KR101972657B1 - Composition comprising extract of Angelica decursiva Franchet et Savatieras or compounds isolated therefrom for preventing or treating osteoporosis - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for prevention and treatment of bone metabolic diseases comprising Angelica decursiva Franchet et Savatieras extract and nodakenetin and nodakenin isolated therefrom as an active ingredient, . The extract according to the present invention and the active ingredient isolated therefrom can be effectively used as a fundamental therapeutic agent for bone metabolic diseases by inhibiting osteoblast proliferation and differentiation promoting activity and osteoclast differentiation at the same time.

Description

A composition for treating or preventing osteoporosis, which comprises extracts of Magnetophyceae or a compound isolated therefrom, the composition for preventing or treating osteoporosis,

The present invention relates to a composition for improving, treating or preventing a bone metabolic disease comprising Angelica decursiva Franchet et Savatier extract or an active ingredient isolated therefrom.

Bone tissue is a dynamic tissue that is constantly absorbed and formed to maintain bone mass and skeletal homeostasis. Bone remodeling involves two types of cells that function in a specific way. While osteoclasts absorb bone, osteoblasts play a role in synthesizing and filling bone matrix.

Thus, bone mass is dependent on the relative function of these cells (Harada S et al (2003) Nature 423: 349-55). In the human body, the process of absorbing old bone and forming a new bone continues, and in adults, about 10% of the bone is regenerated in one year. Therefore, when about 10 years have elapsed from normal bone remodeling, (Matsuo K et al (2008) Arch Biochem Biophys 473: 201-209).

The imbalance between osteoblast and osteoclast activity is due to skeletal abnormalities due to osteoporosis or osteosclerosis. There are various theories and arguments about whether this imbalance of bone metabolism is caused primarily by the functional deterioration of osteoblast cells or by the increase of osteoclast activity or the increase of osteoclast activity. The factors of osteoporosis include aging, (Del Fattore A et al. (2008) Bone disease). In addition, there is a wide range of diseases such as diabetes, lack of exercise, lack of calcium intake, genetic factors, drug use, hormonal abnormalities (menopause in women), inflammatory rheumatoid arthritis and osteosarcoma 42: 19-29).

Osteoporosis, which is a major problem in the aged society, is a disease that can cause bone fracture in a slight impact due to a decrease in the bone mass in the unit volume without a significant change in the chemical composition of the bone. It is known that the elderly, especially postmenopausal women, have the highest incidence of estrogen secretion (Jilka RL et al (1998) Bone 23: 75-81). However, even in males, bone mass tends to decrease with aging (Stein GS et al (1996) Physiol Rev 76: 593-629), and the fracture rate of elementary and middle school students has more than doubled in recent 20 years. In terms of preventing or treating bone diseases caused by imbalance of bone metabolism, it is very important to raise bone mass from a young age. Thus, maintaining bone health is an important issue regardless of gender or age.

Previous studies on osteoporosis focused on inhibition of osteoclast. Most of the drugs such as estrogen and bisphosphonate, which are currently used for the prevention and treatment of osteoporosis, inhibit bone resorption, Therefore, the ultimate goal of osteoporosis can not be prevented completely.

Therefore, studies on the increase of bone formation for the prevention and treatment of osteoporosis have recently been attracting attention, and a scientific approach to the effect on the bone regeneration ability is needed. However, these drugs are associated with the activity of mature osteoclasts, which is problematic for long-term administration. Recently, it has been turned into a study on osteoblast differentiation and activation (Tang DZ et al (2010) J Bone Miner Res 25: 1234-1245).

Wnt / β-catenin signaling plays an important role in bone formation and has received recent attention (Maria P et al (2007) Hormones 6: 279-294). Activation of the Wnt / β-catenin signal transduction system stimulates pluripotent mesenchymal cells to differentiate into osteoblast progenitors and become osteoblasts (Hill TP et al. al. (2005) Dev Cell 8: 727-738). In bone cells, the expression of bone-specific runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 (BMP2) is stimulated during the activation of the ß-catenin signaling pathway (Yan Y et al (2009) J Cell Sci 122: 3566 -3578). BMP2 regulates the expression of Runx2 through Smad1 / 5/8 (Lo YC (2010) J Agr Food Chem 58: 6643-6649).

Natural materials are used for a variety of diseases and have a low side effect, which is a good resource for potential drug development.

Korean Patent Laid-Open Publication No. 2016-0036863 discloses a composition for treating or preventing gastrointestinal diseases containing, as an active ingredient, an extract of Magnetium japonica or a compound isolated therefrom.

Korean Patent Laid-Open Publication No. 2016-0028438 describes the hair loss prevention effect and the hair regeneration effect of Peaucedani Radix extract.

The Peucedani Radix is the Peucedanum praeruptorum Dunn) or Angelica decursiva (Franchet et Savatier). It is used as Peucedani Radix because its name is similar to that of the two plants. However, it has been found that plants differ from each other in that they are different from each other (Kim (2016) Nat Prod Sci 22: 162-167), and the exact origin is unclear in the above-mentioned patent.

None of the prior art discloses or discloses the therapeutic efficacy of an extract of Angelica decursiva Franchet et Savatier or a compound isolated therefrom for the treatment of bone metabolic diseases.

The present invention is directed to a composition for improving, treating or preventing bone metabolic diseases, which comprises extracts of Magnetophyceae or compounds isolated therefrom.

In one embodiment, the present invention provides a pharmaceutical composition for preventing or treating bone metabolic diseases comprising Angelica decursiva Franchet et Savatier extract.

The extracts used in the extract according to the present invention are distinguished in terms of their biological characteristics and activity, etc.

Particularly, in one embodiment, the extracts of the present invention have the effect according to the present invention by using a plant having a root part and a root part including flowers, branches, stems, leaves, leaves, immature fruits or fruits of the prey.

In one embodiment, the Magnetium Phosphate Extract herein is a polar solvent, particularly a water extract.

In another aspect, the present invention also provides a pharmaceutical composition for preventing or treating bone metabolic diseases comprising, as an active ingredient, nodarkenine or nodakenethin, isolated from Magnetium.

The extract according to the present invention, the active ingredient isolated therefrom, or a composition containing the same, has the effect of inhibiting the differentiation of osteoclast, promoting the proliferation, differentiation and bone calcification of osteoblast, And bone metabolic diseases such as osteoporosis, bone cancer, multiple myeloma, Paget's disease, periodontal disease, bone cancer, or degenerative rheumatoid arthritis.

In another aspect, the present invention also provides a health functional food, a food additive, or a food composition for preventing or ameliorating a metabolic disease including a Magnetophobia extract.

In another aspect, the present invention also provides a health functional food, a food additive, or a food composition for preventing or ameliorating a bone metabolic disease comprising nodakenine or nodakenethin as an active ingredient, which is isolated from the extract of Magnetium japonica.

The composition comprising the extract of Magnetophyll of the present invention or the compound isolated therefrom as an active ingredient promotes the proliferation, differentiation and bone calcification of osteoblasts and inhibits the differentiation of osteoclasts, May be usefully used for the prevention, treatment or amelioration of the bone metabolic diseases involved.

FIG. 1 shows that the extract of Magnetophyllum according to one embodiment of the present invention is not cytotoxic in a human kidney cell line.
FIG. 2 shows that nodakenethin, a compound isolated from the extract of Magnetophyllum according to an embodiment of the present invention, is not cytotoxic in a human kidney cell line.
FIG. 3 shows the results of analysis of TCF / LEF transcriptional activation in the human kidney cell line of the extract of Magnetic Resonance Imaging according to one embodiment of the present invention.
FIG. 4 is a graph showing the results of TCF / LEF transcriptional activation assay in human renal cell line of nodakenethin isolated from Magnetic Resonance Assay according to one embodiment of the present invention.
FIG. 5 shows the results of increasing the expression of the lower regulator protein in the human signaling pathway in the human renal cell line of nodakenethin isolated from the extract of Magnetic beads according to one embodiment of the present invention.
FIG. 6 is a graph showing the expression of the signal transduction system-related protein in the human renal cell line of nodakenethin isolated from the extract of Magnetic beads according to one embodiment of the present invention.
FIG. 7 is a graph showing that nodakenethin isolated from Magnetic Resonance Extract according to an embodiment of the present invention is not cytotoxic in mouse osteoblast cells.
FIG. 8 shows the results of stimulating ALP activation in mouse osteoblast cell line of nodakenethin isolated from Magnetic Resonance Extract according to one embodiment of the present invention.
FIG. 9 shows the results that nodakenethin isolated from Magnetic Resonance Extract according to an embodiment of the present invention promotes mineralization in a mouse osteoblast cell line.
FIG. 10 shows the results that nodakenethin isolated from Magnetic Resonance Extract according to one embodiment of the present invention promotes the expression of β-catenin gene in mouse osteoblast cell line.
FIG. 11 shows the results of promoting BMPs gene expression in mouse osteoblast cell line by nodakenethin isolated from Magnetic Resonance Extract according to one embodiment of the present invention.
FIG. 12 shows that the nodakenethin isolated from the extract of Magnetophyllum japonica according to one embodiment of the present invention increases the expression of Wnt signal transduction system related proteins and sub-regulators in mouse osteoblast cells.
FIG. 13 shows the results of promoting BMPs protein expression in mouse osteoblast cell line of nodakenetin isolated from Magnetic Resonance Probe extract according to one embodiment of the present invention.
FIG. 14 shows the morphological changes of bone in animal models of water, ethanol extracts, and nodarketin and nodarkethene isolated therefrom according to one embodiment of the present invention. As a result, the extract according to the present invention and the And that the separated compound can be effectively used for the improvement, treatment or prevention of bone metabolic diseases.
FIG. 15 is a graph showing changes in bone volume ratio, number of cancellous bone, space between cancellous bone and bone density in an animal model of nodarketin and nodarkethene isolated from water, ethanol extracts, and magnetocapsule according to one embodiment of the present invention , The extract according to the present invention and the compound isolated therefrom can be effectively used for the improvement, treatment or prevention of bone metabolic diseases.

The present invention relates to a method for inhibiting osteoclast differentiation and inhibiting osteoclast differentiation by promoting osteoblast proliferation, differentiation and osteocarcinosis of naturally occurring extracts and the compounds separated therefrom, nodakenetin and nodakenin, It is based on the discovery that the disease can be effectively prevented or treated.

In one embodiment, the present invention relates to a pharmaceutical composition for preventing or treating a bone metabolic disease, which contains Nodakenine or Nodakenethin as an active ingredient, which is a Magnetophobia extract or a compound isolated and isolated therefrom.

The term " Angelica decursiva Franchet et Savatier" as used herein refers to a body herb belonging to the Umbelliferae. Young seeds eat as herbs. It uses roots as herbal medicine in one room, and it is effective for cold, cough, It is distributed in Korea, Japan, and China.

The Peucedani Radix is the Peucedanum praeruptorum Dunn or Angelica decursiva Franchet et Savatier. It is used as Peucedani Radix because its name is similar to that of the two plants.

However, Baekhwa-gun and Jahwa-gun were different plants (Kim (2016) Nat Prod Sci 22: 162-167).

Also, the extract according to the present invention is used in a plant including a flower part, a branch part, a stem part, a leaf part, an immature fruit part, or a top part including roots and roots.

As used herein, the term " extract " means an active ingredient isolated from a natural product, i.e., a substance exhibiting the desired activity. The extract may be obtained by an extraction process using a polar or non-polar solvent containing water or an appropriate organic solvent such as methanol, ethanol, or a mixed solvent thereof. The extract may be obtained by extracting, Includes all forms that have been formalized. The above-mentioned extract also includes an extract obtained by fractionating the above extract.

The solvent may be a polar, nonpolar solvent or an organic solvent including water or a mixture thereof. Specific examples of the solvent include water, a lower alcohol having 1 to 4 carbon atoms, such as ethanol, butanol, propanol and isopropanol, a polar solvent such as chloroform, acetone, propylene glycol, ethyl acetate and butylene glycol And organic solvents such as ether, hexane or dichloromethane, or mixtures thereof, especially water, alcohols having from 1 to 4 carbon atoms or mixtures thereof, more particularly water, from about 50 to 100 % Alcohol such as methanol, ethanol or the like having 1 to 4 carbon atoms.

In one embodiment according to the present application, a polar solvent, particularly water, is used and exhibits excellent effects.

The extract of the present invention can be produced by using a common general production method of a conventional plant extract, and is not particularly limited. May be for example, a method for producing by a well-known method by the ultrasonic extraction, reflux extraction, hot water extraction method, or the solvent in the extraction solvent (for example, Nomura T. Phenolic compounds of the mulberry tree and related plants. In : Herz, W .; Grisebach, H .; Kirby, GW; Tamm, Ch. (Eds.) Fortschritte der chemie organischer naturstoffe Springer-Veriag, Wien, 53: 87-201 (1988)). Further, the extracted extract can be further fractionated with a solvent selected from the group consisting of hexane, methylene chloride, acetone, ethyl acetate, chloroform, and mixtures thereof. Two or more kinds of solvents may be used in the fractionation step, or they may be used individually or in combination. Depending on the polarity of the solvent, an extract of each solvent may be sequentially used. The extract preparation temperature may be about 4 to 120 DEG C, but is not limited thereto. The extraction time is not particularly limited, but may be about 10 minutes to 30 days, and conventional extraction equipment, ultrasonic pulverization extractors or fractionators may be used. The produced extract can then be subjected to vacuum filtration and / or lyophilization to remove the solvent. For example, the solvent is added to the heating circulation type extraction tank at a weight ratio of about 1: 1 to 1:20 by weight, and purified water or edible alcohol is added thereto at about 80 to 100 DEG C for about 6-24 hours, And the same procedure can be repeated several times by adding purified water or edible alcohol to the residue. The filtrate may be used as it is, or it may be concentrated under reduced pressure with a vacuum concentrator, and then the dried product obtained by hot air or lyophilization may be pulverized by a pulverizer.

In one embodiment of the invention, the Magnetophosphate Extract of the present invention can be prepared as follows. The dried magnetized film is washed with water containing purified water after the washing and sublimation, a lower alcohol having 1 to 4 carbon atoms, one or more mixed solvents selected from the group consisting of chloroform, acetone, propylene glycol, ethyl acetate, butylene glycol, ether and chloroform , Followed by ultrasonic extraction, hot water extraction, room temperature extraction or reflux extraction for 30 minutes to 48 hours, preferably 1 hour to 12 hours at a temperature of 30 to 150 ° C, preferably 50 to 100 ° C , The extract obtained by repeating the ultrasonic extraction method for about 1 to 20 times, preferably 2 to 10 times, is filtered, concentrated under reduced pressure and dried to obtain the magnetically bound extract of the present invention.

The active ingredients contained in the extract according to the present invention can be isolated as follows.

The compound isolated from the magnetically bound extract of the present invention is used in an amount of about 0.0005 to 0.005 times, preferably 0.05 to 0.5 times (v / v) of the weight of the non-purified extract, preferably 10 to 90% w%) of water, followed by fractionation using ethyl acetate and butanol to obtain a non-polar solvent-soluble fraction extracted with n-hexane, methylene chloride, chloroform and ethyl acetate; And a polar solvent-soluble fraction extracted in a polar solvent such as butanol and water; The non-polar solvent-soluble extractable fraction, preferably the chloroform-soluble fraction, obtained in the above non-polar solvent such as n-hexane, methylene chloride, chloroform, ethyl acetate or the like is subjected to flash column chromatography, RP C18 column chromatography silica gel open column chromatography, Or Diaion HP-20 column chromatography is repeated several times to selectively form nodakenetin and nodakenin compounds of the following formula (1) and (2) Each purified and obtained.

[Chemical Formula 1]

(2)

The present invention relates to a method for promoting TCF / LEF transcription, an increase in the gene and protein of? -Catenin in osteoblast, an increase in c-Myc, Cyclin D1, and Survivin gene expression promoting activity of nadakenetin or nodarkenine (MC3T3-E1) differentiation-promoting activity through the mouse / β-catenin signal transduction system and the activity of increasing the bone-specific differentiation markers Runx2 and BMPs.

The extract according to the present invention or the nodarketin or nodarkenine isolated therefrom has osteoblast proliferation, differentiation promoting activity and osteoclast differentiation inhibiting activity.

Therefore, the above extract of Magnetium japonica or Nodakenethin or Nodarkenin isolated therefrom of the present invention can be effectively used for the prevention, treatment or improvement of bone metabolic diseases.

As used herein, the term " treatment " refers to any action that improves or alleviates the symptoms of a metabolic disease by administering an extract, or a compound separated therefrom, or a composition comprising the same. Those skilled in the art will be able to ascertain, by reference to the data provided by the Korean Medical Association, the precise criteria of the disease for which the composition of the present invention is effective, .

As used herein, " prophylactic " refers to any action that inhibits or delays the onset of a metabolic disease by administration of an extract, or a compound separated therefrom, or a composition comprising the same. It will be clear to those skilled in the art that the extract of the instant invention or the compounds isolated therefrom that have therapeutic effects on bone metabolic diseases can prevent such diseases when taken prior to the appearance of the initial symptoms or symptoms.

Nodakenetin or nodarkenine isolated from the extract according to the present invention has activity of promoting osteoblast proliferation, differentiation and inhibiting osteoclast differentiation, and has various osteoclasts and osteoblasts, Can be effectively used for metabolic diseases.

Herein, " bone metabolic disease " refers to a disease caused by an imbalance of osteoclast and osteoblast in terms of bone strength, bone mass or bone structure disease, or mechanism caused by mineral imbalance and deficiency of vitamin D in terms of bone function. Disease. For example, the activity of osteoblast is significantly reduced compared to osteoclast, and osteoporosis, osteoporosis, osteoporosis such as colorectal cancer or breast cancer metastasizing to the bone, bone metastasis, bone metastasis, Paget ' s disease caused by genetic causes, periodontal disease destroyed by oral bacteria, or degenerative rheumatoid arthritis.

The composition of the present invention may contain 0.01 to 99% by weight of the above magnetically bound extract or nodarkethene or nodarkenine isolated therefrom, based on the total weight of the composition.

However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

The pharmaceutical composition comprising the extract according to the present invention or a compound isolated therefrom can be administered simultaneously or sequentially and these mixtures can be administered alone or in combination with other pharmaceutically active ingredients for the treatment of bone metabolic diseases.

The pharmaceutical composition comprising the extract according to the present invention or the active ingredient compound isolated therefrom may be in the form of an appropriate carrier, diluent, preservative, stabilizer, wetting agent, emulsifier, solubilizer, sweetener, colorant, Antioxidants, and the like. Specific examples include lactose, dextrose, sucrose, sorbitol, mannitol xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone Water, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium, stearate, mineral oil and the like.

The method of administration of the pharmaceutical composition comprising the extract according to the present invention or the active ingredient compound isolated therefrom can be easily selected according to the formulation and can be administered to mammals such as livestock, human, etc. in various routes. For example, it may be formulated in the form of powders, tablets, pills, granules, dragees, hard or soft capsules, liquids, emulsions, suspensions, syrups, elixirs, external preparations, suppositories, sterilized injection solutions, Orally or parenterally. In particular, oral administration is preferred.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid formulations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used diluents, various excipients such as wetting agents, sweeteners, have.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol, gelatin and the like can be used.

Further, the pharmaceutical compositions comprising the extract according to the present invention may be prepared using the appropriate methods known in the art or by methods disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA) And the like.

The dosage of the pharmaceutical composition comprising the extract according to the present invention or the active ingredient compound isolated therefrom may vary depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, The effective dose of the extract is usually about 1 to 100 g / day, especially about 10 to 50 g / day, more preferably about 30 g / day for an adult (60 kg). It will be apparent to those skilled in the art that doses may be additive or subtracted, as the dosage can vary depending on various conditions, and thus the dose is not intended to limit the scope of the invention in any way.

The number of administrations can be administered once or several times a day within a desired range, and the administration period is not particularly limited. In addition, the extract of the present invention or a composition comprising the active ingredient compound isolated therefrom may be added to any food in addition to oral administration as it is, and may be routinely ingested.

The pharmaceutical composition comprising the extract according to the present invention or the active ingredient compound separated therefrom not only provides an excellent therapeutic effect on bone metabolic diseases but also can be safely used for long term administration without toxicity and side effects caused by drugs.

Accordingly, the extract according to the present invention or the active ingredient compound isolated therefrom can be used as a main ingredient, a food ingredient, and a food additive.

In addition, the present invention is a natural vegetable material and can be utilized as a health functional food material for alleviating or improving bone metabolic diseases.

The health functional food comprising the extract of the present invention or the active ingredient compound isolated therefrom can be variously used for foods and beverages for improving or alleviating bone metabolic diseases. Examples of foods to which the extract or compound of the present invention can be added include various foods, beverages, gums, tea, vitamin complexes, health supplements and the like, and they are in the form of powders, granules, tablets, capsules or drinks Can be used.

&Quot; Health functional food " as defined herein means food prepared and processed using raw materials or ingredients having functionality useful to the human body in accordance with Law No. 6727 on Health Functional Foods. &Quot; Functional " Structure and function of the nutrient to control or physiological effects, such as to obtain a beneficial effect for health is intended to eat.

The health functional food according to the present invention contains 0.01 to 95% by weight, preferably 1 to 80% by weight, of the extract or the compound, based on the total weight of the composition.

For the purpose of improving or alleviating bone metabolic diseases, a pharmaceutical dosage form such as powders, granules, tablets, capsules, pills, suspensions, emulsions and syrups, or a health functional food in the form of tea bags, Manufacturing and processing are possible.

In addition, the present invention provides a health supplement having the object of improving or alleviating bone metabolic diseases.

The present invention also provides a food or food additive having an effect of improving or alleviating bone metabolic diseases.

In addition, the above health functional foods may further include food additives, and whether or not they are suitable as "food additives" may be added to the relevant items in accordance with the general provisions of the Food Additives Ordinance approved by the Food and Drug Administration Shall be determined according to the relevant standards and standards.

Examples of the products listed in the above-mentioned "food additives" include natural products such as ketones, chemical products such as glycine, potassium citrate, nicotinic acid and cinnamic acid, coloring matter, licorice extract, crystalline cellulose, guar gum, Sodium laurate, sodium glutamate preparation, noodles-added alkaline agent, preservative agent, tar pigment preparation and the like.

Examples of the functional food containing the extract of the present invention or the active ingredient compound isolated therefrom include confectionery ice cream such as bread, rice cakes, dried fruits, candy, chocolate, chewing gum and mackerel, ice cream products such as ice cream, ice cream powder, , Processed milk products such as low fat milk, lactose decomposed milk, processed oil, goat milk, fermented milk, butter oil, concentrated oil, milk cream, butter oil, natural cheese, processed cheese, milk powder, Meat products such as hamburgers Fish meat products such as fish meat products such as fish cakes, ham, sausage, bacon, etc. Noodles such as ramen, dried noodles, fresh noodles, hot noodles, luxury dried noodles, improved noodles, frozen noodles, Beverage such as vegetable drink, carbonated drink, two oil, yogurt, beverage such as mixed drink, soy sauce, miso, kochujang, chunjang, chonggukjang, mixed bowl, vinegar, sauce, tomato Be seasoned food margarine, shortening, and pizza as concubines, curry dressing, but is not limited thereto.

The health functional beverage composition of the present invention has no particular limitation on the other components other than the above-mentioned purified product as essential components in the indicated ratios and may contain various flavors or natural carbohydrates as additional components such as ordinary beverages . Examples of the above-mentioned natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.); Disaccharide, (e.g., maltose, sucrose, etc.); And polysaccharides (for example, dextrin, cyclodextrin and the like), and sugar alcohols such as xylitol, sorbitol and erythritol. As natural flavors other than those described above, natural flavors (such as tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin)) and synthetic flavors (saccharin, aspartame, etc.) have. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the extract and the compound of the present invention can be added to food or beverage for the purpose of preventing the objective disease. At this time, the amount of the extract and the compound in the food or drink may be 0.01 to 15% by weight of the total food, and the health beverage composition may be added in a proportion of 0.02 to 5 g, preferably 0.3 to 1 g, .

The extracts and compounds according to the present invention added to foods containing beverages in the course of manufacturing the health functional food can be appropriately added or decreased as needed.

Hereinafter, embodiments are provided to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

Example  One. Magnetization number  Preparation of extracts and isolation of active ingredient compounds

Example  1-1. Magnetization number  Preparation of extract

1-1-1. Preparation of 70% ethanol extract

In the dry state of the magnetization ( Angelica decursiva Franchet et Savatier) was received from the Korea Food and Drug Administration (KFDA) Quality Standardization Research Center for Herbal Medicines, washed several times, finely ground, added with 2 L of 70% ethanol, and sonicated at 85 ° C for 3 hours After extraction, the supernatant was recovered by vacuum filtration. After the extraction, the solvent was sufficiently removed by distillation under reduced pressure, and then lyophilized to obtain 171 g of a 70% ethanol extract of Mizuho pref. (Hereinafter referred to as "AD70E"). This extract was dissolved in 0.5% CMC (sodium carboxymethyl cellulose, Cat # 419303, Sigma-Aldrich) and used in the experiment.

1-1-2. Preparation of water extract

500 g of Angelica decursiva Franchet et Savatier containing the above-ground and roots were received from the Institute for Quality Standardization Research for Herbal Medicines at the Food and Drug Administration's Office, washed several times, finely crushed, and 2 L of water was added thereto at 95 ° C After extraction with an ultrasonic wave extractor (8510 DHT; CT, USA) for 3 hours, the supernatant was recovered by vacuum filtration. The extract was distilled under reduced pressure to sufficiently remove the solvent, and then lyophilized to obtain 155 g of a water extract of Magnetophyceae (hereinafter referred to as "ADW"). This extract was dissolved in 0.5% CMC (sodium carboxymethyl cellulose) and used in the experiment.

Example  1-2. Magnetization number  From the extract Of nodakenetin  detach

For the identification of the active substance, A. decursiva (5 kg) was repeatedly extracted 3 times with methanol (MeOH) for 3 hours, and the extract was concentrated under vacuum to obtain methanol extract MeOH extract (1.21 kg). This extract was suspended in distilled water and fractionated as dichloromethane (CH2Cl2), ethyl acetate (EtOAc) and n-butanol (n-BuOH). CH2Cl2 (150 g), EtOAc (22 g) and n-BuOH (290 g) ). The double EtOAc fractions were subjected to silica gel column chromatography to obtain nodakenetin (3.5 g). The remaining BuOH fractions were subjected to silica gel column chromatography to obtain nodakenin (48.5 g). The two compounds were identified as nodecenen and nodecenin by NMR and MS spectroscopic methods.

Nodakenetin

Nodakenin

Example  2. Magnetization number  The extract and Nodakenetin HEK293  Cytotoxicity analysis of cell lines

In order to confirm the cytotoxicity of HEK293 cells, which are human embryonic kidney cell lines, in the test tube of the sample obtained in Example 1, experiments were conducted as follows (Lee SK et al (2008) Chem Biol. Interact 115: 215-28).

HEK293 was distributed from the American Type Culture Collection (ATCC; Manassas, Va., USA), a US cell line bank (CCL-247).

HEK293 used in the present invention was subcultured in DMEM medium (# 12800-017, Gibco, Grand Island, NY) containing 10% FBS, 1% PSF and the like. To each well of a 96-well plate, 10 μl of a sample dissolved in 10% DMSO and 190 μl (6 × 10 ⁴ cells / ml) of the cell suspension were added and cultured for 3 days. 190 μl of the cell suspension was added to at least 16 wells and incubated for 30 minutes, and used as a zero-day control before the experiment. The cultured cells were fixed with 10% TCA (trichloroacetic acid), stained with SRB solution (S9012, Sigma), dissolved in a 10 mM Tris base, and then absorbed at 515 nm. 10% DMSO was used as a control, and the cell survival rate according to the treatment of each test substance was measured using the following equation (1).

[Equation 1]

When the control group without the sample was taken as 100%, the value of the sample treatment group was expressed as a percentage of the control group, and the treatment of each test substance was calculated by the mean value ± SEM of the double or triple test.

As shown in Figs. 1 and 2, the extract obtained in Example 1 or the compound isolated therefrom showed a cell survival rate of 90% or more up to a concentration of water extract, 50 μg / ml and nodecane tin, 100 μM, .

Example  3. Magnetic Phone Extract and Molecular Mechanism Analysis of the Active Ingredients Extracted Therefrom According to the Present Invention

Example  3-1. β- Catenin  Introduced HEK293  In the cell line Magnetization number  The extract and Nodakenetin TCF / LEF luciferase  Promotes activity-promoting efficacy

The mechanism of bone metabolism of the extracts or isolated active compounds according to the present invention was investigated. For this purpose, β-catenin was introduced into HEK293 cells, and the molecular mechanism was evaluated by treating the extract with Magnetophobia. Park et al. (E-j. Park et al (2009) Bioorg Med Chem Lett 19: 2282-2284).

First, in order to confirm whether or not the extracts of Magnetium japonica were related to the transcriptional regulation of TCF / LEF, a transcription factor of β-catenin, HEK293 cells were transfected with plasmids containing TCF4 and pcDNA β-catenin and TCF binding sites ) Was transfected, and then the effect of the extracts of Magnetium japonica on the activity of luciferase activity according to the increase of transcriptional activity of TCF was tested as follows.

HEK293 cells in the 10% FBS adjusted so that a 1.5 ⅹ 10 ⁴ per well in a DMEM medium 48 well plate containing the cultured 24 hours at 37 ℃, 5% CO ₂ conditions, and have a TRE binding site attached to the luciferase plasmid Invitrogen, Grand Island, NY) and pRL-SV40 vector (E2231, Promega, Madison, WI) and lipofectamine 2000 (# 11668-019, Invitrogen, Grand Island, (6.25, 12.5, 25, 50 μg / ml) pre-diluted in DMEM medium containing 10% FBS and nordakenethin (6.25, 12.5, (E1910, Promega, Madison, WI). After the supernatant was removed, the supernatant was removed and the supernatant was suspended in 1X Passive Lysis Buffer (E194A, Promega, Madison, WI) Luciferase activity was measured with a luminometer (Cetro LB 960, Berthold, Germany), and the transfection efficiency The rate was calibrated with pRL-SV40 luciferase activity.

As shown in the following Table 1 and Figures 3 and 4, when TCF4, pcDNA beta -catenin and TOPFlash were introduced into HEK293 cells, the luciferase activity was increased compared to the control group in which TOPFlash alone or TOPFlash and TCF4 plasmids were introduced together . (6.25, 12.5, 25, 50 μg / ml) and nodakenethin (6.25, 12.5, 25, 50 μM) were added to HEK293 cells and TCF / LEF transcription Activity was analyzed statistically (* P <0.05; ** P <0.01), and the transcriptional promoting activity was dependent on the concentration. In detail, water extracts showed 6.25, 12.5, 25 and 50 μg / 120.2, 130.2, and 185.3% of TCF / LEF transcriptional activity was promoted at 6.25, 12.5, 25, and 50 μM, respectively, in the case of nordakenethin.

When the plasmid (FOPFlash) with mutation of the TCF binding site was transfected using the water extract of Magnetization and nodakenethin, TCF4 and pcDNA β-catenin were introduced into the cells without effect on luciferase activity , The extract of N. japonica, or the compound isolated therefrom, showed excellent TCF / LEF transcription promoting activity.

[Table 1]

Example  3-2. HEK293  In the cell line Nodakenetin Wint / β- Catechin  Identification of effects on signal transduction pathway protein regulation

In order to investigate the effect of the sub-regulatory factors on the protein factor in the Wint / β-catenin signal transduction pathway, the following experiment was conducted using the described method (Hong JY et al (2011) J Nat Prod 74: 2102-2108 ).

HEK293 cells were cultured in DMEM medium containing 10% FBS at a concentration of 1 × 10 ⁵ in a 100 mm culture dish, cultured at 37 ° C. in 5% CO ₂ for 24 hours, and then incubated with phosphate buffered saline (PBS) for 2 Lt; / RTI &gt; 10 ml of a DMEM medium containing 10% FBS diluted by concentration in advance was added and cultured for a predetermined time. Cells that were not attached to the cell culture medium and attached cells were collected and washed twice with PBS, and then boiled 2 × sample loading buffer (250 mM Tris-HCl (pH 6.8), 4% SDS, 10% glycerol, 0.006% bromophenol blue , 2% β-mercaptoethanol, 50 mM sodium fluoride and 5 mM sodium orthovanadate), and the cells were incubated at 100 ° C. for 5 minutes. After cooling, the cells were stored at 20 ° C. and dissolved at 37 ° C. Was used in Young Dong. 80 μg of the protein was electrophoresed at 138 V for 2 hours using 5-12% SDS-polyacrylamide gel (# 456-1036, Bio-Rad, Hercules, Calif.). The separated proteins were transferred to a PVDF membrane (ISEQ15150, Millipore, Bedford, MA) for 1 hour at 100 V, washed twice with TBST and blocked with 5% Albumin from Bovine Serum in PBS containing 0.1% Tween- )) And cultured in a shaker at room temperature for 1 hour. After washing with TBST 3 times for 5 minutes, the monoclonal antibody was diluted with TBST in 3% Albumin from Bovine Serum (BSA (A9647, Sigma, St Louis, WI)) and incubated with membrane at 4 ° C for 18 hours Lt; / RTI &gt; After washing the membranes (Membrane, ISEQ 1S150, Millipore, Bedford, MA) three times for 5 minutes with TBST, the HRP (horseradish peroxidase) -binding secondary antibody was diluted with TBST to a ratio of 1: 1,000-1: And reacted with the membrane at room temperature for 3 to 4 hours. After washing with TBST three times for 5 minutes, the western blotting substrate (WEST-ZOL PLUS (16021, Intron)) was treated to confirm protein expression level on LAS-3000 (Fuji Film Corp., Japan).

Specifically, HEK293 cells were treated with 12.5, 25, 50, and 100 μM of nordakenethin, cultured for 24 hours, and analyzed for protein expression. As a result, the activity of the ω-catenin signal transduction system was examined in HEK293 cells of nordakenetin. As shown in Fig. 6, the expression of c-Myc, Cyclin D1 and Survivin protein in the downstream signal transduction system was increased in a concentration-dependent manner. In Fig. 6, DKK1, an inhibitor of Wnt / Respectively.

In conclusion, it was concluded that the extracts of Zygosporium japonica L. and the active ingredients isolated therefrom have an effect on DKK1 and β-catenin, activating the Wnt / β-catenin signaling system.

Example  4. Nodakenetin  Cytotoxicity test on osteoblast

Example  4-1. Osteoblast cell culture

As osteoblast cells, MC3T3-E1 was distributed from the American Type Culture Collection (ATCC; Manassas, Va.). The MC3T3-E1 cells were cultured in MEM-α (minium essential medium alpha, # A1049001, Gibco, Grand Island, NY) containing 10% FBS, 100 U / ml penicillin and 100 μg / ml and without ascorbic acid And cultured at 37 ° C and 5% CO ₂ .

Example  4-2. Cytotoxicity test on osteoblast

The cultured MC3T3-E1 cells were seeded on a 96-well plate at 1.5 × 10 ⁴ cells per well and cultured in MEM-α medium for 24 hours. The medium was removed, and the cell survival rate was measured by MTT assay after culturing in MEM-α medium containing nodecane tin at a certain concentration. The result is shown in FIG. As shown in Fig. 7, the nadakenethin of Example 1 exhibited a cell viability of 80% or more when treated at the highest concentration of 100 μM. As a result, there was no cytotoxicity.

Example  5. Through ALP assay Nodakenetin  Confirm osteoblast activity

In this experiment, the activity of the basic phosphatase was measured using the cultured MC3T3-E1 cells to evaluate the effect of the extract of Magnetized Phytophthora and Nodakenine in Example 1 on osteoblast activation. In general, the basic phosphatase is known to be a specific marker of early osteoblast, since its activity is evident at the early stage of osteoblast differentiation.

MC3T3-E1 cells were added to a 48-well plate at 0.6 x 10 ⁴ per well, cells were formed into a monolayer, treated with vitamin C and? -Glycerophosphate to induce differentiation of osteoblasts, Lt; RTI ID = 0.0 &gt; MEM-a &lt; / RTI &gt; medium. As a comparative example, 17? -Estradiol currently used as a therapeutic agent for osteoporosis was treated at a certain concentration. After 6 days, the cells were lysed with 1 × passive lysis buffer, and 10 μl of lysates and 190 μl of 0.5 M Tris-HCl (pH 9.9) buffer containing 6 mM p-nitrophenyl phosphate and 1 mM MgCl ₂ were incubated at 37 ° C. for 1 hour Lt; / RTI &gt; The p-nitrophenol (PNP) produced by the enzyme reaction was quantitated by measuring the absorbance at 405 nm using the PNP standard curve, and the protein was quantified using the Bradford method. ALP activity is shown in Figure 8 as PNP produced / min / mg protein.

As shown in FIG. 8, the ALP activity was not significantly increased in the untreated control group, whereas the ALP activity was significantly increased in the nodecenethin-treated group of Example 1 of the present invention in a concentration-dependent manner, &Lt; / RTI &gt;

Example  6. Mineralization  Through staining Nodakenetin  Confirm osteoblast activity

After inducing differentiation into osteoblasts as in Example 5, the nadakenethin of Example 1 was cultured in a MEM-α medium containing a certain concentration. As a comparative example, 17 [beta] -estradiol currently used as a therapeutic agent for osteoporosis was treated at a certain concentration. After 10 days, the cells were washed once with PBS, fixed with 4% formaldehyde for 10 minutes, and stained with 40 mM alizarin red solution (Alizarin red S, pH 4.2) for 30 minutes at room temperature. The solution was washed with third distilled water and PBS several times, and dissolved in 10% cetylpyridinium chloride solution dissolved in 10 mM sodium phosphate (pH 7.0). The absorbance at 570 nm was measured and shown in FIG.

As shown in FIG. 9, the untreated control group did not undergo much mineralization, whereas the group treated with nodecenethin of Example 1 of the present invention showed a significant increase in the concentration of mineralization in a concentration-dependent manner, And the effect of increasing bone formation by calcium production.

Example  7. Nodakenetin  The osteoclast- Catechin , BMP2  Identification of effects on gene regulation

To confirm the effect of β-catenin and osteoblast differentiation-related gene expression on the osteoblast of the sample obtained in Example 1, an experiment was conducted as described below (Kang YJ et al (2012) Mol Pharm 82: 168-177).

MC3T3-E1 cells (8 × 10 ⁴ cells / ml) were cultured in MEM-α medium containing 10% FBS in a 100 mm dish at 37 ° C and 5% CO ₂ for 24 hours. The diluted samples were then added to the medium Time. Cells were washed twice with PBS, and then cells were disrupted using TRI reagent (TRIZol (# 15596-026, Invitrogen, Grand Island, NY)). RNA was extracted by adding CHCl ₃ and isopropyl alcohol Precipitated. RNA precipitates were washed with 70% ethanol, dried in air and then dissolved in nuclease-free water (AM9937, Ambion, Austin, Tex.), Heated at 55 ° C for 10 min and heat- treated at 70 ° C for 5 min. single strand state. Total RNA was quantitated with NanoDrop (ND-1000, Dae Myung Sceince, Seoul, Korea) and diluted to 1 μg / μl. The avian myeloblastosis virus (AMV) reverse transcriptase (M5108, Promega, Madison, WI) ) 15 primer (C110B, Promega, Madison, WI). The target gene was amplified using the MiniOpticon ™ Real-time PCR Detection system (CFB-3120, Bio-Rad) using iQ ™ SYBR® Green Supermix (# 170-8880, Bio-Rad, Hercules, CA) and target gene- , Hercules, Calif.). Real-time PCR conditions were denaturation at 95 ° C for 20 seconds, followed by denaturation at 95 ° C for 20 seconds, annealing at 56 ° C for 20 seconds, and elongation at 72 ° C for 40 cycles Followed by a final step at 95 ° C for 1 min and at 55 ° C for 1 min. Ct values were determined by the method of Leak et al. (Livak KJ et al (1996) Method 25: 402-408) using MJ Opticon Monitor software (Opticon Monitor 3.1.32, Bio-rad, Hercules, CA) Actin, and the expression of β-catenin and BMP2 gene was reduced by nodecane-tin, as shown in FIG. 10 and FIG.

[Table 2] Real-time PCR primer

Example  8. MC3T3 -E1 cell line Wint / β- Catechin  Measurement of effect on signaling pathway protein regulation

In order to investigate the effect of the sub-regulatory factors on the protein factor in the Wint / β-catenin signal transduction pathway, the following experiment was conducted using the described method (Hong JY et al. (2011) J Nat Prod 74: 2102-8 ).

MC3T3-E1 cells (8 X 104 cells / ml) were cultured in MEM-a medium containing 10% FBS for 24 hours at 37 ° C and 5% CO ₂ in a 100 mm dish and washed twice with PBS. 10 ml of MEM-α medium containing 10% FBS diluted by concentration in advance was added and cultured for a certain period of time. Cells that were not attached to the cell culture medium and attached cells were collected and washed twice with PBS, and then boiled 2 × sample loading buffer (250 mM Tris-HCl (pH 6.8), 4% SDS, 10% glycerol, 0.006% bromophenol blue , 2% β-mercaptoethanol, 50 mM sodium fluoride and 5 mM sodium orthovanadate), and the cells were incubated at 100 ° C. for 5 min. After cooling, the cells were stored at 20 ° C. and dissolved at 37 ° C. for protein quantification and electrophoresis Lt; / RTI &gt; 80 μg of the protein was electrophoresed at 138 V for 2 hours using 5-12% SDS-polyacrylamide gel (# 456-1036, Bio-Rad, Hercules, Calif.). The separated proteins were transferred to a PVDF membrane (ISEQ15150, Millipore, Bedford, MA) for 1 hour at 100 V, washed twice with TBST and blocked with 5% Albumin from Bovine Serum in PBS containing 0.1% Tween- After washing with TBST three times for 5 minutes, the monoclonal antibody was diluted with TBST in 3% Albumin from Bovine Serum (BSA, Sigma, St Louis, MD) After washing the membrane (Membrane, ISEQ 1S150, Millipore, Bedford, MA) with TBST three times for 5 minutes, HRP (horseradish peroxidase) -binding secondary antibody was incubated with TBST for 3 hrs. (BSA) at a ratio of 1: 1,000 to 1: 2,000 and reacted with the membrane at room temperature for 3 to 4 hours. After washing with TBST three times for 5 minutes, a Western blotting substrate (WEST-ZOL PLUS 16021, Intron, Seongnam, South Korea) was treated with LAS-3000 (Fuji Film Corp., Japan) And the degree of protein expression was confirmed.

In detail, MC3T3-E1 cells were treated with 12.5, 25, 50 and 100 μM of nodecane tin, cultured for 24 hours, and analyzed for protein expression to confirm the activity of the mouse / β-catenin signal transduction system in MC3T3-E1 cells As a result, as shown in Fig. 12, DKK1, which is an inhibitor of the &lt; RTI ID = 0.0 &gt; winter / beta -catenin &lt; / RTI &gt; signal transduction was decreased and beta -catenin was increased and the subadrenergic c-Myc, cyclin D1 and Survivin protein expression was increased in a concentration- As shown in FIG. 13, BMP2 and BMP4 increased. In conclusion, the extracts of Magnetophyllum japonica affect DKK1 and β-catenin, activating the wint / β-catenin signal transduction system, and promoting osteoblast differentiation by increasing BMP2 and BMP4.

Example  9. Measurement of morphological changes of bone in animal models

The ovariectomized animal model was prepared by ovariectomy (OVX group) of 8-week-old ICR female mice (28-30g) or ovariectomy (Sham group). After 1 week recovery period, , Nodakenine, and nadakenetin were administered at the indicated concentrations five times a week for 12 weeks from week 9 to week 21, and administration of 17β-estradiol (10 μg / kg / day), the osteoporosis treatment agent currently used as a comparative substance, (E2 group).

All groups were weighed at 4-week intervals to evaluate changes in body weight with osteoporosis and sample efficacy. Females were separated and used in the following experiments after 12 weeks of sample administration.

In order to determine the morphological changes of the bones, the extracts of Magnetophyceae (100 mg / kg / day), nodakenine and nodecaneethin were administered to the ovariectomized mice at a dose of 50 and 100 mg / kg / The femur was separated and scanned with micro-computed tomography (microCT, Skyscan 1076). The scanned data was processed into a second cross-sectional image using NRrecon software (Skyscan). The bone volume / total volume of the femur (BV / TV, bone volume ratio), trabecular number (Tb.N.), trabecular separation (Tb. Sp.) And structure model index (SMI) Bone Mineral Density (BMD) changes were measured, and 200 sections were selected from the femoral end. The third reconstruction image of the implemented femoral end was used to evaluate the morphological changes of osteoporosis and cortical bone by the administration of the sample. The characteristics of the three-dimensional fine cancellous bone and cortical bone structure in each group are shown in Fig. 14, and the body weight of the ovariectomized group (OVX group) is lower than that of the ovariectomized group (Sham group) Respectively. On the other hand, it was confirmed that the bone microstructure was recovered in the drug administration group.

FIG. 15A shows that the bone volume ratio BV / TV of the OVX group was 4.9%, which was 42.4% lower than that of the Sham group (8.5%). In addition, BV / TV of the extracts of Magnolia sieboldii (water, ethanol extract, 100 mg / kg / day), nodarkenine and nodakenethine (50 and 100 mg / kg / day, respectively) The NODKENIN 50 and 100 mg / mL / day were 1.5 times higher than the OVX group, and the NODKENETIN 50 and 100 mg / mL / day were higher than BV / TV increased by 1.9 times and 2.2 times. BV / TV in the E2 group, positive control group, was 10.8%, 1.3 times more than Sham group.

The number of cancellous bone (Tb.No) was 46.8% lower in the OVX group than in the Sham group, and the extracts of Magnetium succinate (water, ethanol extract concentration 100 mg / ml / day), nodakenine and nodakenethin 100 mg / kg / day), Tb. NODAKENIN 50 and 100 mg / mL / day increased 1.5 times and 1.6 times, respectively, and NODKENETIN 50, 100 mg / mL / day, day is Tb. No. increased by 1.9 times and 2.1 times. Number of cancellous bone in E2 group, positive control group, Tb. No. 1 was increased 1.1-fold compared to the Sham group (Fig. 15B).

In the sponge-bone interval (Tb. Sp.), The OVX group was 1.4 times higher than that of the Sham group, and the magnetized water and ethanol extracts (100 mg / ml / day) were decreased by 25.8% and 9.9% NODAKENIN 50 and 100 mg / mL / day were decreased by 9.5% and 17.2%, respectively. Nodakenethin 50 and Tb 100 mg / mL / day were decreased. Sp., Decreased by 15.5% and 20.4% (Fig. 15C).

The bone mineral density (BMD) of the ovariectomized group (OVX group) was 60.4% lower than that of the Sham group as shown in Fig. 15D. The water and ethanol extracts of the ovariectomized group were significantly lower than those of the OVX group BMD was increased 2.5 and 1.7 times, respectively. Nodakenine 50 and 100 mg / ml / day were increased 1.8 and 2.1 times, respectively. Nodakenethin 50 and 100 mg / ml / day were increased 2.2 and 2.4 times, respectively Respectively. The bone mineral density of the E2 group, a positive control group, was increased by 10% compared with that of the Sham group. Therefore, it is considered that the extracts of N. japonica, nodokenine and nodecaneethyne are useful for the prevention and treatment of osteoporosis.

Hereinafter, formulation examples of the composition containing the compound of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Formulation example  One. Sanje  Produce

Magnetophyceae extract (including nodakenine and nodakenethin) 200 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

Magnetophyceae extract (including nodakenine and nodakenethin) 200 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3. Preparation of capsules

Magnetophyceae extract (including nodakenine and nodakenethin) 200 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed in accordance with a conventional method for producing a capsule, and filled in a gelatin capsule to prepare a capsule.

Formulation example  4. Preparation of injections

Magnetophyceae extract (including nodakenine and nodakenethin) 200 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ , 12H ₂ O 26 mg

(2 ml) per 1 ampoule according to the usual injection preparation method.

Formulation example  5. Liquid  Produce

Magnetophyceae extract (including nodakenine and nodakenethin) 200 mg

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and then purified water was added thereto. The whole was adjusted to 100 ml by adding purified water, To prepare a liquid agent.

Formulation example  6. Of health food  Produce

Magnetophyceae extract (with nodakenine, nodakenethin) 1000 mg

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

0.5 mg vitamin B6

0.2 [mu] g vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

24.8 mg of magnesium chloride

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7. Manufacture of health drinks

Magnetophyceae extract (with nodakenine, nodakenethin) 1000 mg

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated for about 1 hour at 85 DEG C with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, &Lt; / RTI &gt;

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

While the present invention has been described in connection with what is presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

Claims (9)

A pharmaceutical composition for preventing or treating osteoporosis, which comprises, as an active ingredient, nodarkenine or nodakenethin isolated from Angelica decursiva Franchet et Savatier extract.
The method according to claim 1,
The pharmaceutical composition for preventing or treating osteoporosis according to claim 1, wherein the extract is a supernatant comprising a root part and a root part including flowers, branches, stems, leaves, immature fruit or fruit.
The method according to claim 1,
The pharmaceutical composition for prevention or treatment of osteoporosis, which is a polar solvent extract.
A health functional food for preventing or ameliorating osteoporosis comprising nodakenine or nodakenethin as an active ingredient, which is isolated from the extract of Matsukushima prefecture. delete delete delete delete delete

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