KR102127712B1 - Composition for the preventing and treating female menopausal disease comprising the extract of Sophora japonica L. fruits - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of climacteric disease in women containing the extract of lumps as an active ingredient, more specifically, genistein diglucoside, sophoravioside, and sophoricoside 1: 2.5 to 3.5: 3.5 It relates to a pharmaceutical composition and a food composition for the prevention or treatment of climacteric disease of a woman comprising a lump extract containing as a weight ratio of ~4.5 as an active ingredient. The ingot extract having a specific component ratio of the present invention has little cytotoxicity, is safe, and has excellent antioxidant and anti-inflammatory effects compared to other solvent extracts, so as a pharmaceutical composition and food composition for preventing, improving or treating climacteric disease in women It can be useful.

Description

Composition for the preventing and treating female menopausal disease comprising the extract of Sophora japonica L. fruits}

The present invention relates to a composition for the prevention and treatment of climacteric disease in women containing the extract of lumps as an active ingredient, more specifically, genistein diglucoside, sophoravioside, and sophoricoside 1: 2.5 to 3.5: 3.5 It relates to a pharmaceutical composition and a food composition for the prevention or treatment of climacteric disease of a woman comprising a lump extract containing as a weight ratio of ~4.5 as an active ingredient.

Menopause refers to the end of menstruation due to gradual deterioration of ovarian function. At this time, hormonal changes such as irregular menstrual cycle and decreased estrogen and increased follicle stimulating hormone appear as major signals. It is known to increase the risk of osteoporosis by accelerating it, and in normal body state, estrogen inhibits bone resorption and increases the amount of osteoclast precursors in the bone marrow, thereby promoting a decrease in inflammatory cytokines that increase bone resorption. have.

With the recent development of civilization and advances in science, humans living in the modern age have been gradually extended, and in the case of women, the average life expectancy is 80.4 years in 2002. Need special attention to healthy life. Estrogen replacement therapy has been mainly used to improve menopausal symptoms as a treatment, since the biggest cause of menopausal symptoms is lack of estrogen. However, only 35-40% of women with menopausal symptoms use estrogen replacement therapy, even most of them avoid using this method continuously. This is because estrogen replacement therapy is based on artificial hormone administration, which is followed by adverse reactions and side effects. As a side effect of estrogen replacement therapy, many studies have reported that the risk of uterine bleeding, stroke, heart attack, breast cancer, and uterine cancer may be increased.

Because of these problems, interest in replacing estrogen therapy with natural methods taken in food form is increasing, and there is a need for the development of a new menopausal symptom improvement agent that has an excellent effect of alleviating symptoms of menopause without side effects. Research to find natural plant components such as phytoestrogen as a substitute has been actively conducted.

Among isoflavones, especially genistein and daidzein have attracted attention for their anticancer and antioxidant effects, and are highly anticipated for the prevention and treatment of various cancers as well as various adult diseases. It has been found to inhibit the action of enzymes involved in the proliferation of cancer cells, and in particular, the possibility of inhibiting carcinogenesis such as inhibition of prostate cancer has been reported in various aspects. In addition, studies have shown that it inhibits the development of breast cancer cells that require estrogen activity by weakly binding to the estrogen receptor.

Since genistein, which is a non-glycoside form that exhibits physiological activity, is contained in a very small amount in the ingot, it is necessary to convert the glycoside form of genistein in the ingot to the non-glycoside genistein form in the body. It took a lot of time and there was a need for a technique to ingest the glycosides itself and allow the body to convert and absorb naturally.

Some researchers have developed a technique for converting sophoricosides into non-glycoside genistein in vitro using microorganisms, but these techniques need to undergo additional processes using yeast and lactic acid bacteria (World J Microbiol Biotechnol (2015) ) 31:187--197)

The glycoside form of genistein is known to be naturally converted to genistein by about 10% in the small intestine when ingested, and the rest is converted to genistein by microorganisms in the large intestine. (Journal of Nutritional Biochemistry 17 (2006) 257-264)

In addition, non-glycoside genistein is rapidly absorbed in the small intestine, but has low bioavailability due to low solubility in water. Therefore, it has been reported that it is excellent in terms of bioavailability to induce conversion in the body after ingesting the glycoside of genistein (International Journal of Pharmaceutics 337 (2007) 148-154).

However, the results of profiling what type of glycoside of genistein is present in the ingot extract have not been found, and a study on the optimal ratio of profiled glycosides present in the ingot extract showing excellent effects against female menopausal disease Also, no progress has been made.

As a result, the present inventors profiled the glycoside form converted to genistein in the lump extract, and identified genistein diglucoside, sophoravioside, and sophoricoside, and these specific glycosides (genistein diglucoside, sophoravioside) , And Sopolicoside), when having a specific component ratio and more than a specific content, the snail extract that satisfies such characteristics was confirmed that the improvement and treatment of women's menopausal disease is excellent compared to other sgot extracts. Completed.

Therefore, the object of the present invention, the genistein diglucoside, sophoravioside, and sophoricoside containing 200mg / g or more, genistein diglucoside, sophoravioside, and sophoricoside 1: 1: 2.5 ~ 3.5: It is to provide a pharmaceutical composition for the prevention or treatment of climacteric disease, including women in a weight ratio of 3.5 to 4.5.

Another object of the present invention, containing genistein diglucoside, sophoravioside, and sophoricoside 200mg / g or more, genistein diglucoside, sophoravioside, and sophoricoside 1: 2.5 to 3.5: It is to provide a food composition for preventing or improving menopausal disease in women, comprising a weight ratio of 3.5 to 4.5.

Another object of the present invention, the genistein diglucoside, sophoravioside, and sophoricoside containing 200mg / g or more, genistein diglucoside, sophoravioside, and sophoricoside 1: 2.5 to 3.5 : To provide an antioxidant food composition containing an ingot extract contained in a weight ratio of 3.5 to 4.5 as an active ingredient.

Another object of the present invention, the genistein diglucoside, sophoravioside, and sophoricoside containing 200mg / g or more, genistein diglucoside, sophoravioside, and sophoricoside 1: 2.5 to 3.5 : It provides an anti-inflammatory food composition or a pharmaceutical composition containing an ingot extract containing a weight ratio of 3.5 to 4.5 as an active ingredient.

In order to achieve the above object, it contains genistein diglucoside, sophoravioside, and sophoricoside at least 200mg/g, and genistein diglucoside, sophoravioside, and sophoricoside 1:2.5~ 3.5: provides a pharmaceutical composition for the prevention or treatment of menopausal diseases, including in a weight ratio of 3.5 to 4.5.

In order to achieve another object of the present invention, it contains at least 200mg/g of genistein diglucoside, sophoravioside, and sophoricoside, and genistein diglucoside, sophoravioside, and sophoricoside are 1:2.5. ~ 3.5: provides a food composition for preventing or improving menopausal disease, including in a weight ratio of 3.5 to 4.5.

In order to achieve another object of the present invention, the present invention contains genistein diglucoside, sophoravioside, and sophoricoside at least 200 mg/g, genistein diglucoside, sophoravioside, and sophoricoside It provides a food composition for antioxidant comprising a lump extract containing as a weight ratio of 1: 2.5 ~ 3.5: 3.5 ~ 4.5 as an active ingredient.

In order to achieve another object of the present invention, the present invention contains genistein diglucoside, sophoravioside, and sophoricoside at least 200 mg/g, genistein diglucoside, sophoravioside, and sophoricoside It provides a food composition for anti-inflammatory, or a pharmaceutical composition comprising a lump extract containing as a weight ratio of 1: 2.5 to 3.5: 3.5 to 4.5 as an active ingredient.

Hereinafter, the present invention will be described in detail.

The ingot (Sophora japonica L.) is the fruit of the painting tree, which is a deciduous tree belonging to the legume family. As a country of origin, China and China are distributed all over the country, and are used for ornamental, industrial, edible, and medicinal purposes. The main components of the ingot are nine flavonoids and isoflavonoid compounds, which include sophoraflavonolosidegenistein, sophorabioside, kaemprerol, and so on. Rutin, and glucoside-C, etc., and the content of rutin in young fruit reaches 1.76%.

The ingot extract of the present invention is characterized in that it contains genistein diglucoside, sophoravioside, and sophoricoside in a weight ratio of 1: 2.5 to 3.5: 3.5 to 4.5, and also genistein diglucoside and sophoravioside. And, characterized in that it contains more than 200mg / g of sophoricoside.

In this regard, the present inventors have confirmed that in one embodiment, the ingot extract having the weight ratio and content of the components is safe without cytotoxicity, and specifically exhibits the best antioxidant and anti-inflammatory effects. In addition, it was confirmed that it exhibits a remarkably excellent preventive and/or therapeutic effect on various symptoms of climacteric disease in vivo .

In a further preferred embodiment, the ingot extract of the present invention may contain an isoflavone glycoside of 300 mg/g or more, and preferably contain 300 mg/g to 400 mg/g. The isoflavone glycoside is composed of genistein diglucoside, sophoravioside, sophoricoside, glycoside camphorol-glucoside, and sophoraflavonoloside represented by the following formula.

The genistein diglucoside (Genistein 4'-O,7-diglucoside) may be preferably represented by the following formula (1).

[Formula 1]

The sophoravioside (sophorabioside) may be preferably represented by the following formula (2).

[Formula 2]

The sophoricoside may preferably be represented by Formula 3 below.

[Formula 3]

The glycoside camphorol glucoside may be preferably kaempferol-3-o-beta-d-glucopyranoside-(1→3)β-D-glucopyranosyl-7-O-α-rhamnopyranoside.

The ingot extract of the present invention may include a pre-treatment process for the raw material in order to increase the extraction efficiency in the manufacture of the extract, for example, after drying the ingot can be used in detail.

The ingot extract may be extracted by a known natural product extraction method. For example, it can be extracted by cold immersion extraction, room temperature extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, and heat extraction, and can be repeatedly extracted once to 10 times.

The extract of the present invention may be extracted using one or more selected from the group consisting of water and an organic solvent having 1 to 6 carbon atoms. In the present invention, the extraction solvent may be edible singular or plural types of solvents, but preferably water, alcohol, and alcohol having 1 to 6 carbons [methanol (C1), ethanol (C2), propanol (C3), butanol ( C4), pentanol (C5), hexanol (C6)]. In a more preferred embodiment, in the present invention, at least one selected from the group consisting of water, alcohol, and alcohol having 1 to 4 carbons [methanol (C1), ethanol (C2), propanol (C3), butanol (C4)] It may be used as.

More preferably, it may be to use alcohol. The alcohol may preferably have a concentration of 1% (v/v) to 99% (v/v), more preferably a concentration of 40% (v/v) or more, and most preferably 50 % (v/v) to 70% (v/v).

The extract of the present invention can be used as a liquid by filtration and/or concentration, and can be used by solidifying through a common drying process such as spray drying or freeze drying. In the drying process, dextrin may be mixed and dried before spray drying or freeze drying.

The disease targeted in the present invention, menopause (climacterium), also referred to as menopause, refers to the period of menstrual abolition as a sign of loss of reproductive function in women. During menopause, the amount or cycle of menstruation becomes irregular due to a lack of ovarian function, menstruation is abolished due to the decrease in follicular hormone (estrogen) over several months to 3 years, and it acts on the autonomic nervous center of the brain, causing the autonomic nervous system to fail. Causes menopausal disorder. In addition, the adrenal cortex function is enhanced by the adrenal cortex function, showing masculinity, and the effects of thyroid hormone cause abnormalities in the thyroid function, leading to obesity. For example, hot flashes (一過性熱感), winter (motion: heartbeat is worse than usual), dizziness, tinnitus, high blood pressure, digestive problems, headaches, decreased memory, depression, etc. Symptoms of may appear.

Therefore, female menopausal diseases of the present invention include hot flashes, sweating, dry skin, vaginal dryness, vaginal atrophy, lower urethral atrophy, vaginitis, cystitis, urination, urinary distress, concentration disorder, short-term memory disorder, anxiety, nervousness, memory loss, It may be one or more selected from the group consisting of muscle pain, joint pain, and osteoporosis, but is not limited thereto.

In addition, the present invention contains at least 200mg/g of genistein diglucoside, sophoravioside, and sophoricoside, and genistein diglucoside, sophoravioside, and sophoricoside 1: 2.5-3.5: 3.5-4.5. It provides an antioxidant food composition comprising an ingot extract contained in a weight ratio of as an active ingredient.

The antioxidant means an effect of slowing, blocking or preventing the oxidation process. More specifically, it may mean an active oxygen species radical scavenging ability.

In addition, the present invention contains at least 200mg/g of genistein diglucoside, sophoravioside, and sophoricoside, and genistein diglucoside, sophoravioside, and sophoricoside 1: 2.5-3.5: 3.5-4.5. It provides an anti-inflammatory composition (food composition, or a pharmaceutical composition) containing the lump extract contained in a weight ratio of as an active ingredient.

In one embodiment, the present invention contains genistein diglucoside, sophoravioside, and sophoricoside at least 200 mg/g, and genistein diglucoside, sophoravioside, and sophoricoside 1: 2.5- 3.5: Provides a pharmaceutical composition for preventing and/or treating inflammation comprising an ingot extract contained in a weight ratio of 3.5 to 4.5 as an active ingredient.

In one embodiment, the present invention contains genistein diglucoside, sophoravioside, and sophoricoside at least 200 mg/g, and genistein diglucoside, sophoravioside, and sophoricoside 1: 2.5- 3.5: Provides a food composition for preventing and/or improving inflammation comprising an ingot extract contained in a weight ratio of 3.5 to 4.5 as an active ingredient.

In a further embodiment, the present invention contains genistein diglucoside, sophoravioside, and sophoricoside at least 200 mg/g, and genistein diglucoside, sophoravioside, and sophoricoside 1: 2.5- 3.5: It may be to provide a pharmaceutical composition for the prevention and / or treatment of inflammatory diseases, including as an active ingredient ingot extract containing a weight ratio of 3.5 to 4.5.

In another further embodiment, the invention contains at least 200 mg/g genistein diglucoside, sophoravioside, and sophoricoside, and genistein diglucoside, sophoravioside, and sophoricoside 1: 2.5 to 3.5: It may be to provide a food composition for the prevention and/or improvement of inflammatory diseases, including an ingot extract contained in a weight ratio of 3.5 to 4.5 as an active ingredient.

In the present invention, the inflammatory disease is not particularly limited in its kind, but in one embodiment, the inflammatory disease may be hepatitis, hepatitis, allergic rhinitis, coronary artery disease, rheumatoid arthritis, but is not limited thereto.

The pharmaceutical composition according to the present invention may contain the ingot extract of the present invention alone, or may further contain one or more pharmaceutically acceptable carriers, excipients or diluents. "Pharmaceutically acceptable" as used herein is a non-toxic composition that is physiologically acceptable and does not inhibit the action of the active ingredient when administered to humans and does not normally cause an allergic or similar reaction such as gastrointestinal disorders, dizziness, or the like. Says

Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, the carrier for parenteral administration may include water, a suitable oil, saline, aqueous glucose and glycol, and the like, and may further include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-parabens and chlorobutanol. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, etc. in addition to the above components. Other pharmaceutically acceptable carriers and formulations can be referenced as described in the following documents (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The composition of the present invention can be administered in any way to mammals, including humans. For example, it can be administered orally or parenterally. The parenteral administration method is not limited to this, but intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal administration. Can be

The pharmaceutical composition of the present invention may be formulated as a formulation for oral administration or parenteral administration according to the administration route as described above.

In the case of preparations for oral administration, the compositions of the present invention may be formulated using methods known in the art as powders, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. Can. For example, oral preparations can obtain tablets or dragees by combining the active ingredient with a solid excipient, then grinding it and adding a suitable adjuvant to the granule mixture. Examples of suitable excipients are sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including cellulose starch, wheat starch, rice starch and potato starch, cellulose, etc. Fillers such as cellulose, gelatin, polyvinylpyrrolidone and the like may be included, including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose. In addition, in some cases, crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may further include an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and a preservative.

For formulations for parenteral administration, injections, creams, lotions, external ointments, oils, moisturizers, gels, aerosols and nasal inhalants can be formulated by methods known in the art. These formulations are described in Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA, 1995, a prescription generally known to all pharmaceutical chemistries.

The total effective amount of the composition of the present invention can be administered to a patient in a single dose, and can be administered by a fractionated treatment protocol that is administered for a long time in multiple doses. The pharmaceutical composition of the present invention can vary the content of the active ingredient according to the degree of disease. Preferably, the preferred total dose of the pharmaceutical composition of the present invention may be about 0.01 μg to 1,000 mg per kg of body weight of the patient per day, most preferably 0.1 μg to 500 mg per kg of body weight per patient. However, the dosage of the pharmaceutical composition is determined by considering various factors such as the formulation method, the route of administration and the number of treatments, as well as the patient's age, weight, health status, sex, disease severity, diet and excretion rate, etc. Considering this, one of ordinary skill in the art will be able to determine an appropriate effective dosage of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, route of administration and method of administration as long as it shows the effects of the present invention.

Furthermore, the pharmaceutical composition of the present invention can be administered in parallel with a known compound having an effect of preventing and treating women's menopausal disease.

Furthermore, it can be preferably formulated in accordance with each disease or ingredient using methods disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA or by appropriate methods in the art.

The food composition of the present invention includes all forms of functional food, nutritional supplement, health food, and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

For example, as a health food, the ingot extract of the present invention may be prepared in the form of tea, juice, and drink to be consumed or granulated, encapsulated, and powdered. In addition, the ingot extract of the present invention can be prepared in the form of a composition by mixing with known substances or active ingredients known to be effective against female menopausal disease. For example, the food composition of the present invention may further contain trace minerals, vitamins, lipids, saccharides, and components having known anti-allergic activity, etc., in addition to the extract component of the ingot. The minerals may contain nutrients necessary for growth, such as calcium and iron, and vitamins may include vitamin C, vitamin E, vitamin B1, vitamin B2, and vitamin B6. Lipids may include alkoxyglycerol or lecithin, and sugars may include fructooligosaccharides.

In addition, functional foods include beverages (including alcoholic beverages), fruits and processed foods thereof (e.g. canned fruits, bottled foods, jams, marmalades, etc.), fish, meat and processed foods (e.g. ham, sausage corn beef) Etc.), breads and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), juice, various drinks, cookies, syrup, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein , Retort food, frozen food, various seasonings (eg, miso, soy sauce, sauce, etc.) can be prepared by adding the extract of the ingot of the present invention.

In addition, in order to use the ingot extract of the present invention as a food additive, it can be prepared and used in the form of a powder or a concentrate.

The preferred content of the ingot extract in the food composition of the present invention may be contained in a ratio of 0.01 to 90%, preferably 0.1 to 50%, based on the total weight of the food relative to the total weight of the food composition.

The ingot extract having a specific component ratio of the genistein glycoside according to the present invention as well as having a specific content or more has little cytotoxicity, is safe, and has excellent antioxidant and anti-inflammatory effects compared to other solvent extracts, thereby preventing female menopausal disease , It can be usefully used as pharmaceutical compositions and food compositions for improvement or treatment.

Figure 1 shows the results of LDH cytotoxicity test of the lump extract by solvent.
Figure 2 shows the results of the DPPH scavenging ability test of the solvent-specific ingot extract.
Figure 3 shows the results of the hydroxy radical scavenging ability test of the ingot extract for each solvent.
Figure 4 shows the result of analyzing the expression level of TGF-β, a bone formation promoting factor, by RT-PCR, by treatment with sgot water extract or sgot enzyme decomposition extract using the same (RG: sgot water extract treatment group, RA: sgot enzyme) Degradation extract treatment group, SS: soybean x powder treatment group, E: 17-β estradiol treatment group).
Figure 5 shows the results of confirming the pattern change in the trabecular bone area of the tibia by treatment with lump water extract or lump enzymatic decomposition extract using the menopausal animal model (ovary extraction mouse) (RG: lump water extract treatment) Group, RA: lump enzymatic decomposition extract treatment group, SS: soybean x powder treatment group, E: 17-β estradiol treatment group).
FIG. 6 shows the results of confirming the lumbar spinal column area change pattern by treatment with lump water extract or lump enzymatic decomposition extract using the same in menopausal animal model (ovary extraction mouse) (RG: lump water extract treatment group, RA: lump Enzymatic digestion treatment group, SS: soybean x powder treatment group, E: 17-β estradiol treatment group).
Figure 7 is a comparison of the ability to inhibit the expression of iNOS gene extract by solvent.
Figure 8 is a comparison of the ability to inhibit the expression of GM-CSF gene by lump extract by solvent.
Figure 9 shows the results of the hydroxy radical scavenging ability test of the lump extract by alcohol concentration.
Figure 10 shows the IL-6 gene expression inhibitory ability of the extract of the ingot by alcohol concentration.
11 shows the ability to inhibit the expression of nitric oxide synthase gene by ingot extract by alcohol concentration.
Figure 12 shows the results of the nitrogen monoxide production test of the ingot extract by alcohol concentration.
13 comparatively shows the effect of the ingot extract (Rex) peculiar to the present invention on the symptoms of menopausal vasomotor symptoms.
FIG. 14 is an experiment for examining the effect of the ingot extract (Rex) peculiar to the present invention on the menopausal urinary system epithelial cell change, and shows an image of vaginal epithelial cells observed.
FIG. 15 is a quantitation of the number of keratinocytes observed in FIG. 14, and a high proportion of keratinized epithelial cells in the vaginal mucosal cells is considered to decrease the level of vaginal keratinization and to make the vaginal mucosa flexible.
Figure 16 shows a comparative effect of the effect of the ingot extract (Rex) unique to the present invention on the ALT level change indicating hepatitis or fatty hepatitis during climacteric blood factor change.
FIG. 17 shows the effect of the ingot extract (Rex) unique to the present invention comparatively on the change in LDL level that interferes with blood circulation during climacteric blood factor changes.
Figure 18 shows the comparative effect of the ingot extract (Rex) unique to the present invention on the RANKL expression change among the major gene expression changes associated with menopausal bone disease.
Figure 19 shows the comparative effect of the lump extract (Rex) unique to the present invention on the TGF-β expression change, which is an osteoblast promoting factor, among the major gene expression changes associated with menopausal bone disease.
FIG. 20 shows comparatively the effect of the ingot extract (Rex) peculiar to the present invention in the change in Leptin expression related to obesity among changes related to menopausal metabolic disease.
FIG. 21 shows the effect of the ingot extract (Rex) unique to the present invention comparatively on the expression change of the inflammation-causing cytokine IL-6 among the changes related to menopausal metabolic disease.
FIG. 22 shows the effect of the ingot extract (Rex) unique to the present invention comparatively on the expression change of the inflammatory-causing cytokine TNF-α among the changes related to menopausal metabolic disease.

Hereinafter, the present invention will be described in detail.

However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Example 1: Analysis of the components of each solvent in the extract of ingot

The content of Genistein, which is a representative substance of isoflavone glycoside and non-glycoside Isoflavone aglycon, was confirmed as active ingredients of the coniferous tree (lump).

First, 20 g of painting tree fruit (lump) was pulverized to a size of 50 mesh using a grinder. The crushed ingot was placed in a flask, and each of the solvents listed in Table 1 below was added to 9 times (180 g) of the raw material weight, refluxed for 4 hours, cooled to 40° C., and then used a filter paper (75 μm cartridge). Filter. The filtrate was concentrated under reduced pressure (55 to 58°C) to remove residual solvent and concentrated to 60 brix. The residue was sterilized at 95° C. for 30 minutes, and then spray dried (liquid temperature 75 to 80° C., blowing temperature 180° C., atomizer 18,000 rpm) to prepare an extract powder of prickly pear fruit.

Table 1 and Table 2 show the results of component content analysis per 1 g of powder for the ingot extract by solvent type. It is the result of three repeated tests.

number menstruum exam Genistein glycoside content (mg/g)** G-1 S-7 S-8 One Alcohol 95% Average 32.79 106.24 147.23 ratio 1.0 3.24 4.50 2 Alcohol 85% Average 31.25 96.53 140.61 ratio 1.0 3.09 4.50 3 Alcohol 75% Average 34.60 109.53 153.03 ratio 1.0 3.17 4.42 4 Alcohol 65% Average 35.64 105.45 143.89 ratio 1.0 2.96 4.04 5 Alcohol 55% Average 25.22 78.01 101.97 ratio 1.0 3.09 4.04 6 Alcohol 45% Average 25.00 84.42 112.62 ratio 1.0 3.38 4.50 7 Alcohol 35% Average 18.19 65.40 94.42 ratio 1.0 3.60 5.19 8 Alcohol 25% Average 18.62 57.76 69.11 ratio 1.0 3.10 3.71 9 water Average 25.23 70.70 55.20 ratio 1.0 2.80 2.19 10 Methanol Average 39.09 110.77 156.98 ratio 1.0 2.83 4.02 11 Acetone Average 5.59 62.39 115.56 ratio 1.0 11.16 20.67 12 Ethyl acetate Average 0.71 30.92 68.13 ratio 1.0 43.35 95.96 13 chloroform Average N/D 0.30 0.55 ratio - - 14 Nucleic acid Average N/D 0.40 0.63 ratio - -

^{* The} experimental value is the average value of the 3 repetition results, and the relative proportion of other components based on G-1 is calculated by rounding off the 3rd decimal place.

^** G-1: Genistein 4'-O,7-diglucoside

S-7: Sophorabioside

S-8: Sophoricoside

number menstruum Isoflavone glycoside content (mg/g) ^** Genistein (mg/g) ^*** (G-1)+(S-7)+ Sum of (S-8) (mg/g) yield(%) One Alcohol 95% 422.33 0.36 286.26 9.8 2 Alcohol 85% 394.49 0.32 268.39 10.4 3 Alcohol 75% 435.91 0.39 297.16 10.1 4 Alcohol 65% 422.36 0.38 284.98 11.2 5 Alcohol 55% 305.56 0.35 205.20 10.8 6 Alcohol 45% 327.25 0.40 222.04 10.2 7 Alcohol 35% 253.74 0.49 178.01 10.5 8 Alcohol 25% 221.46 0.59 145.49 9.5 9 water 242.07 0.34 151.13 - 10 Methanol 449.30 0.36 306.84 - 11 Acetone 235.39 1.98 183.54 - 12 Ethyl acetate 103.88 2.18 99.76 - 13 chloroform 2.49 N/D 0.85 - 14 Nucleic acid 2.76 N/D 1.03 -

^* Experimental value is the average value of 3 repetitions

^** Glycoside Isoflavone glycoside content is the sum of Genistein 4'-O,7-diglucoside (G-1), glycoside Kaempferol-glucoside, Sophoraflavonoloside, Sophorabioside (S-7), and Sophoricoside (S-8).

As a result of profiling what a glycoside with a structure that converts to genistein in the lump extract and how the structure is, the structures of genistein diglucoside, sophoravioside, and sophoricoside present in glycoside form It was confirmed that it has a structure that is naturally converted to genistein by an enzyme in the body.

Example 2: Cytotoxicity test of ingot extract by solvent

LDH (lactate dehydrogenase) measurement method was used to confirm the cytotoxicity of each extract. One of the most widely used indicators of cell necrosis is the disruption of cell membranes. Therefore, the degree of cell necrosis can be determined by measuring how much the cell membrane is damaged. LDH is a stable enzyme present in the cytoplasm inside the cell and is released to the outside of the cell depending on the degree of damage to the cell membrane. In other words, by comparing and measuring the amount of LDH a cell has according to the toxicity of the cell, it is possible to infer the degree of damage to the cell.

Specifically, in order to evaluate cytotoxicity, cells (10 ⁵ /96-well) are cultured in an incubator in which 95% oxygen and 5% carbon dioxide are injected at 37° C. for 4 hours. After cultivation, in order to measure the amount of LDH in the cell culture solution of the standard group, control group and test group, the reaction was performed using an LDH analysis solution (product number: G1780) manufactured by the American Promega company, and then SpectraMax Plus 384 Microplate Reader ( Molecular Devices, USA).

Subject of cytotoxicity and antioxidant efficacy test number Extraction solvent One water 2 Acetone 3 Alcohol -95% 4 Alcohol-65% 5 Alcohol-45% 6 Ethyl acetate 7 Hexane

As a result, as shown in FIG. 1, #2 (acetone extract), #6 (ethyl acetate extract), and #7 (hexane extract) were measured to have a mild to moderate cytotoxicity by reducing intracellular LDH. It is judged that it is not suitable for use as a safe herbal material.

Example 3: Comparison of antioxidant effect of lump extract by solvent

Example 3-1: DPPH scavenging activity evaluation

2,2-Diphenyl-1-picrylhydrazyl (DPPH) is a chemically stabilized water-soluble free radical that is a purple compound that exhibits characteristic light absorption at 517 nm, and this radical is very stable in organic solvents such as alcohol and has antioxidant activity. It is a representative antioxidant activity test method that can easily observe the oxidizing activity with the naked eye by changing the color from the purple color of the initial solution to yellow while giving an electron when it encounters a substance. Prepare a methanol solution containing 0.3 mM DPPH, mix 10, 25, and 50 μg/mL test substances (see Table 3), react for 10 minutes at room temperature, measure absorbance at 517 nm to measure radical scavenging activity between test substances. Comparative analysis. As a positive control, ascorbic acid (150 μM) having excellent antioxidant effect was used.

As a result, as shown in FIG. 2, #1, #3, #4, and #5 showed significant antioxidant efficacy from a low dose of 10 μg/mL, and a dose of 50 μg/mL was equivalent to ascorbic acid used as a control substance. The radical scavenging ability of was confirmed. Furthermore, in the case of the #4 sample, it was shown that the DPPH radical was inhibited by 66% when treated with a 25 μg/mL dose, and it was confirmed that the overall analysis had better antioxidant efficacy than other test substances.

In particular, estrogen acts as an antioxidant to inhibit cardiovascular disease by inhibiting the oxidation of low-density lipoprotein cholesterol. #4 The strong antioxidant effect of the test substance not only lowers the risk of cardiovascular disease, but also removes free radicals. It is thought to effectively control osteoclast differentiation from efficacy to effectively control bone-related diseases such as osteoporosis.

On the other hand, #2, #6, and #7 showed little radical scavenging capacity at 10 μg/mL doses, and #6 and #7 did not show radical scavenging ability even when treated at high doses, so it was confirmed that they did not exhibit an antioxidant effect. .

Example 3-2: Hydroxyl radical scavenging assay

As a method of confirming the effect of an antioxidant that protects a protein or an enzyme from damage caused by reactive oxygen species (ROS) using a metal ion catalytic reaction, produced by reacting Cu ^{2 +} (1mM), H ₂ O ₂ (10mM) After mixing and reacting the test substance (see Table 3) with the hydroxyl radical, BSA (bovine serum albumin, Sigma-Aldrich, USA) was applied as a target protein. This was electrophoresed on 10% sodium dodecyl sulfate (SDS)-polyacryamide gel to confirm the level of BSA protein destruction inhibition. As a positive control, ascorbic acid (150 μM) having excellent antioxidant effect was used.

Hydroxy radicals (hydroxy radicals, OH·), a strong reactive oxygen species resulting from cellular respiration, are classified as very harmful substances for bone health and aging progression, including menopausal symptoms. It is known to cause various diseases through peroxidation and protein denaturation. Therefore, it is very useful to have a hydroxy radical scavenging ability among physiological activities of a sample.

As shown in Figure 3 , the results of protein protection efficacy analysis of each sample for hydroxy radicals generated by the reaction of Cu ^{2 +} (1mM), H ₂ O ₂ (10mM), a standard concentration of 25 μg except for #7 When the /mL reaction was observed, a full level of protein protection was observed, and when reacted with a lower concentration of 10 μg/mL, excellent protein protection was observed only in samples #1, #3, #4, and #5, resulting in excellent antioxidant power. The results showed.

Therefore, in addition to exhibiting toxicity according to the cytotoxicity and antioxidant test results, except for #2 (acetone extract), #6 (ethyl acetate extract), and #7 (nucleic acid extract), which have little antioxidant effect, the following Examples The difference in their efficacy with respect to the water extract and the alcoholic extract from the field was closely analyzed.

Example 4: Ghost Menopause of water extract Bone metabolism Check the effect on the disease

Example 4-1: Preparation of sgot water extract and sgot enzyme decomposition extract using the same

First, when the ingot extract was prepared using water, the most commonly used solvent (ie, ingot water extract), it was examined what effect it has. In addition, an enzyme was added to the ingot water extract and reacted to prepare an ingot enzymatic decomposition extract, and the difference in effect from the ingot water extract was confirmed by using this. The method for producing the lump enzymatic decomposition extract is specifically as follows. To the crushed ingot 20g, 9 times of the weight of the original water was added, refluxed for 4 hours, cooled to 40° C., and then filtered using filter paper (75 μm cartridge). To the filtrate thus obtained, amylase was added to a concentration of 0.5% (v/v) to perform enzymatic reaction at 50° C. for 16 hours. The reaction solution was concentrated to a volume of 1/5 using a concentrator to prepare a lump enzymatic digestion extract. This was spray-dried using a spray dryer in the same manner as above, and powdered into an experiment. In the same manner as in Example 1, the component content analysis per 1 g of powder was performed, which is shown in Table 4 below.

division exam Genistein glycoside content (mg/g) Dividend sum
(mg/g) Genistein
(mg/g) G-1* S-7** S-8*** Ghost
Enzymatic digestion extract 3 repeat average 31.87 74.61 42.78 149.26 10.25 Weight ratio One 2.34 1.34

*G-1: Genistein 4’-O, 7-diglucoside

**S-7: Sophorabioside

***S-8: Sophoricoside

( ^{* The} experimental value is the average value of the 3 repetition results / The relative proportion of other components based on G-1 is calculated by rounding off to the third decimal place / The sum of glycosides is (G-1)+(S-7)+(S- 8) value)

Example 4-2: Confirmation of the effect on the expression of TGF-β, a factor for promoting bone formation

TGF-β is known to stimulate osteoclast replication and improve collagen and matrix synthesis. In particular, TGF-β is known to inhibit osteoclast function and promote osteoclast apoptosis, and bone resorption decreases as TGF-β increases.

Each of MG-63 human osteoblast-like cells was treated with a concentration of 10 ^-8 % (w/v) after diluting each of the lump water extract (RG group) and the lump enzymatic digestion extract (RA group) with a cell culture medium. After 72 hours, the expression level of TGF-β was measured through RT-PCR. At this time, as a comparative group, soybean X powder (SS group, purchased from Sindongbang) and estradiol (E) were treated in the same way, and a cell culture medium was added to the control group.

MG-63 human osteoblast-like cells were purchased from the Korea Cell Line Bank of Seoul National University College of Medicine and used after passage. The frozen MG-63 human osteoblast-like cells were thawed in a 37° C. water bath for about 1 minute, centrifuged at 1300 rpm for 5 minutes, and the supernatant was removed. The obtained pellet was resuspended in DMEM medium to which 10% FBS was added, and then cultured by dispensing it into a 25 cm3 culture flask. For the stabilization of the cells, a culture period of about 2 weeks was allowed, and it was used in the experiment after confirming under a microscope whether the cells stably formed a monolayer.

RT-PCR was carried out briefly as follows. Total RNA was extracted by TRIZOL method from MG-63 human osteoblast-like cells treated with a concentration of 10 ^-8 % (w/v) for each sample. Each complementary DNA was prepared by performing a reverse transcription reaction of 2 μl of the total RNA. Complementary by adding 5 μl of total RNA and 1 μl of 10 pM primers to 12.85 μl of DEPC distilled water, denaturing at 72° C. for 10 minutes, adding 0.15 μl of reverse transcriptase (5U) to it, and performing a reaction at 42° C. for 10 minutes. DNA was prepared. Polymerase chain reaction was performed using the complementary DNA as a template. At this time, a one-stop RT-PCR premix (Accupower, Bioneer) was used for reproducibility and consistency of the experiment. Using the PCR system (Dual-bay DyadTM thermal cycler system, MJ Research), the reaction solution was 35 cycles of 5 minutes at 95°C, 30 seconds at 95°C, 60 seconds at 60°C, and 60 seconds at 72°C as 1 cycle. The cycle was run. GAPDH was used as a standard control. The amplified PCR product was quantified using a gel analyzer (gell documentation system), and the expression level was expressed in% relative to the control group. The primer sequence used for RT-PCR in the above is as follows. Sense primer of TGF-β: 5'-CGC CCT GTT CGC TCT GGG TAT-3', antisense primer of TGF-β: 5'-AGG AGG TCC GCA TGC TCA CAG-3'.

The expression level of TGF-β was expressed in% relative to the control group.

As shown in FIG. 4, TGF-β was found to be very highly expressed in the treatment group of the lump water extract (R-G group) and the treatment group of the lump enzymatic digestion (R-A group), compared to the estradiol treatment group (Group E).

In addition, in the additional experiments, the degree of osteoblast proliferation by treatment with the test substances was compared, and as a result, osteoblasts having a similar degree in the treatment group of the lump water extract (RG group) and the treatment group of the lump enzymatic digestion extract (RA group). A proliferative effect was shown (data not shown).

Example 4-3: in vivo Identification of bone-related effects in climacteric animal models

The effect of preventing or treating osteoporosis of each test substance was investigated through animal experiments. After extracting the ovaries of the experimental rats to induce osteoporosis, each test substance was administered to examine the bone-related properties. ANOVA test was used for comparison between all experimental groups, and comparison between specific groups was statistically significant when the confidence interval (p value) was less than 0.05 after statistical processing using the Student's T-test. It was judged.

Specifically, as a test animal, a female rat weighing 230-250 g (Sprague-Dawley) was purchased and used from the Hallym Experimental Zoo (Gyeonggi-do). The experimental animals were kept under the conditions of temperature 23±1°C, humidity 40-60% and contrast cycle 12 hours, and basic feed (solid feed, Hallym Laboratory Animal Research Institute) and drinking water were supplied without limitation. However, only the drinking water was supplied the day before blood collection. Ovariectomy was performed by sterile removal of hair from the dorsal area with a razor after ether inhalation anesthesia in a 12-week-old rat, followed by sterilization of the surgical site with 70% alcohol. The skin tissue was incised about 2 to 3 cm along the vertebral line in the lower part of the lateral dorsal region, and then the muscle and peritoneum where the ovaries were located were incised 1.5 cm to expose the ovaries. After ligation of the fallopian tube with silk, the ovaries were excised and the peritoneum, muscles and skin were sutured using silk. Ovaries were also extracted from the opposite side in the same manner. As a control group, the same procedure was performed up to the peritoneum, and the ovary was performed with a sham operation to reseal the ovaries without being removed. He had a recovery period of one week after surgery.

Samples were administered to each of the experimental groups in the doses shown in Table 5 below, and the administration period of the samples was 9 weeks until 22 weeks of age using 13-week-old rats 1 week after ovarian extraction surgery.

group Sample dosage and method Normal group (Early consumption group) No sample administration Control 1 (most surgical group) Drinking water 1ml/day, oral administration Control group 2 (ovary extraction group) Drinking water 1ml/day, oral administration Group E 1 μg/kg/day, intraperitoneal injection R-G group 0.556g/kg/day, oral administration R-A group 0.556g/kg/day, oral administration S-S group 0.556g/kg/day, oral administration

Among the bone-related characteristics, FIGS. 5 and 6 show results of measuring the trabecular bone area of the tibia and lumbar vertebrae. In menopausal bone disease, it is very important to control bone density characteristics. The trabecular bone is a place where bone metabolism occurs most actively, and is a place where bone formation and bone absorption by external effects react most rapidly. Therefore, it is possible to determine the osteoporosis or osteoporosis-inducing inhibitory effect according to the increase or decrease in the area by measuring the area of the shochu bone (Faugere MC. et al., American Physiological Society, E35-E38, 1986).

Specifically, in order to investigate the effect of the extract of lumps of water (R-G group) and its enzymatic decomposition (R-A group) on bone density of bone, animals of each experimental group were sacrificed, and then tibia and lumbar bone were taken and fixed in 10% formalin solution. Demineralization was performed in formic acid and the area to be observed in the bone tissue was cut with a surgical knife. After a step-by-step dehydration process from 70% alcohol to 100% alcohol and acetone, it was clarified with xylene and embedded in paraffin. The embedded bone tissue was cut into 5 microns with a microtome and then hematoxyline eosin (H&E) staining was observed with an optical microscope (Olympus BH-2). Quantitative and morphometric measurements of the lumbar and tibial bone ends It was measured.

The measurement method was a Polaroid digital camera, and after obtaining the image through the 1X objective lens of an optical microscope (Olympus BH-2), a program that was automatically calculated by drawing the contour of each shochu bone on the computer was used. Among the bony ends, all of the shochu bones located in the secondary ossification region of the growth plate were measured. The area was automatically calculated from the computer and analyzed using an image analysis system (Optimas ver 6.2, Media Cybernetics. Inc.). The average of these numbers was statistically analyzed to quantify and analyze the area occupied by the shochu bone in the total area of the measurement site.

As a result, in the case of tibia, it was found that the degree of reduction in the area of small bone was significantly improved in the group administered with the extract of lump water (RG group) and the group treated with extract of enzymatic enzyme (RA group) compared to the control group 2 (ovary extraction group) administered with drinking water. It was found to maintain a bone density similar to or higher than that of the estradiol administration group (E group) (see FIG. 6). In addition, in the case of the lumbar vertebrae, the degree of reduction in the area of small bone was smaller in the group administered with the extract of lump water (R-G group) and the group treated with the extract of lump enzyme (R-A group) than the control group.

Overall, it was confirmed that the effect of the lump water extract and the lump enzymatic digestion extract administration group (R-A group) on menopausal bone disease such as osteoporosis was similar.

Example 5: Comparison of anti-inflammatory efficacy of sgot water extract and sgot spirit extract

Inflammatory response inducer lipopolysaccharide (LPS) and each test substance are treated with macrophages RAW264.7 cells within 24 hours after treatment with the osteoclast-associated gene (granulocyte-macrophage colony-stimulating factor (GM-CSF)) and menopausal cardiovascular By comparing and analyzing the expression of the related gene (endothelial nitric oxide synthase), we observed whether the test substance (see Table 3) can help improve menopausal symptoms (cardiovascular).

For RT-PCR, total RNA was extracted from the cells of each group using a trizol reagent (Invitrogen, USA). That is, 1 mL of trizolic reagent was added to lyse the cells, allowed to stand at room temperature for 5 minutes, and then 200 μL of chloroform was added, followed by centrifugation at 13500 rpm for 15 minutes. The clear supernatant (500 μL) was taken and transferred to a new tube, the same amount of isopropyl alcohol was added, followed by centrifugation at 13500 rpm for 10 minutes to precipitate RNA. The precipitated RNA pellet was washed with 0.75 mL of 75% ethanol diluted with diethyl pyrocarbonate (DEPC) and then dried in air to be used as a reverse transcription sample. For cDNA synthesis, 1 μg of total RNA, Improm-II reverse transcription system (Promega, USA), and oligo dT primers were used to prepare a total amount of 20 μL, followed by reverse transcription reaction, and the primers shown in Table 6 (Bioneer, Korea) were used. PCR was performed.

primer Sequence Sequence number Access number iNOS Forward 5'-TGCCCCTGGAAGTTTCTCTT-3' One NM_010927 Reverse 5'-ACTGCCCCAGTTTTTGATCC-3' 2 IL-1β Forward 5'-GTGTCTTTCCCGTGGACCTT-3' 3 XM_006498795 Reverse 5'-ATGGGAACGTCACACACCAG-3' 4 IL-6 Forward 5'-TCCATCCAGTTGCCTTCTTG-3' 5 NM_031168 Reverse 5'-CCACGATTTCCCAGAGAACA-3' 6 GM-CSF Forward 5'-ACATGACAGCCAGCTACTAC-3 7 NM_009969 Reverse 5'-CTTCCTCATTTTTGGCCTGG-3 8 β-actin Forward 5'-TACAGCTTCACCACCACAGC-3' 9 NM_007393 Reverse 5'-AAGGAAGGCTGGAAAAGAGC-3' 10

Accordingly, as a result of analyzing the expression level of the inflammatory cytokine gene (GM-CSF, iNOS) that treats each test substance in the inflammatory-induced macrophage cell line Raw264.7 cell line and increases bone resorption after a 24-hour reaction, #1 (water extract) ) Did not show the inhibitory effect of GM-CSF and iNOS gene expression, but it was evaluated that it was possible to develop a functional substitute that can compensate for estrogen deficiency because a significant level of gene expression inhibitory effect was observed in the case of alcoholic extract. (See FIGS. 7 and 8)

Therefore, it was confirmed that the extract of alcohol rather than the extract of ingot water was a useful material as a therapeutic agent for climacteric disease in women, and the antioxidant and anti-inflammatory effects were evaluated based on the extract of ingot alcohol.

Example 6: Comparison of antioxidant efficacy and anti-inflammatory efficacy by alcohol concentration

The antioxidant efficacy and anti-inflammatory efficacy were compared in order to compare the efficacy of the ingot alcohol extract by alcohol concentration (see Table 7). As the experimental method, the same methods as those of the antioxidant and anti-inflammatory efficacy test methods of Examples 3 and 4 were used.

number(#) Extraction solvent One Alcohol -95% 2 Alcohol-65% 3 Alcohol-25%

Example 6-1: Hydroxyl radical scavenging assay by alcohol concentration

As shown in FIG. 9, as a result of analyzing the protein protection efficacy of each sample for the generated hydroxy radical, excellent protein protection efficacy was observed in the #2 (65% alcoholic extract) sample when reacted with 10 μg/mL of each test substance. It was identified as a useful material with excellent antioxidant power. On the other hand, 25% alcoholic extracts have a weak protein protection effect, making it difficult to see as an excellent antioxidant.

Example 6-2: Anti-inflammatory efficacy evaluation by alcohol concentration

Various osteoclast-associated genes (interleukin-6 (IL-6)) within 24 hours after treatment of macrophages RAW264.7 cells with lipopolysaccharide (LPS), which is an inducer of inflammatory reaction of ingot extract at each alcohol concentration, and each test substance together , And comparing the expression of nitric oxide synthase and nitric oxide production to determine whether the test substance can help improve menopausal symptoms (cardiovascular) of the test substance. It is done.

Nitrogen monoxide was measured using Griess reagent (Sigma-Aldrich, USA). That is, lipopolysaccharide (LPS) and each test substance were treated with macrophages, RAW264.7 cells, 24 hours after treatment, and the cell culture solution was taken and reacted with the same amount of Griess reagent to specify absorbance at a wavelength of 540 nm within 10 minutes. The amount of nitrogen monoxide produced was comparatively analyzed through a standard calibration curve.

As shown in FIGS. 10 and 11, in the case of 25% alcohol extract (#3), the IL-6 inhibitory effect was hardly observed, and when compared to the 65% alcohol extract, it was confirmed that the iNOS inhibitory effect was hardly exhibited.

On the other hand, it was confirmed that the #95 and 65% alcohol extracts #1 and #2 had significantly better inflammatory cytokine inhibitory effects than the 25% alcohol extract.

In addition, as shown in Fig. 12, in the case of the 25% alcohol extract, it was confirmed that the amount of nitrogen monoxide produced was significantly higher than that of other extracts, and thus had little anti-inflammatory effect. On the other hand, it was confirmed that in the case of alcoholic extracts 95% and 65%, the increased nitrogen monoxide was significantly reduced due to the inflammatory reaction.

Therefore, based on the experimental results confirming the antioxidant and anti-inflammatory efficacy, it was determined that the 25% alcohol extract was not suitable for use as a therapeutic agent for female menopausal disease.

Thus, the present inventors specifically analyzed the common characteristics of the extract of the lump extract showing the therapeutic effect of female menopausal disease, ① Genistein diglucoside, sophoravioside, and sophoricoside 1: 2.5 ~ 3.5: 3.5 ~ It is included in a weight ratio of 4.5, ② the content of genistein diglucoside, sophoravioside, and sophoricoside is 200 mg/g or more, and ③ as an additional feature, the content of isoflavone glycoside is 300 mg/g or more.

Therefore, as it was determined that the extract of lumps having a specific component ratio of the present invention can most effectively treat menopausal disease, while containing more than 200mg/g of genistein diglucoside, sophoravioside, and sophoricoside,

It was confirmed that the lump extract containing genistein diglucoside, sophoravioside, and sophoricoside in a weight ratio of 1: 2.5 to 3.5: 3.5 to 4.5 can be usefully used as a functional material for treating menopausal disease in women.

Example 7: Confirmation of disease improvement/treatment effect on climacteric animal model of peculiar composition extract of the present invention

Experiment method

The ingot alcohol extract (Rex) having the composition of the present invention derived from Example 6-2 and the ingot hydrothermal extract enzymatic decomposition product (Rex-AG, the same substance as in Example 4 RA) prepared in Example 4-1 ) For in vivo activity.

Efficacy studies were conducted by establishing an OVX rat model (an ovarian extraction mouse model), and after confirming estrogen depletion after OVX, a dietary feed prepared with each test substance was consumed for a total of 6 weeks, and the experiment was conducted with the following protocol. In order to derive the accuracy and professional results of the experiment, the diet and observation for 6 weeks were performed in the animal room at the College of Life Sciences, Gangneung-Wonju University, and the tissue staining and blood tests, etc. Requested quickly to minimize data errors.

The animals used in this study were 12 weeks old female, Sprague-Dawley (SD) rats (220-240 g) purchased from KOATECH (KOATECH, Gyeonggi, Korea) and used for the test. The breeding conditions were maintained at a temperature of 21±2℃ and a humidity of 40 to 60%. Contrast was composed of 12L/12D, and the group was separated through randomization of each individual after a period of purification for one week. This study was conducted after the approval of the experimental plan (GWNU-2018-21) from the Animal Experimental Ethics Committee of Gangneung-Wonju University, and ovaxectomy (OVX) was performed after an additional adjustment period of 1 week after group separation.

That is, after induction of anesthesia in rats, surgery was performed in the ovarian resection group (n=21) and the sham surgery group (Sham, n=4). The ovarian resection group removed the ovaries after removing both ovaries by cutting the skin and both muscles in the middle of the back after epilating the back, and the Sham group proceeded in the same way as ovarian resection, but under the same stress without ovarian resection. After exposure to, surgery was performed to reseal, and after confirming that no specific change appeared with the control group, it was included or not included in the analysis target group. After the surgery, the surgical site was exploded and the inflammatory reaction was observed to observe the specific situation, and the successfully completed subjects were applied to this experiment. Using the AIN-76A pellet optimized for the menopause experiment model for feeding, the production of feed pellets for each test material according to the purpose of the experiment was used after being commissioned by a specialized company.

The experimental group includes the sham group (non-ovariectomized control, Sham), the ovarian ablation group (Ovariectomized control, OVX), the ovarian ablation group and the positive control group (Ovariectomy + 100 μg/kg 17β-estrogen, PC), ovarian ablation and the present invention. The groups were divided into 5 groups (Ovariectomy + 150 mg/kg Rex), ovarian ablation and enzymatic lysate (Ovariectomy + 150 mg/kg Rex-AG) (see Table 8 below). Requests to manufacture prepared feed mixed with samples according to food intake (DAEHAN BIOLINK, Chungbuk, KOREA) to provide diet (Based AIN-76A diet), and Sham and OVX groups are AIN-76A (Harlan teklad, Madison, WI, USA) ) As an autonomous meal. The body weight was measured twice a week at the same time, and the pellet intake amount and the negative amount were measured periodically.

No. Group Diet base Dose (mg/kg) One Sham AIN-76A - 2 OVX - - 3 Positive control (PC) 0.1 4 Invention of the present application (Rex) 150 5 Enzyme Enzyme Extract (Rex-AG) 150

Experiment result

Example 7-1: Vasomotor symptoms of menopause

Vasomotor symptoms are symptoms that appear in the early stages of menopausal women, and the typical symptom of vasomotor symptoms is facial flushing, which is characterized by the sudden redness of the skin on the face, neck, and chest, accompanied by unpleasant sensations and sweating in the whole body. . This is known to be due to various causes, but one of the hypotheses is that the elasticity of blood vessels decreases due to a decrease in estrogen levels, which delays the skin vascular response to changes in internal body temperature. Therefore, in this experiment, the rectal temperature was measured every day 2 days before the autopsy to observe the symptoms of vasomotor movement. Measurement of rectal temperature 15 minutes at a rate of 15 m/min, immediately after a forced running on an electric treadmill (MK-680S, Muromachi Kikai, Tokyo, Japan), the rectal temperature was measured for 120 minutes at 10-minute intervals and the net change was measured. The original temperature at each time point (average temperature for 20 minutes prior to forced running) was calculated as the divided value. The rectal temperature was measured with an electronic thermometer (MT200, Microlife, Taipei, Taiwan) to reduce the deviation.

The amount of change in vascular movement was expressed by dividing the amount of change based on the starting temperature. This is one of the symptoms of menopause, and is a result that can provide a correlation against facial flushing and increase in body temperature in humans. As shown in FIG. 13, when the recovery time and the amount of change were compared with the OVX group as a control after artificially increasing body temperature, it was confirmed that the whole returned normally without a large change width, and the Rex group (specific ingot extract) Compared to the Rex-AG group, the change in rectal temperature was found to be significantly less, which showed a pattern similar to that of the positive control group. Through this, it was confirmed that the Rex group (the present invention) had a more remarkable effect on representative facial flushing among vasomotor symptoms as one of the symptoms of menopausal women.

Example 7-2: Menopausal urinary system epithelial cell changes (keratinization test)

Vaginal dryness is one of the most common symptoms of menopause. Therefore, in this experiment, we observed the change in the urinary epithelial cells of each group to determine whether the test substance helps to relieve menopausal symptoms. That is, just before the autopsy, the vaginal walls of each experimental group were rubbed with a cotton swab, and the vaginal epithelial cells were coated and stained to measure the number of nucleated cells, keratinized epithelial cells and keratin, and then the percentage of keratinized epithelial cells was shown. A high proportion of keratinous epithelial cells in the vaginal mucosal cells considers the level of vaginal keratinization to decrease and the vaginal mucosa to be flexible.

14 and 15, it was confirmed that the ratio of keratinized epithelial cells was higher in the Rex group (specific ingot extract of the present invention) than in the Rex-AG group through comparison of keratinocytes. Through this, the level of vaginal keratinization is reduced and the vaginal mucosa becomes flexible, so it can be said that the Rex group (the present invention) is more effective for vaginal dryness.

Example 7-3: Analysis of climacteric blood factor change

A comparative analysis of changes in the concentration of total lipids (cholesterol, HDL, LDL), which is a liver function test ALT and a lipid test for evaluating cardiovascular disease, is a blood-related factor for menopausal symptoms due to menopause. In particular, ALT (Alanine Aminotransferase) is a liver function test that indicates hepatitis or fatty hepatitis if the level is high, and may imply autoimmune hepatitis, chronic viral hepatitis, hemochromatosis, etc. Increased risk of arteriosclerosis and heart disease increases as low density lipids (LDLs) interfere with blood circulation.

As a result of hematological marker analysis of each test group after OVX surgery for 6 weeks, as shown in FIGS. 16 and 17, the ALT level indicating hepatitis or fatty hepatitis in the Rex group (specific ingot extract of the present invention) and LDL interfering with blood circulation It was confirmed that the value was significantly lower than the Rex-AG group. This shows that the Rex group (the present invention) has a more beneficial effect on liver function and cardiovascular disease in postmenopausal women.

Example 7-4: Analysis of the expression of major bone cell-related genes (bone marker genes) in menopausal bone disease

Bone is a hard connective tissue that supports the body of animals belonging to the axonal animal gate, and builds a skeleton to support the body's structures and prevent damage to other body organs. In healthy bones, balanced regulation of osteoblasts that produce bones and osteoclasts that destroy bones is key. Osteoporosis is a disease in which bone is easily broken due to decreased bone strength. Osteoporosis is a disease that occurs as osteoblasts decrease and the effects of osteoclasts increase, thereby increasing the frequency of bones being destroyed. Therefore, by determining the specific gravity of osteoblasts and osteoclasts present in the bone by gene expression, it is possible to confirm the health status of the bone.

After menopause, estrogen deficiency increases the expression of IL-6 and TNF-α, which are bone resorption promoters, leading to an increase in blood calcium levels. Accordingly, the expression of IGF-1, TGF-β, which is a factor for promoting bone formation, decreases, and bone formation becomes insufficient, resulting in osteoporosis. Estrogen induces apoptosis of osteoclasts, and when deficient, osteoclasts increase. At this time, the cytokine that activates osteoclasts is RANKL, which binds to the RANK receptor to regulate osteoclast formation and activity.

The rats to which the test substance was administered for 6 weeks were euthanized by ether inhalation anesthesia, and then the femurs were extracted to separate bone marrow cells. The extracted femur was immediately washed twice with sterile phosphate buffered saline (PBS), once in 70% ethanol, and twice again with PBS.The shank portion of the femur was cut using a bone cutter and placed in a microcentrifuge tube and centrifuged. Isolation (5000 × g, 5 minutes) to isolate bone marrow cells (rat bone marrow-derived cells, rBMs). Red blood cells were removed by adding 5 mL of red blood cell lysis buffer (Invitrogen, CA, USA) to the separated bone marrow cells, and cell filtration was performed using 100 μm nylon mesh to remove other non-myeloid-derived cells.

Reverse transcriptional polymerase chain reaction (RT-PCR) was performed to confirm the reaction of bone-related cells to the test substance. To this end, total RNA of bone marrow cells was extracted using Trizol (Thermo Fisher Scientific, MA, USA), and reverse transcription using Oligo dT primer and TOP scriptTM Reverse Transcriptase (Enzynomics, Daejeon, Korea) for 2 μg total RNA Through the reaction, cDNA to be used as a template for PCR was synthesized. PCR was performed using the primers shown in Table 9 using cDNA and 2X DreamTaq PCR Master Mix (Thermo Fisher Scientific), and the PCR product was electrophoresed with 1.0% agarose gel to confirm the degree of gene expression. Afterwards, gene expression was quantified by normalizing with β-actin, a house-keeping gene, using GelQuant software (MiniBIS Pro, Jerusalem, Israel). Table 9 below is a list of Primers used for bone cell RT-PCR.

Gene Primer Sequence Accession No. β-actin 5'primer 5’-CATCAAAGAGAAGCTGTGCT NM_001101.4 3'primer 5’-GAAGGAAGGCTGGAAAAGAG RANKL 5'primer 5’-CAGCATCGCTCTGTTCCTGT NM_011613.3 3'primer 5’-CCAGAGTCGAGTCCTGCAAA TGF-β 5'primer 5’-GGAGACGGAATACAGGGCTT NM_011577.2 3'primer 5’-GGTCCCAGACAGAAGTTGGC

18 and 19, as a result of examining the expression of the osteoporosis-related gene RANKL and the bone formation promoting factor TGF-β gene, the Rex group (specific ingot extract) inhibits the expression of the osteoporosis gene RANKL and , It was confirmed that the expression of TGF-β, a factor for promoting bone formation, was more effective than Rex-AG for the symptoms of osteoporosis, one of menopausal symptoms.

Example 7-5: Gene expression analysis of fatty tissue and liver tissue related to menopausal metabolic disease

Post-menopausal metabolic disease studies lead to changes such as increased total cholesterol and triglycerides, high-density lipoprotein cholesterol, increased visceral fat, and decreased vascular function. It causes metabolic changes due to changes in hormones caused by menopause and increases insulin resistance, acting as risk factors for metabolic syndrome. For this reason, when the prevalence of metabolic syndrome in men and women according to changes in age is compared, it is higher than that of men after age 55 when women become menopause. After menopause, body fat build-up accelerates weight gain. Furthermore, the production and secretion of substances that are markers of inflammatory reactions in adipocytes is increased in obese patients, thereby defining obesity as a low-level systemic inflammation. Obesity-related inflammation increases the number of macrophages in fat and increases the secretion of inflammation-causing cytokines. Various inflammation-causing cytokines secreted through macrophages activate vascular endothelial cells in fat, and TNF-α, IL-6, etc. are inflammation-causing cytokines involved in the activation of vascular endothelial cells. Leptin is a hormone composed of 167 amino acids secreted from adipose tissue, and is a fat-related gene that plays a major role in the regulation of food intake and energy consumption. When the Leptin receptor is expressed in the hypothalamus, it is known to inhibit food intake and increase energy consumption, thereby participating in obesity control.

After a portion of the tissue collected at the autopsy, total RNA of bone marrow cells was extracted using Trizol (Thermo Fisher Scientific, MA, USA), and Oligo dT primer and TOP scriptTM Reverse Transcriptase (2 μg total RNA) were targeted. Enzynomics, Daejeon, Korea) was used to synthesize cDNA to be used as a PCR template. PCR was performed using the primers shown in Table 10 below using cDNA and 2X DreamTaq PCR Master Mix (Thermo Fisher Scientific), and the PCR product was electrophoresed with 1.0% agarose gel to confirm the degree of gene expression. Table 10 below is a list of Primers used for adipocyte RT-PCR.

Gene Primer Sequence Accession No. β-actin 5'primer 5’-CATCAAAGAGAAGCTGTGCT NM_001101.4 3'primer 5’-GAAGGAAGGCTGGAAAAGAG Leptin 5'primer 5’-AGCTGCAAGGTGCAAGAAGA NM_008493.3 3'primer 5’-ACCGACTGCGTGTGTGAAAT IL-6 5'primer 5’-CCTTCCTACCCCAACTTCCA NM_012589.2 3'primer 5’-AGCACACTAGGTTTGCCGAG TNF-α 5'primer 5’-GATTATGGCTCAGGGTCCAA NM_013693 3'primer 5’-GAGACAGAGGCAACCTGACC

As shown in FIGS. 20, 21 and 22, the Rex group (specific ingot extract of the present invention) had a better inhibitory effect on the expression of Leptin, an obesity related gene, and gene expression of IL-6 and TNF-α, which are markers of inflammation. Was significantly suppressed.

As described above, the present invention relates to a composition for the prevention and treatment of climacteric disease in women, which contains the lump extract as an active ingredient, and more specifically, genistein diglucoside, sophoravioside, and sophoricoside 1: 2.5 to 3.5: It relates to a pharmaceutical composition and a food composition for the prevention or treatment of climacteric disease in women, which includes as an active ingredient an extract of lumps containing a weight ratio of 3.5 to 4.5.

The ingot extract having a specific component ratio of the genistein glycoside according to the present invention as well as having a specific content or more has little cytotoxicity, is safe, and has excellent antioxidant and anti-inflammatory effects compared to other solvent extracts, thereby preventing female menopausal disease , It can be usefully used as a pharmaceutical composition and a food composition for improvement or treatment, and thus has high industrial applicability.

<110> NOVAREX Co., Ltd. <120> Composition for the preventing and treating female menopausal disease comprising the extract of Sophora japonica L. fruits <130> NP18-0057P <150> KR 10-2018-0124707 <151> 2018-10-18 <160> 24 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> iNOS forward primer <400> 1 tgcccctgga agtttctctt 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> iNOS reverse primer <400> 2 actgccccag tttttgatcc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-1beta forward primer <400> 3 gtgtctttcc cgtggacctt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-1beta reverse primer <400> 4 atgggaacgt cacacaccag 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-6 forward primer <400> 5 tccatccagt tgccttcttg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-6 reverse primer <400> 6 ccacgatttc ccagagaaca 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GM-CSF forward primer <400> 7 acatgacagc cagctactac 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GM-CSF reverse primer <400> 8 cttcctcatt tttggcctgg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta-actin forward primer <400> 9 tacagcttca ccaccacagc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta-actin reverse primer <400> 10 aaggaaggct ggaaaagagc 20 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TGF-beta forward primer <400> 11 cgccctgttc gctctgggta t 21 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TGF-beta reverse primer <400> 12 aggaggtccg catgctcaca g 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta-actin forward primer (table 9, table 10) <400> 13 catcaaagag aagctgtgct 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta-actin reverse primer (table 9, table 10) <400> 14 gaaggaaggc tggaaaagag 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RANKL forward primer (table 9) <400> 15 cagcatcgct ctgttcctgt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RANKL reverse primer (table 9) <400> 16 ccagagtcga gtcctgcaaa 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TGF-beta forward primer (table 9) <400> 17 ggagacggaa tacagggctt 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TGF-beta reverse primer (table 9) <400> 18 ggtcccagac agaagttggc 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Leptin forward primer (table 10) <400> 19 agctgcaagg tgcaagaaga 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Leptin reverse primer (table 10) <400> 20 accgactgcg tgtgtgaaat 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-6 forward primer (table 10) <400> 21 ccttcctacc ccaacttcca 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-6 reverse primer (table 10) <400> 22 agcacactag gtttgccgag 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha forward primer (table 10) <400> 23 gattatggct cagggtccaa 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha reverse primer (table 10) <400> 24 gagacagagg caacctgacc 20

Claims (10)

It contains at least 200 mg/g of genistein diglucoside, sophoravioside, and sophoricoside,
Prevention of women's menopausal disease accompanied by vaginal dryness, which includes as an active ingredient an extract of gangrene containing genistein diglucoside, sophoravioside, and sophoricoside in a weight ratio of 1: 2.5 to 3.5: 3.5 to 4.5, or As a therapeutic pharmaceutical composition,
The women's menopausal disease consists of hot flashes, sweating, skin dryness, vaginal atrophy, lower urethral atrophy, vaginitis, cystitis, urination, urinary distress, concentration disorder, short-term memory disorder, anxiety, nervousness, memory loss, muscle pain, joint pain and osteoporosis. Pharmaceutical composition, characterized in that at least one selected from the group and vaginal dryness.
delete delete The pharmaceutical composition according to claim 1, wherein the alcohol is a alcohol having a concentration of 40% (v/v) or more.
delete It contains at least 200 mg/g of genistein diglucoside, sophoravioside, and sophoricoside,
Prevention of women's menopausal disease accompanied by vaginal dryness, which includes as an active ingredient an extract of gangrene containing genistein diglucoside, sophoravioside, and sophoricoside in a weight ratio of 1: 2.5 to 3.5: 3.5 to 4.5, or As a food composition for improvement,
The women's menopausal disease consists of hot flashes, sweating, skin dryness, vaginal atrophy, lower urethral atrophy, vaginitis, cystitis, urination, urinary distress, concentration disorder, short-term memory disorder, anxiety, nervousness, memory loss, muscle pain, joint pain and osteoporosis. Food composition, characterized in that at least one selected from the group and vaginal dryness. delete delete delete delete

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