KR102588771B1 - COMPOSITION COMPRISING EXTRACT Colpomenia sinuosa OR Colpomenia peregrina Sauvageau AS AN ACTIVE INGREDIENT FOR ALLEVIATING OR IMPROVING SYMPTOM BY ESTROGEN REDUCING - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for controlling, suppressing, preventing, improving or treating diseases related to changes in estrogen levels, containing an extract of Colpomenia sinuosa or Colpomenia peregrina Sauvageau as an active ingredient. Specifically, a pharmaceutical composition for the control, prevention or treatment of symptoms related to changes in estrogen levels, and a food composition for the improvement, containing as an active ingredient an extract of Malt vulgaris or Malt vulgaris, which has an estradiol-increasing effect in an estradiol-specific cell model. , relates to quasi-drug compositions.

Description

A composition for relieving and improving symptoms caused by estrogen reduction, comprising extracts of Malt vulgaris or Malt vulgaris as active ingredients {COMPOSITION COMPRISING EXTRACT Colpomenia sinuosa OR Colpomenia peregrina Sauvageau AS AN ACTIVE INGREDIENT FOR ALLEVIATING OR IMPROVING SYMPTOM BY ESTROGEN REDUCING}

The present invention relates to a composition for alleviating and improving symptoms caused by a decrease in estrogen, comprising as an active ingredient an extract of Colpomenia sinuosa or Colpomenia peregrina Sauvageau, which has the effect of improving the decrease in estrogen levels.

In women, when estrogen levels decrease due to decreased ovarian function, various symptoms appear along with the decrease in estrogen levels in the body. Representative examples include facial flushing, vaginal dryness, sleep disorders, psychological symptoms such as depression and anxiety, and cardiovascular disease. and the risk of chronic diseases such as osteoporosis increases. It is known that more than 50% of women during a period when estrogen levels decrease experience symptoms such as vaginal dryness and itching due to urogenital atrophy, and 75% of women during a period when estrogen secretion ceases experience vasomotor symptoms, especially facial flushing. Most women experience it for about 1-2 years, and symptoms may persist for 10 years or more. In addition, the menstrual cycle gradually becomes irregular and the amount of bleeding begins to change, and various negative physical changes are experienced. After the period when estrogen secretion stops, the phenomenon of osteoporosis due to bone loss becomes noticeable.

Hormone replacement therapy, including synthetic estrogen, is being used to reduce these problems after the secretion of estrogen is stopped. However, in long-term follow-up studies, it is reported that it actually increases not only breast cancer and venous thromboembolism, but also coronary artery disease and stroke. , In the case of representative calcium preparations, if more calcium is consumed than necessary, urinary stones may occur due to increased calcium excretion, so caution is required if there is a history of urinary stones. In addition, estrogen, a representative drug used as a bone resorption inhibitor, raises the risk of coronary artery disease and stroke, as well as a risk to the cardiovascular system. Calcitonin requires injection or nasal spray and is resistant to long-term use. This can occur, so a break of several months is needed. Bisphosphonate, a representative treatment, can cause gastrointestinal symptoms such as nausea, vomiting, and indigestion, and amino-bisphosphonate can cause esophagitis, so take it in a large cup with at least a glass of plain water and avoid lying down for at least 30 minutes to 1 hour. Caution is required when taking it. In the case of Strontium, which was recently developed and widely used as a treatment, the most common side effects are diarrhea and creatinine kinase levels exceeding twice the upper limit of normal are observed in some patients. Therefore, it is necessary to suggest a management plan that can alleviate symptoms caused by the reduction or cessation of estrogen, which is an important factor in reducing women's quality of life, and prevent the occurrence of chronic diseases, and prevent side effects from such synthetic hormone treatment. There is an increasing need to develop materials for improving disorders caused by the reduction or cessation of estrogen derived from natural products that can be reduced.

On the other hand, Colpomenia sinuosa and Colpomenia peregrina Sauvageau are brown algae of the Cypress family and are native seaweeds widely distributed on Jeju Island and throughout the country in Korea. They are spherical or irregular in shape, about 4 to 10 cm in diameter, and have a body structure. The cortex is made of 1-2 layers of square or polygonal cells, and the inner layer is made of 2-5 layers of round cells. Recently, continuous research is being conducted on the feasibility of using Bulegimal and Banjil Bulegimal.

1. KR 10-2012-0091995 A

The present inventors have made efforts to find marine biological resources that are effective in preventing and treating symptoms caused by reduction or cessation of estrogen levels, and as a result, extracts from seaweeds such as Colpomenia sinuosa and Colpomenia peregrina Sauvageau are not toxic. The present invention was completed by confirming that it had the effect of increasing estradiol levels in an estradiol-specific cell model.

One object of the present invention is to provide a pharmaceutical composition for controlling, preventing, improving, or treating estrogen levels, which contains extracts of the horse vulgaris or banjil vulgaris as an active ingredient.

Another object of the present invention is to provide a use for the composition.

Another object of the present invention is to provide a method of treating decreased estrogen levels, comprising administering the pharmaceutical composition to a subject suspected of having decreased estrogen levels.

Another object of the present invention is to provide a food composition for controlling, preventing, or improving the decrease in estrogen levels, which contains extracts of the vulgaris or banjil vulgaris as an active ingredient.

Another object of the present invention is to provide a quasi-drug composition for controlling, preventing, or improving the decrease in estrogen levels, which contains extracts of the horse vulgaris or banjil vulgaris as an active ingredient.

The present invention relates to a pharmaceutical composition for controlling, preventing, ameliorating or treating symptoms related to decreased estrogen levels, which contains an extract of Malt vulgaris or Banjil vulgaris as an active ingredient, and has an effect of improving estrogen levels in an estradiol-specific cell model. It can be usefully used. In particular, unlike some seaweed extracts, the extract of the seaweed or banjil seaweed has the advantage of being non-toxic and derived from a natural product, so that it can be used to treat the above diseases without side effects.

Figure 1 shows the results of confirming the cytotoxicity (A) and changes in estrogen activity (B) of extracts of Bolegifolia or Banjil Bolegifolia depending on the ethanol content.
Figure 2 confirms the effect of the extract of vulgaris horse on phosphorylation regulation of estrogen signaling molecules, with Figure 2 (A) confirming this in MCF-7 cells and Figure 2 (B) confirming this in T47D cells.
Figure 3 shows the results of confirming the change in body temperature that appears when an animal model is treated with Banjilbulregi extract, etc.
Figure 4 shows the results of an experiment related to bone metabolism in an ovariectomized female menopausal animal model according to the treatment concentration of the Banjilbulae horse extract, and Figure 4 (A) shows the femur micro bone metabolism in the ovariectomized female menopausal animal model according to the treatment concentration of the Banjil bullae horse extract. CT image, Figure 4 (B) is bone volume/total volume (BV/TV), Figure 4 (C) is trabecular thickness (Tb.Th), Figure 4 (D) is trabecular number (Tb.N), and Figure 4 (E) shows the measurement results of trabecular bone mineral density (BMD).
Figure 5 shows the results of confirming changes in bone metabolism biomarkers in a female menopausal animal model according to the treatment concentration of Banjilbulregi horse extract. Specifically, Figure 5 (A) shows the results of ELISA analysis of RANKL, Figure 5 (B) shows osteoprotegerin (OPG), and Figure 5 (C) shows the RANKL/OPG ratio.
Figure 6 shows the results of confirming changes in bone metabolism biomarkers and hormonal changes in a female menopausal animal model according to the treatment concentration of Banjilbulregi horse extract. Specifically, Figure 6 (A) shows the ELISA analysis results at the main biomarker analysis stage of alkaline phosphatase (ALP) and Figure 6 (B) CTX, and Figure 6 (C) shows the ELISA analysis of follicle stimulating hormone (FSH). It shows the results.
Figure 7 relates to the activity of serotonin and norepinephrine, which are neurotransmitters, in the entire brain of an ovariectomized mouse model of the extract of the end of Banjilbulregi by concentration.
Figure 8 shows the results of comparing the activity of extracts from Malt vulgare or Banjil vulgaris and other brown algae extracts.

One aspect of the present invention for achieving the above object provides a pharmaceutical composition for controlling, improving, or treating a decrease in estrogen levels, which contains an extract of the vulgaris or banjil vulgaris as an active ingredient, and a use of the composition.

In the present invention, the term “ Colpomenia sinuosa ” is a brown algae of the Colpomenia family and has been reported to be effective for metabolic diseases such as diabetes, hyperlipidemia, and obesity. However, the Colpomenia sinuosa extract is used for controlling and treating decreased estrogen levels. There is nothing known about it, and it was first identified by the present inventors. In the present invention, the Buleggi mal can be purchased commercially or used when collected from nature.

In the present invention, the term " Colpomenia peregrina Sauvageau" is a brown algae of the Cypress family and has been reported to be effective in preventing and treating sarcopenia or treating diabetes. However, there is no known use of the Banjilbulregi extract for controlling or treating decreased estrogen levels, and it was first identified by the present inventors. In the present invention, the vulgaris can be purchased commercially or collected from nature.

In the present invention, the term "Bulegimal or Banjilbulegimal extract" refers to an extract obtained by extracting Bulleggimal or Banjilbulegimal. The extract refers to all the results obtained by extracting and processing the dried or half-baked dried fruit, such as extract, diluted or concentrated liquid of the extract, dried product obtained by drying the extract, crude or purified extract, or mixtures thereof, etc. , includes the extract itself and all formulations of the extract that can be formed using the extract.

The extract of the dried vulgaris or semi-alcoholic malts is about 2 to 20 times the weight of the dried quince or semi-porous quince, specifically, about 3 to 5 times the volume of water, methanol, ethanol or butanol with a carbon number of 1 (C). A polar solvent of lower alcohols ₁ ) to 4 (C ₄ ) or a mixed solvent thereof can be used as an elution solvent. The elution solvent may be specifically 30 to 95% ethanol, 30 to 90% ethanol, or 30 to 80% ethanol, more specifically 30 to 70% ethanol, even more specifically 50 to 70% ethanol, Most specifically, 70% ethanol can be used.

The extraction may be performed at 20 to 100°C, specifically at room temperature, with an extraction period of about 4-12 hours to 4-7 days, using extraction methods such as hot water extraction, cold immersion extraction, reflux cooling extraction, or ultrasonic extraction. , any method for controlling the decrease in estrogen levels or extracting substances with therapeutic activity can be used without limitation. Specifically, extraction is performed 1 to 5 times in succession, filtered under reduced pressure, and the filtered extract is concentrated under reduced pressure using a vacuum rotary concentrator at 20 to 100°C, more specifically at room temperature, to produce a vulcanic substance soluble in water, lower alcohol, or a mixed solvent thereof. It may be the result of obtaining an extract of Gigi or Vanjilbulae Gigi, but is not limited thereto, as long as it can control and treat the effect of reducing estrogen levels according to the present invention, and may be obtained by drying the extract, diluted or concentrated extract of the extract, or dried extract. This includes all dried products obtained, or their crude or purified products. The extracts of the Malt officinalis or Banjil of the Extract can be extracted from natural, hybrid, or varietal plants, and can be used alone or in combination with other pharmaceutically active extracts, fractions, or compounds, as well as in appropriate combinations. In an example of the present invention, ethanol was added to powdered or semi-powdered bulaegi and extracted (Example 1).

The concentration of the extract of the Malt vulgaris or the Malt vulgaris may be 10 to 80 μg/ml, specifically 10 to 60 μg/ml, and more specifically, 10 μg/ml, 30 μg/ml, or 60 μg/ml. At extract concentrations outside the above range, the desired effect cannot be achieved.

The composition of the present invention may include extract of the extract of the extract of the extract of the extract of the extract of the extract of the extract of the extract of the extract of the extract of the extract of the extract of the extract of the extract of the extract of the extract or mixtures thereof. The composition may specifically include Banjilbulregi mal extract. In one embodiment of the present invention, it was confirmed that the extract of Banjilbulregi malae has an estrogen activity effect that is about 5 times more excellent than that of other brown algae such as seaweed and kelp. In particular, it was confirmed that the Banjilbulgi extract had an excellent effect in improving estrogen levels even compared to the Banjilbulgi extract extracted with the same ethanol concentration (see Figure 8).

In the present invention, the term "prevention" refers to all actions that control changes in estrogen levels or delay the onset of changes by administering the composition, and "treatment" refers to improving symptoms caused by changes in estrogen levels by administering the composition. It refers to all actions that are beneficially changed.

In the present invention, the term 'estrogen level reduction' refers to a disease related to a decrease in estrogen level, and specifically refers to symptoms or diseases caused by menopause, endometriosis, uterine fibroids, uterine leiomyosarcoma, endometrial cancer, uterine cancer, ovarian cancer, breast cancer, It may mean any one or more of uterine bleeding, vaginal bleeding, menorrhagia, cervical intraepithelial neoplasia, adenomyosis, and menopausal metabolic bone disease. More specifically, it may mean menopausal metabolic bone disease.

Additionally, the composition of the present invention can be used to improve, prevent, or treat female menopausal symptoms or diseases related to decreased estrogen levels. The above female menopausal symptoms or diseases may specifically be diseases caused by decreased estrogen secretion, for example, facial flushing, sweating, heart palpitations, dizziness, tinnitus, high blood pressure, obesity, hyperlipidemia, digestive disorders, headaches, memory disorders, and depression. One or more days selected from the group consisting of fatigue, anxiety, nervousness, sleep disturbance, decreased libido, vaginal infection, vaginal atrophy, vaginal dryness, vaginitis, cystitis, pain during urination, urgent urination, skin atrophy, dry skin, muscle pain, joint pain, and osteoporosis. You can.

The pharmaceutical composition containing the extract of the anthurium vulgaris or the anthurium vulgaris of the present invention as an active ingredient may further include appropriate carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. Specifically, it can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, and sterile injectable solutions. At this time, the content of the Banjilbulregi extract included in the composition is not particularly limited, but may be included in 0.0001 to 10% by weight, more specifically, 0.001 to 1% by weight, based on the total weight of the composition.

The pharmaceutical composition includes carriers, excipients, and diluents such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, and methyl. It may contain cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulating the composition, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and these solid preparations may contain one or more excipients, such as starch, calcium carbonate, It can be prepared by mixing sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral use may include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives are included. may be included. Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.

The composition of the present invention can be administered in a pharmaceutically effective amount.

In the present invention, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the individual, age, gender, and condition of the disease. It can be determined based on factors including the type, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and can be easily determined by a person skilled in the art. The preferred dosage of the composition of the present invention varies depending on the patient's condition and weight, degree of disease, drug form, administration route, and period, but for desirable effects, the extract of the Malt vulgaris or Banjyl vulgaris extract of the present invention should be administered in an amount of 0.0001 to 0.0001 per day. It can be administered at 1000 mg/kg, preferably at 0.001 to 1000 mg/kg, more specifically at 30 to 120 mg/kg, more specifically at 50 to 100 mg/kg, most specifically at 50 or 100 mg. /kg can be administered. Administration may be administered once a day, or may be administered several times. The composition can be administered to various mammals such as rats, livestock, and humans by various routes, and the method of administration includes without limitation any method conventional in the art, for example, orally, rectally, intravenously, intramuscularly, or intravenously. It may be administered by subcutaneous, intrauterine, intrathecal, or intracerebrovascular injection. The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. Usually, a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating it using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art. Alternatively, it can be manufactured by placing it in a multi-capacity container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, or capsule, and may additionally contain a dispersant or stabilizer.

In a specific example of the present invention, it was confirmed that the extract of the horse vulgaris or banjil vulgaris has the ability to change toxicity and estrogen activity (Figure 1).

Another aspect of the present invention provides a food composition for controlling, preventing, or improving a decrease in estrogen levels, comprising an extract of the end of the day or the end of the day as an active ingredient.

Specifically, the food composition according to the present invention can be formulated in the same way as the pharmaceutical composition and used as a health functional food or added to various foods.

When using the extract of the present invention as a food additive, the above-mentioned extract of Bolegifolia or Banjilberchia can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The amount of active ingredients mixed can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment), and foodologically acceptable food supplements may be additionally included. Since the composition of the present invention uses extracts derived from natural products as an active ingredient, there is no problem in terms of stability, so there is no significant limitation on the mixing amount. In addition, the food composition of the present invention can include all foods in the conventional sense, and can be used interchangeably with terms known in the art such as functional food and health functional food.

The term "functional food" in the present invention refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with Act No. 6727 on Health Functional Food, and "functional" refers to food that is useful for the human body. It means ingestion for the purpose of controlling nutrients for structure and function or obtaining useful health effects such as physiological effects. In addition, “health functional food” refers to a food manufactured or processed using specific ingredients as raw materials or by extracting, concentrating, refining, or mixing specific ingredients contained in food ingredients for the purpose of health supplementation. It refers to food designed and processed to fully exert bioregulatory functions on the living body, such as biodefense, regulation of biorhythm, prevention and recovery of disease, etc. The composition for health food has functions related to prevention of disease and recovery from disease. can be performed.

There are no restrictions on the types of food in which the composition of the present invention can be used. In addition, a composition containing the extract of the vulgaris root of the present invention or the root of the vulgaris extract of the present invention as an active ingredient can be prepared by mixing known additives with other appropriate auxiliary ingredients that may be contained in foods according to the selection of a person skilled in the art. Examples of foods that can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and There are vitamin complexes, etc., and they can be manufactured by adding them to juices, teas, jellies, juices, etc. prepared using the extract according to the present invention as a main ingredient.

In addition, foods that can be applied to the present invention include, for example, special nutritional foods (e.g., infant formula, infant and toddler food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g., ramen, noodles, etc.), and health supplements. , seasoned foods (e.g. soy sauce, soybean paste, red pepper paste, mixed paste, etc.), sauces, confectionery (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi, pickles, etc.) ), beverages (e.g. fruit, vegetable drinks, soy milk, fermented beverages, etc.), natural seasonings (e.g. ramen soup, etc.), etc.

When the health functional food composition of the present invention is used in the form of a beverage, it may contain various sweeteners, flavoring agents, or natural carbohydrates as additional ingredients like ordinary beverages. In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, and glycerin. , alcohol, and carbonating agents used in carbonated beverages. In addition, it may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks.

Another aspect of the present invention provides a quasi-drug composition for controlling, preventing, or improving a decrease in estrogen levels, comprising an extract of the end of the day or the end of the day as an active ingredient.

In the present invention, the term "quasi-drug" refers to fibers, rubber products, or similar products used for the purpose of treating, alleviating, treating, or preventing diseases in humans or animals, or devices that have a weak effect on the human body or do not directly act on the human body, or devices. Or, it is an article that is not a machine or similar, or an agent used for sterilization, insecticide, and similar purposes to prevent infection, and is used for the purpose of diagnosing, treating, alleviating, treating, or preventing diseases in humans or animals. It refers to articles other than instruments, machines, or devices among articles used for the purpose of having a pharmacological effect on the structure and function of humans or animals, excluding articles for external use on the skin and personal hygiene. Also includes supplies.

When added to a quasi-drug composition for the purpose of controlling, preventing, or improving the decrease in estrogen levels, which contains the extract of the end of the perennial or half-drug of the present invention as an active ingredient, the extract may be added as is or together with other fractions or other quasi-drug ingredients. It can be used, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use.

The skin external preparation is not particularly limited thereto, but is preferably prepared and used in the form of ointment, lotion, spray, patch, cream, powder, suspension, gel or gel. The personal hygiene products are not particularly limited, but are preferably soap, cosmetics, wet tissues, toilet paper, shampoo, skin cream, face cream, toothpaste, lipstick, perfume, makeup, foundation, blush, mascara, eye shadow, and sunscreen. It may be a lotion, hair care product, air freshener gel, or cleaning gel. Additionally, other examples of the quasi-drug composition of the present invention include disinfectant cleaners, shower foams, garnishes, wet tissues, detergent soaps, hand washes, humidifier fillers, masks, ointments, or filter fillers.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrating the present invention in more detail, and the scope of the present invention is not limited to these examples.

Example 1: Preparation of extract from the end of the vulgaris or the end of the vultures

Buleggi Mal or half-jilbulgi Mal was washed thoroughly with water, dried in the shade, and then powdered with Waring brand. Add 500 g of powdered or semi-powdered dried ginseng into 5 liters of ethanol, extract at 70°C for 5-10 hours through an extractor, filter under reduced pressure with filter paper (Whatman, USA), and then extract the filtered extract at room temperature using a vacuum rotary concentrator. After removing the ethanol solvent, the extracted residue was obtained as an extract of vulgaris mal or banjil vulgaris mal.

Experimental Example 1: Confirmation of cytotoxicity and estrogen activity of the extract according to the change in ethanol content of Bolegioma or Banjilbeogi.

To confirm toxicity, T47D-KBluc cells were seeded at 3Х10 ⁴ cells/well in a 96 well plate and left overnight. Afterwards, the extract was treated for 24 hours and reacted with MTT reagent for 3 hours, the supernatant was removed, the resulting salt was dissolved in DMSO, and the absorbance was measured. The T47D-KBluc cell line was used to evaluate the estrogen activity of the extract from Example 1. The stabilized cells were replaced with assay media before seeding, cultured in an incubator for 24 hours, and seeded at 3x10 ⁴ cells/well in a 96 well plate overnight. Afterwards, the cells were treated with the extract of the lysicum or seminiferous root, cultured in the incubator for another 24 hours, removed the supernatant, lysed for 30 minutes with 60 μl/well of 1X lysis buffer, and treated with 25 μl of cell lysate on a 96 well white plate using a luminescent microplate reader. detect. At this time, the conditions are set to substrat volume of 25 μl, duration of 1 s, and counting time of 2 s.

As a result, as confirmed in Figure 1, no toxicity was observed in the extracts of the extract of the extract of the extract of the extract of the extract of the extract of the extract of the extract of the ethanol content at any ethanol content (Fig. 1 (A)). However, it was confirmed that there was a large change in estrogen activity in the extract or half-length vulgaris extract extracted using ethanol at a concentration of 70% or higher (Figure 1 (B)).

Experimental Example 2: Confirmation of the effect of phosphorylation regulation of estrogen signaling molecules by Extract of the vulgaris root

To measure the phosphorylation of signaling molecules, MCF-7 cells and T47D cells were seeded in a 6-well plate at 1 Х10 ⁶ cells/well, stabilized, replaced with serum free medium, and cultured for 12 hours. Afterwards, the extract was treated for 15 and 30 minutes and harvested. Proteins were extracted and quantified from the obtained cells, and phosphorylation of Akt and Erk was confirmed through Western blot.

As a result, phosphorylation of Akt and Erk molecules in the vulgaris extract was confirmed in MCF-7 (Figure 2(A)) and T47D cells (Figure 2(B)). It was confirmed that the phosphorylation of Akt was increased by treatment with the vulgaris extract. However, the phosphorylation of Erk decreased at low concentration compared to the control group, but it was confirmed that the phosphorylation of Erk tended to increase in the high concentration group.

Experimental Example 3: Changes in body temperature in animal models caused by Banjilbulregi horse extract

To evaluate the effect of Banjilbulregi mal extract on alleviating menopausal symptoms, an ovariectomy model using C57BL/6 mice was used. The ovariectomy model can induce menopause because there are no hormones secreted by the ovaries. Six-week-old C57BL/6 mice had a recovery period for one week after ovariectomy, and then menopause was induced for six weeks.

Mice in which menopause was induced were orally administered ethanol extract of the quinceafolium and estradiol every day for 6 weeks, and their body temperature was measured every week.

As a result, when measuring the body temperature of the lower abdomen in the animal model, the overall body temperature was found to be higher in the extract-treated group compared to the OVX group (Figure 3). This means that the metabolic rate increases compared to the OVX group.

Experimental Example 4: Image analysis related to bone metabolism of Banjilbulregi extract

The femur of the menopausal model mouse prepared in Experimental Example 3 was removed. The extracted femur was fixed in 10% formalin for 24 hours and placed in a polystyrene tube filled with PBS for micro CT analysis. The analysis conditions are as follows. Axial rotation was measured up to 360.36° in 0.42° increments, and a 0.5mm aluminum filter was used. The voltage and current were 88 kV and 112 μA to obtain an image with 9 μm resolution (Figure 4(A)). As a result, compared to the state where the inside of the bone was empty due to ovariectomy, the phenomenon of internal bone filling was confirmed through treatment with Banjilbulregi mal extract.

In addition, the obtained image was analyzed between 0.5mm and 2.5mm below the growth plate of the femur through an analysis program to determine bone volume/total volume (BV/TV) (Figure 4(B)) and trabecular thickness (Tb.Th) (Figure 4(B)). C)), trabecular number (Tb.N) (Figure 4(D)), and trabecular bone mineral density (BMD) (Figure 4(E)) were confirmed. As a result, it was confirmed that the thickness, content, and density increased when treated with the Banjilbulregi extract.

Experimental Example 5: Confirmation of the alleviating effect related to bone metabolism biomarkers through serum analysis of ovariectomized female menopausal animal model of Banjilbulregi horse extract

Blood was collected from the heart of the menopausal model mouse prepared in Experimental Example 3, the collected blood was processed in a SST (serum separate tube), and then centrifuged at 5000 RPM for 5 minutes to obtain serum. CTX (c-terminal telopeptide) bone resorption marker and ALP (alkaline phosphatase) bone formation marker in serum were measured using immunoassay. This is an experiment in which the marker molecules present in the sample bind to a plate coated with an antibody that recognizes each specific marker, and the antibody bound to HRP (horseradish peroxidase) binds to the marker molecules remaining in the plate and measures them. did.

Results of ELISA analysis of RANKL (FIG. 5(A)), osteoprotegerin (OPG) (FIG. 5(B)), and RANKL/OPG ratio (FIG. 5(C)). In the analysis step of major biomarkers related to bone formation, the extract The control effect was confirmed through increase and decrease during treatment.

Experimental Example 6: Confirmation of alleviating effect and hormonal changes related to bone metabolism biomarkers through serum analysis of ovariectomized female menopausal animal model of Banjilbulregi horse extract

Serum analysis was performed in the same manner as in Experimental Example 5.

As a result, as shown in Figures 6 (A) and (B), it was confirmed that the levels of ALP and CTX in the serum of mice whose ovaries were removed were increased compared to mice whose ovaries were not removed. This is a symptom of osteoporosis and the microstructure of the bone deteriorates due to increased bone turn over. When ovariectomized mice were treated with Banjilbulregi mal extract, the levels of ALP and CTX were confirmed to decrease compared to untreated mice. In particular, it was confirmed that at high concentrations (100 mg/kg - less than 500 mg when applied to the human body), it exhibited a similar effect to the activity of estradiol, a comparison group.

In addition, as a result of ELISA analysis of follicle stimulating hormone (FSH), it was confirmed that the follicle stimulating hormone (FSH) decreased compared to the ovariectomized group by treatment with the Banjilbulregi mal extract (Figure 6 (C)).

Experimental Example 7: Confirmation of the improvement (increase) effect of Banjilbulregi horse extract related to neurotransmitter biomarkers in an ovariectomized female menopausal animal model

The brain extracted from the menopausal model mouse prepared in Experimental Example 3 was pulverized in lysis buffer using a homogenizer and then centrifuged at 12000 RPM for 10 minutes to obtain mouse brain lysate, which was quantified to confirm protein concentration. In addition, the concentrations of norepinephrine and serotonin, which are neurotransmitters in the brain lystate, were measured using an immunoassay and then normalized to the protein concentration of the previously obtained mouse brain lysate.

As a result, it was confirmed that the levels of norepinephrine (Figure 7(A)) and serotonin (Figure 7(B)) in the serum of mice whose ovaries were removed were decreased compared to mice whose ovaries were not removed. And when mice with ovaries removed were treated with Banjilbulregi mal extract, the levels of norepinephrine and serotonin were confirmed to increase compared to untreated mice.

Experimental Example 8: Comparison of activity with other brown algae

The same method as the estrogen activity measurement in Experimental Example 1 was performed.

As a result, as can be seen in Figure 8, when a comparative estrogen activity test was conducted with kelp and seaweed among other brown algae and the brown algae used in this study, the estrogen and half-colored algae showed estrogen activity compared to other materials. It was confirmed that this was very good in a concentration-dependent manner, and in particular, it was confirmed that Banjilbulegimal had superior estrogen activity compared to Buleggimal.

Taken together, the above results suggest that the Banjilbuljeong extract of the present invention can effectively prevent or treat decreased estrogen levels, such as female menopausal symptoms.

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Claims (7)

A pharmaceutical composition for preventing or treating diseases related to decreased estrogen levels, comprising extract of Banjilbullegicum as an active ingredient,
The diseases associated with decreased estrogen levels include endometriosis, uterine fibroids, uterine leiomyosarcoma, endometrial cancer, uterine cancer, ovarian cancer, breast cancer, uterine bleeding, vaginal bleeding, menorrhagia, cervical intraepithelial neoplasia, adenomyosis, or menopausal metabolic bone disease. A pharmaceutical composition.
The pharmaceutical composition according to claim 1, wherein the extract is extracted with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.
The pharmaceutical composition according to claim 2, wherein the lower alcohol is methanol, ethanol, or butanol.
The pharmaceutical composition according to claim 1, wherein the extract is extracted with 30 to 95% ethanol.
The pharmaceutical composition according to claim 1, wherein the extract has a concentration of 10 to 80 μg/ml.
The pharmaceutical composition according to claim 1, wherein the dosage of the extract is 30 to 120 mg/kg.
A food composition for preventing or improving diseases related to decreased estrogen levels, comprising extract of Banjilbulregi horse as an active ingredient,
The diseases associated with decreased estrogen levels include endometriosis, uterine fibroids, uterine leiomyosarcoma, endometrial cancer, uterine cancer, ovarian cancer, breast cancer, uterine bleeding, vaginal bleeding, menorrhagia, cervical intraepithelial neoplasia, adenomyosis, or menopausal metabolic bone disease. A food composition that is.