentration of 1mg/ml.

The medium used for the treatment experiment was phenol-red free DMEM, not normal RPMI1640.

In addition, as FBS, Charcoal-Dextran FBS (CD-FBS), a steroid-free FBS treated with charcoal coated dextran, was used.

Samples were prepared with phenol-red free DMEM containing 10% CD-FBS.

When the seeded cells were cultured for 24 hours, the medium was removed and washed twice with phenol-red free DMEM (no FBS, no PS).

A sample DMEM prepared in advance was dispensed.

Incubated in an incubator (37℃, 5% CO₂) for 144hr.

※ SRB assay

The SRB assay was developed by Skehan et al. to measure drug-induced cytotoxicity and cell proliferation in the field of large-scale drug screening.

Sulforhodamine B, an anionic aminogentene (a dye that binds amino acids), is used to stain cells by binding to basic amino acids in proteins under moderate acidic conditions.

Through this, the growth rate of living cells can be confirmed, and this can be used as a cytotoxicity test.

③ SRB assay

After 144 hrs after the incubation, the medium was removed and washed twice with PBS.

Cold 10% Trichloroacetic acid (4℃, protein precipitant, to stain cells) was dispensed into each well by 500 μl.

The reaction was carried out at 4°C for 30 min.

The supernatant was removed and washed 5-6 times with D.W.

It was dried until the water dried.

Dispense 300 μl of 0.4% SRB solution (in 1% acetic acid) into each well,

The reaction was carried out for 15 min.

The supernatant was removed and washed 5-6 times with 1% acetic acid.

Dry the wells until dry.

200μl of 10mM Trizma base (pH 10.5) was dispensed into each well and shaken to release the dye.

After transferring all of the supernatant to a 96 well plate, absorbance was measured at 490 nm.

As can be seen from FIG. 1 , it was confirmed that the 70% EtOH extract of Chunma, which is the 6th natural product among 10 types of natural products, was the most effective.

Example 2: Concentration-dependent effect of Chunma extract on MCF-7 cells

It was found through E-screen assay that Chunma 70% EtOH extract has cell proliferation ability against MCF-7 cells. In order to check whether the cell proliferation rate is affected by the treatment concentration, the treatment and E -screen assay was performed.

① Cell seeding

To provide sufficient space for MCF-7 cells to attach, seeding was performed in a 24-well plate, and the number of cells was 1*10⁴ cells per well.

The medium was carried out from seeding to phenol-red free DMEM (10% CD-FBS, 1% PS).

After seeding, it was incubated for 24 hours in an incubator (37℃, 5% CO₂).

② Sample processing

The positive control to be used in the present invention is 17β-estradiol (E2, in 100% EtOH, final concentration 10^-10M), and the vehicle control automatically used 100% EtOH.

In the case of EtOH, the sample was prepared by diluting it to an appropriate concentration so that it became 1%, which is the concentration that does not affect the cells when treating the medium.

The sample was treated so that 100mg/ml of 70% EtOH extract of Cheonma (4℃, 12hr extraction) became final concentration of 1000, 750, 500, 250, 125μg/ml.

The medium used for the treatment experiment was phenol-red free DMEM, not normal RPMI1640. In addition, as FBS, Charcoal-Dextran FBS (CD-FBS), a steroid-free FBS treated with charcoal coated dextran, was used.

Samples were prepared with phenol-red free DMEM containing 10% CD-FBS.

When the seeded cells were cultured for 24 hours, the medium was removed and washed twice with phenol-red free DMEM (no FBS, no PS).

A sample DMEM prepared in advance was dispensed.

Incubated in an incubator (37℃, 5% CO₂) for 144hr.

※ SRB assay

The SRB assay was developed by Skehan et al. to measure drug-induced cytotoxicity and cell proliferation in the field of large-scale drug screening.

Sulforhodamine B, an anionic aminogentene (a dye that binds amino acids), is used to stain cells by binding to basic amino acids in proteins under moderate acidic conditions.

Through this, the growth rate of living cells can be confirmed, and this can be used as a cytotoxicity test.

③ SRB assay

After 144 hrs after the incubation, the medium was removed and washed twice with PBS.

Cold 10% Trichloroacetic acid (4℃, protein precipitant, to stain cells) was dispensed into each well by 500 μl.

The reaction was carried out at 4°C for 30 min.

The supernatant was removed and washed 5-6 times with D.W.

It was dried until the water dried.

Dispense 300 μl of 0.4% SRB solution (in 1% acetic acid) into each well,

The reaction was carried out for 15 min.

The supernatant was removed and washed 5-6 times with 1% acetic acid.

Dry the wells until dry.

200μl of 10mM Trizma base (pH 10.5) was dispensed into each well and shaken to release the dye.

After transferring all of the supernatant to a 96 well plate, absorbance was measured at 490 nm.

As can be seen from Figure 2, it was found that as the concentration of the 70% Cheonma EtOH extract increased, the cell proliferation was increased in a concentration-dependent manner.

Through this, it is judged that the estrogenic activity of the 70% EtOH extract of Chunma can be expected by utilizing the appropriate concentration of treatment.

Example 3: Confirmation of TFF1 gene expression level (qRT-PCR)

When the MCF-7 cell receives a signal through the ER, it transmits a cell proliferation signal to the nucleus through a series of metabolic pathways, and the genes involved in the pathway are induced to be transcribed.

Using this, the mRNA expression level of TFF1 (pS2 protein gene), one of the results of the estrogen receptor pathway, was measured using qRT-PCR. It was confirmed that the cells proliferate.

① Cell seeding

In order to provide sufficient space for the MCF-7 cells to attach, seeding was performed with a 60mm dish, and the number of cells was 2*10*?* cells per plate. (In the case of N.C, which grows late due to the characteristics of the slow-growing MCF-7 cells, 8*10*?* cells were seeded.)

The medium was carried out from seeding to phenol-red free DMEM (10% CD-FBS, 1% PS).

After seeding, it was incubated for 24 hours in an incubator (37℃, 5% CO₂).

② Sample processing

The positive control to be used in the present invention is 17β-estradiol (E2, in 100% EtOH, final concentration 10^-10M), and the vehicle control automatically used 100% EtOH.

In the case of EtOH, the sample was prepared by diluting it to an appropriate concentration so that it became 1%, which is the concentration that does not affect the cell when treating the medium.

The sample was treated with 100mg/ml of 70% EtOH extract of Chunma (4℃, 12hr extraction) to have a final concentration of 1mg/ml.

The medium used for the treatment experiment was phenol-red free DMEM, not normal RPMI1640.

In addition, as FBS, Charcoal-Dextran FBS (CD-FBS), which was treated with charcoal-coated dextran to become steroid-free, was used.

Samples were prepared with phenol-red free DMEM containing 10% CD-FBS.

After 24 hr of incubation of the seeded cells, the medium was removed and washed twice with phenol-red free DMEM (no FBS, no PS).

Dispense the pre-made Sample DMEM,

It was incubated in an incubator (37℃, 5% CO₂) for 2 days, 4 days, and 6 days.

③ RNA isolation

After the incubation time was completed, the culture medium was all discarded and washed once with 2 ml of cold PBS.

Discard the PBS, put 1ml of cold PBS again, and scrape off the cells with a cell scrapper,

After transferring to the E-tube, centrifuged at 7500rcf, 5 minutes 30 seconds, 4℃ conditions,

All of the supernatant was removed.

Cells were disrupted using Trizol,

Add 200 μl of Chloroform, shake vigorously for 10 seconds, and leave it for 2 minutes,

Centrifugation was performed at 12000 rcf, 15 minutes 30 seconds, and 4°C.

400 μl of RNA layer was removed with a wide-pore filter tip and transferred to a new e-tube,

Add 400 μl of isopropanol,

After careful inverting, let stand for 10 minutes,

Centrifugation was performed (12,000 rcf, 10 min 30 sec, 4°C).

After removing the remaining supernatant except for the precipitated RNA pellet,

One more brief centrifugation (12,000rcf, 3min, 4℃) was performed to remove all liquid remaining on the e-tube wall.

1ml of 70% EtOH was added, inverted, and centrifuged. (12,000rcf, 5min 30sec, 4℃)

After removing the remaining supernatant except for the RNA pellet,

50 μl of DNase was added, vortexed slightly, and incubated in a water bath (37°C, 1 hr).

After adding 5 μl of inactivator, tapping every 2-3 minutes for a total of 10 minutes,

Centrifugation was performed (12,000 rcf, 3 min 30 sec, 4°C).

Carefully separate only the supernatant so that the bead does not follow, and transfer it to a new e-tube.

The amount of RNA was measured by nano-drop.

④ cDNA synthesis

A mixture was prepared in a PCR tube, and cDNA synthesis was performed according to the composition shown in Table 1.

RNA (500 ng) x μl Nuclease-free water (7-x) μl 5x RT buffer 2 μl Primer mix 0.5 μl Enzyme mix 0.5 μl Total 10 μl

⑤ qRT-PCR

- Mixture was made and added, and qRT-PCR was performed according to the composition of Table 2.

cDNA 1.5 μl Primer(Sense) 0.5 μl Primer (Anti-sense) 0.5 μl SYBR green 5 μl Nuclease-free water 12.5 μl Total 20 μl

The primer sequences used in the PCR of the present invention are as follows:

TFF1 Forward: 5'-GAGAACAAGGTGATCTGCGC -3'

TFF1 Reverse : 5'-TGGTATTAGGATAGAAGCACC -3'

As can be seen in FIG. 3, it was found that the expression level of TFF1 of the Chunma 70% EtOH extract-treated MCF-7 cells increased as 2 days, 4 days, and 6 days passed after the sample treatment of the present invention. Through this, it was found that the extract induces cell proliferation via the estrogen receptor.

Claims (12)

delete delete delete delete delete delete delete delete delete delete Method of increasing the viability of cells by treating 1mg/ml of 70% ethanol extract of Chunma in vitro to MCF-7 cells, which are human female breast cancer cells

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