KR100694570B1 - Composition comprising the extract of ficus erecta. for preventing and treating osteoporosis - Google Patents

Abstract

An extract of Ficus erecta or a fraction thereof are provided to inhibit the expression of IL-6 and COX-2 in a cell where osteoporosis is induced by IL-1beta, and show inhibition effect on TRAP numerical value of an index of osteoclast formation and also excellent inhibition effect on INF-gamma involving with osteoclast formation, thereby being usefully used for medicines and health supplemental foods for preventing and treating osteoporosis. The pharmaceutical composition or health supplemental food for preventing and treating osteoporosis comprises a methanol extract of Ficus erecta or a chloroform fraction thereof as an effective ingredient.

Description

Composition for the prevention and treatment of osteoporosis comprising the narrow leaf scabbard and extract {Composition comprising the extract of Ficus erecta. for preventing and treating osteoporosis}

1 is a view showing a process for producing a narrow leaf cheonnyeol and extracts and fractions,

Figure 2 is a diagram showing the IL-6 inhibitory effect and cell viability of the narrow leaf perennial solvent and fractions by solvent,

Figure 3 is a diagram showing the IL-6 inhibitory effect and cell survival rate by the concentration of the narrow leaf cheoncheon and hexane fraction,

Figure 4 is a diagram showing the IL-6 inhibitory effect and cell survival rate by the concentration of the narrow leaf cheonmyeon and chloroform fraction,

Figure 5 is a diagram showing the IL-6 inhibitory effect and cell survival rate by the concentration of the narrow leaf peridot and ethyl acetate fraction,

Figure 6 shows the expression level of COX-2 mRNA when treated for 1 day at a concentration of 100 ㎍ / ml of the narrow leaf perennial and solvent-specific fractions,

FIG. 7 shows the degree of COX-2 protein expression through Western blotting when treated at 100 μg / ml at 100 μg / ml of a narrow leaf periflorum and solvent fractions.

8 is a diagram showing the osteoclast formation inhibitory effect and cell viability when treated for 4 days at 100㎍ / ml concentration of the narrow leaf perennial and solvent fractions through TRAP assay,

9 is a diagram showing the effect of inhibiting mature osteoclast formation after treatment for 7 days at the concentration of 100 ㎍ / ml of the narrow leaf and the solvent-specific fractions through TRAP staining,

10 is a diagram showing the effect of inhibiting mature osteoclast formation after treatment for 7 days at 50 ㎍ / ml concentration of the narrow leaf perium and the fraction for each solvent through TRAP staining,

FIG. 11 is a diagram showing the effect of inhibiting mature osteoclast formation after treatment for 7 days at 25 ㎍ / ml concentration of the narrow leaf periflorum and solvent fractions through TRAP staining,

12 is a diagram showing the osteoclast formation inhibitory effect and cytotoxicity according to the concentration of the narrow leaf cheonmyeon and CHCl ₃ fraction treatment group through TRAP staining.

The present invention relates to a composition for the prevention and treatment of osteoporosis containing a narrow leaf zenith and extracts and fractions.

Contrary to what we generally know, humans continue to synthesize and break down bones not only during the growth phase but also throughout their lives (Mundy et al., Growth Regul . , 3 (2) , pp124-128, 1993). This breakdown of bones continues during the growing season, but nevertheless, the growth continues because the synthesis occurs more actively than the breakdown of bones.

However, in older age, where the effects of various environmental effects, mental or physical changes in the body, or disintegration are more active than making bones, bone breaks can occur easily with small gunshots. Osteoporosis, which often occurs in postmenopausal women, is a disease caused by the formation of spaces between bones, making it more difficult to make bones, and more active in breaking down bones due to hormone imbalances in the body. to be. In addition, osteopetrosis is a disease in which bone space is calcified when bone breakdown does not occur, and bone development does not occur, or the arms and legs are short, and bone marrow formation, which is the basis of all immune cells, does not occur. This also happens. This osteoporosis also breaks up like bones with osteoporosis because the bones are so hard. However, osteoporosis occurs more frequently than osteoporosis in Korea, and osteoporosis is emerging as a post-menopausal problem worldwide (Teitelbaum SL. Et al., Journal of Bone and Mineral Metabolism, 18 (6)). , pp344-349, 1996).

Bone is a specialized tissue that combines a hardened calcified surface with an internal cellular component called the bone marrow. The combination of these two physiologically different structures is due to the life-long process of bone remodeling, which is caused by the bones of osteoclasts in the bone marrow by hormones or physical stimuli applied to the bone. It is explained by the bone resorption that breaks down and destroys the bone as it digs into the surface and the bone formation of bone matrix by osteoblasts that gathered at the site where the absorption took place (Park JS., Natl Nutr., 215 , pp 25-31, 2000).

Osteoblasts located on the bone surface have glycoprotein enzyme ALP (alkaline phosphatase) on the cell membrane, and collagen type I, osteocalcin, osteopontin and bone sialoprotein. It plays a role in the secretion and calcification of bone matrix substances (Collier FM, Huang WH et al ,, Endocrinology, 139 , pp1258-1267, 1998), which is parathyroid hormone (PTH), calcitonin, active vitamin D, and sex hormones (women) Hormones estrogen), insulin-like growth factor-1 (ITG-1), transforming growth factor-β (TGF-β), IL-1 (interleukin-1), and IL-6 (secreted by systemic hormones such as insulin and bone tissue) interleukin-6), etc. (Kang Soon Ah, Jang Ki-ho et al., J. Arche, 8 , pp 44-48, 2001).

This work in the bones is carried out by the sharing of two cells: osteoclasts responsible for bone destruction and osteoblasts responsible for bone synthesis. These two cells have different origins as opposed to what they do. Osteoclasts are multinucleated giant cells that exist only in bone and have the ability to absorb bone. They are hematopoietic cells derived from the colony-forming unit granulocyte macrophage (CFU-GM), but differentiate differently from the monocyte / macrophage family. In contrast, osteoblasts originate from stem cells that form the mesenchyma. (Takahashi et al , 1999)

Recently, it has been found that osteoblasts, bone marrow stromal cells or activated T cells are required for differentiation between osteoblasts, stromal cells, activated T cells and osteoclasts (Kong YY, Toshida). H. et al., Nature, 397 , pp 304-309, 1999). Osteoclast differentiation factor (ODF) / osteoprotegerin ligand (OPGL) / TNF-related activation-induced cytokine (TRN) / receptor activator of NF-kB ligand (RANKL) expressed in these types belong to the Tumor Necrosis Factor (TNF) ligand family The membrane transmembrane protein of II (transmembrane protein) binds to the receptor activator of NF-kB (RANK) of the osteoclast precursor cells (osteoclast precusor) to induce the formation of osteoclasts. In this process, RANKL acts not as a growth factor but as a differentiation factor. Osteoclasts are called osteoclastogensis, a process formed by the fusion of a monocyte / macrophage mononuclear progenitor. OPG produced in osteoblast / stromal cells inhibits the binding of RANKL and RANK, inhibiting the formation of osteoclasts (Simonent WS, DL Lacey et al., Cell, 89 , pp309-319). , 1997).

Osteoclasts first recognize the RGD sequence, such as osteopontin in the bone matrix, integrin, the adhesion molecule on the cell membrane, and adheres to the bone surface. A wrinkled film is formed on the bone surface surrounded by integrins (Teitelbaum SL. Et al., Journal of Bone and Mineral Metabolism, 18 (6) , pp344-349, 1996). The cytoplasm of osteoclasts has high tartaric acid-resistant phosphatase activity and deoxidative dehydratase activity involved in atomic (H +) production. The atoms (H +) produced by the activity of deoxidation dehydratase are discharged out of the cell by a proton pump. In microenvironments, acidic conditions of around pH 4 are maintained and bone mineral dissolution begins. Furthermore, the secretion of lysosomal enzymes, such as cathepsin K, degrades the organic bone matrix. Under acidic conditions, cathepsin K also degrades into collagen (Vaananen HK, Horton M. et al., Journal of Cell Science , 108 (Pt8) , pp2729-2732, 1995).

The function of osteoclasts is regulated by many hormones and cytokines. However, all of the receptors for vitamin D3, PTH, IL-1, TNF-α and phosphaglandin E2, which are known as bone resorption factors, do not exist in osteoclasts. These factors act on osteoblasts first, and then a humoral factor called osteoclast activating factor is produced by osteoblasts, and it is estimated that osteoclast activation occurs through this factor. (Ducy P, Schinke T et al., Science , 289 (5484) , pp1501-1504, 2000) On the other hand, there are many receptors for calcitonin secreted by the thyroid gland, and the binding of these hormones stops the action of the membrane Bone resorption capacity is sharply lowered. On the other hand, bone absorption is performed by osteoblasts in addition to bone absorption by osteoclasts, and since the osteoblasts are located inside the bone, absorption occurs slowly. Some of the osteoblasts spread into the bone matrix and become osteocytes, while others remain on the surface of the bone to become glandular cells (Kong YY, Feige U et al., Nature , 402 (6759) , pp304-309, 2000).

Bone resorption and bone formation by bone cells are balanced in normal subjects, but changes in all-signal hormones and local factors caused by genetic, physiological and environmental factors relatively increase the activity of osteoclasts and chemical changes in bone. Without this, bone uptake increases, leading to lower bone density and osteoporosis. Confucianism is caused by a number of causes and is generally referred to as secondary (primary) osteoporosis when there is a history of osteoporosis, and when it occurs after menopause without this cause, it is called primary (primary) osteoporosis (KIM KS, The Women's News, seoul, p 10-11, 1998). The incidence of osteoporosis, especially in postmenopausal women, can be explained by the rapid increase in bone loss due to decreased secretion of the female hormone estrogen after menopause (Kim KS, Min BK et al., Kor. J. Soc. Menopause, 6 (1) , pp 36-42, 2000).

Bone resorption inhibitors such as estrogen, calcitonin, and bisphosphonate, which are the current treatments for osteoporosis, are commonly used. Drugs that promote bone formation include fluorine and parathyroid hormone, and vitamin D metabolites and calcium are used. There is no satisfactory performance yet (Rogers J., N. Engl. J. Med. , 280 , pp 364-367, 1967). Estrogen is basically used to treat osteoporosis in postmenopausal women, in which bone mass is maintained or bone loss is reduced. However, when it is used for the purpose of prevention in people who are at high risk of developing osteoporosis, it is reported that it has a good effect on the increase and maintenance of bone mass, and it is suggested that prevention using bone resorption inhibitors such as estrogen before menopause is more important than treatment after osteoporosis. It is becoming. (KIM KS, The Women's News, seoul, p31-62, 1998) However, long-term use of estrogen has been reported to cause endometrial and breast cancer, including side effects such as nausea, headache, weight gain, breast pain, and irregular uterine bleeding. There is a need to develop new therapeutic means that can be used safely and long-term to avoid use.

Recently, researches on alternative therapies using active ingredients of natural medicines, such as herbal medicines and foods, have been actively conducted to supplement the risks caused by the administration of estrogen. (John JB et al., Bailliere's Clinical Endocrinnology and Metabolism, 12 (4) , pp543-557, 1998) As a representative alternative therapy for the prevention of postmenopausal osteoporosis, dietary addition of phytoestrogen or food containing large amounts of Ingestion has been attempted (Kenneth D, R. setchell et al., J. Nutr., 129 , pp758s-767s, 1999). Phytoestrogens are high in grains such as alfafa, peas, oatmeal, red beans, rice and soybeans. As for phytoestrogens, research on isoflavones is being actively conducted. Among these, daidzein and genistein are natural products with an estrogen-like structure, but have a lower affinity for ERα than estrogen in cells but bind to ERα (Kuiper GGJM, Enmark E et al., Proceedings of the National Academy of Science of USA , 93 , pp5925-5930, 1996) Not only does it have an estrogen effect but also an anti-estrogen effect, so it does not cause side effects from estrogen supplementation therapy. I am in the limelight.

Amid growing interest in natural products, these natural active substances are expected to be effective in preventing and treating menopausal osteoporosis.

In Jeju Island, the continental and marine climates are clearly distinguished according to the seasons, and the slopes are surrounded by the sea, and the variety of tropical plants, temperate plants, and Korean boreal forests are distributed according to the topographical conditions and special climatic conditions, centering on Mount Halla at 1950m above sea level. The plant has been reported (Yang, Kim WH et al., Principle of Oriental medicine , pp107-111, 2002, Seoulsungbosa). The distribution is also clearly distinguished, making it an important ecological and botanical research site.

Ficus erecta. Is a plant belonging to the fig and is called a thin-leaf snail, and is a tree that grows mainly in Jeju Island and the southern coast of Korea, especially Jeju Island. It grows at the foot of the seashore and is 2 ~ 4m high. The bark is white with ash gray, dark brown cork, and hairless. The leaves are alternate, the lanceolate with the narrower bar, and the length is 10-20cm. The tip is pointed and the edge is flat or coarse. Side veins are 5 ~ 6 pairs, petiole is 1 ~ 4cm long (Lee Chang-bok, 1979, Korea Plant Book). The thin leaf line has been used in the private sector for a long time in 補 中 (보), 益氣 (cooking), 健脾 (dry), (濕), 强筋 壯骨 (ganglia), 消腫 (small sarcoma), 活血 ( Live blood), detoxification. Rheumatoid arthritis, 中 氣 虛弱 (medium weakness), 氣血 衰微 (deafness), Sadisan (powerless and dull limbs), 筋骨 不利 (muscle bone), bruises, 經 閉 (menopause),, Postpartum milk deficiency has been used for treatment. However, there are few reports on the physiological activity of such components.

In this regard, the present inventors treated narrow leaf paragons, extracts and fractions with MG-63 cells, a human osteoblast cell line stimulated with IL-1β (10 ng / ml), to produce IL-6 and COX-2, which are the main agents of osteoporosis. The differentiation of osteoclasts was confirmed by differentiation into osteoblasts using RAW 264.7 cells, a macrophage cell line in rats, and TRAP staining to inhibit the formation of mature osteoclasts. It was confirmed that it suppresses the TRAP level which is an indicator of. In addition, S.D. ovaries were extracted to induce osteoporosis. After artificially administering the extract of the present invention to the white paper, the blood and the femur were separated and the bone weight was measured. As a result, the bone weight loss inhibitory effect was confirmed, and the inhibitory effect of INF-γ involved in osteoclast formation was confirmed. The invention has been completed.

It is an object of the present invention to provide a composition for preventing and treating osteoporosis in a narrow leaf pericardium extract and fraction having IL-6 production and COX-2 expression inhibitory effect and osteoclast formation inhibition.

In order to achieve the above object, the present invention provides a composition for the prevention and treatment of osteoporosis, including extracts and fractions of the narrow leaf snails ( Ficus erecta. ).

The extract includes an extract selected from water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, preferably a mixed solvent of methanol and water.

In addition, the present invention provides a composition for the prevention and treatment of osteoporosis comprising a fraction with a narrow leaf peripaea.

The fraction refers to an extract soluble in water, methanol, butanol, ethyl acetate, hexane and chloroform or a solvent selected from them, preferably ethyl acetate, hexane and chloroform.

Method for separating the extracts and fractions from the narrow leaf of the present invention is as follows.

Hereinafter, the present invention will be described in detail.

For example, the narrow leaf scabbard extract of the present invention is pulverized by narrowing the narrow leaf scabbard, and then about 1 to 20 times the weight of the dried sample, preferably about 5 to 15 times the amount of water, ethanol, C ₁ to C ₄ lower alcohols such as methanol or the like, or a mixed solvent of water and methanol having a mixing ratio (kg / L) of about 1: 0.1 to 1:10, preferably 1: 0.2 to 1: 5, at room temperature Using an extraction method such as stirring extraction, hot water extraction, cold extraction, reflux cooling extraction or ultrasonic extraction for about 1 to 48 hours, preferably 20 to 30 hours, preferably the extract obtained after stirring extraction is filtered, Concentrating or drying under reduced pressure to obtain an extract soluble in a polar solvent;

A second step of suspending the extract in water to sequentially dissolve it in a nonpolar solvent such as hexane, chloroform and ethyl acetate, and then fractionating and filtering with a separating funnel to obtain a hexane fraction, a chloroform fraction and an ethyl acetate fraction;

A third step of sequentially fractionating and filtering the aqueous layer using a polar solvent such as butanol, water, etc. to obtain a butanol fraction and a water fraction;

The hexane fraction, chloroform fraction, ethyl acetate fraction, butanol fraction and water fraction comprises a separation step consisting of a fourth step of drying by concentrated under reduced pressure.

In addition, conventional fractionation processes can also be carried out (Harborne JB, Phytochemical methods: A guide to modern techniques of plant analysis, 3rd Ed. , Pp 6-7, 1998).

The narrow leaf periphery, extracts and fractions obtained by the above method showed an inhibitory effect on the expression of IL-6 and COX-2 and inhibition of TRAP levels, which are indicative of osteoclast formation, in osteoporosis-induced cells with IL-1β. Excellent inhibitory effect on the INF-γ involved in cell formation, it was confirmed that exhibits the prevention and treatment of osteoporosis.

The present invention contains a narrow leaf zenith and extract and fractions obtained by the production method as an active ingredient, and provides a pharmaceutical composition effective for the prevention and treatment of osteoporosis.

The composition for preventing and treating osteoporosis of the present invention comprises the extract and fractions in an amount of 0.1 to 50% by weight based on the total weight of the composition.

Pharmaceutical compositions comprising extracts and fractions of the invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

The pharmaceutical composition comprising the narrow leaf zenith and extracts and fractions according to the present invention, powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. It can be formulated and used in the form of sterile injectable solutions. Carriers, excipients and diluents that may be included in the composition comprising the extract may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate , Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the narrow leaf periphery and extracts and fractions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The narrow leaf periphery and extracts and fractions of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention provides a dietary supplement comprising the above-mentioned narrow leaf zenith and extracts and fractions and food acceptable food additives exhibiting the effect of preventing and treating osteoporosis. Foods to which extracts and fractions can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, health functional foods, powders, granules, tablets, capsules or beverages.

It may also be added to foods or beverages for the purpose of preventing and treating osteoporosis. At this time, the amount of the extract and fractions in the food or beverage may be added in 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml Can be added.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the narrow leaf zenith and extracts and fractions as essential ingredients in the indicated ratios, and additional ingredients such as various flavors or natural carbohydrates, such as ordinary drinks. It may contain as. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the narrow leaf zenith and extracts and fractions of the present invention are a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, such as flavoring agents, coloring and neutralizing agents (cheese, chocolate, etc.), pectic acid and Salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the extracts and fractions of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited by the following Experimental Examples.

Example 1. Preparation of a narrow leaf zenith and extract (see FIG. 1)

1-1. Crude Extract Preparation

The leaves of Ficus erecta. Native to Jeju Island were collected, dried, and ground to fine grinding. 50 g of the fine powder sample was extracted twice with 80% MeOH, filtered and concentrated under reduced pressure to obtain 23.8 g of methanol extract (hereinafter referred to as FEM).

1-2. Fraction preparation by solvent

1-2-1. Preparation of n-Hexane Fractions from Narrow Leaf Lines

       5.44 g of the methanol extract of Example 1-1 was suspended in 500 ml of distilled water, and then extracted and fractionated three times with 500 ml of n-hexane, the n-hexane fractions were combined, and the solvent was evaporated with a rotary evaporator to n-hexane fractions. 0.5931 g (hereinafter referred to as FEH) was obtained.

1-2-2. CHCl with Narrow Leaf Lines ₃ Preparation of Fractions

The remaining layer (supernatant) except for the n-hexane fraction of Example 1-2-1 was extracted and fractionated three times with CHCl ₃ , the CHCl ₃ fractions were combined, and the solvent was evaporated with a rotary evaporator to remove the CHCl ₃ fraction 0.4586. g (hereinafter referred to as FEC) was obtained.

1-2-3. Preparation of EtOAc Fraction with Narrow Leaf Lines

The remaining layer (supernatant), except for the CHCl ₃ fraction of Example 1-2-2, was extracted and fractionated three times with EtOAc, the EtOAc soluble layers were combined, and the solvent was evaporated with a rotary evaporator to give 0.111 g of EtOAc fraction (hereinafter, Named FEE).

1-2-4. Preparation of n-BuOH Fractions from Narrow Leaves

       The remaining layer (lower layer), except for the EtOAc fraction of Example 1-2-3, was extracted and fractionated three times with n-BuOH, the n-BuOH fractions were combined, and the solvent was evaporated with a rotary evaporator to n-BuOH fraction 0.4797. g (hereinafter referred to as FEB) was obtained.

1-2-5. H with narrow leaf line ₂ Preparation of 0 Fraction

Example 1-2-4 of the n-BuOH extract three times the remaining layer (lower layer) except for the fraction with H ₂ 0, fraction, H ₂ 0 plus the fraction by evaporating the solvent in the following, a rotary evaporator H ₂ O 3.2091 g (hereinafter referred to as FEW) fractions were obtained.

Reference Example 1. Cell Culture and Reagents

Human osteoblast cell line MG-63 cells and rat macrophage line RAW 264.7 cells were distributed from Korean Cell Line Bank (KCLB) and 100 units / ml penicillin-streptomycin and 10% DMEM medium containing fetal bovine serum (FBS) was incubated in a 37 ° C., 5% CO ₂ incubator. For subculture, MG-63 cells once every 4 days and RAW 264.7 cells once every 3 days Subcultures were performed. It was purchased from Interleukin-1β (Biosource, CA USA) and sRANKL (Biovision, CA 94043 USA), respectively.

Reference Example 2. RNA Isolation

Total RNA extraction was isolated using MRC (TRI-reagent). The cells were homogenized by addition of TRI-reagent and then centrifuged by addition of chloroform. Equivalent amount of isopropanol was added to the supernatant to precipitate RNA, precipitated with RNA, washed with 75% DEPC treated ethanol, dried and dissolved in DEPC treated distilled water. RNA was quantified by measuring the absorbance of 260 nm, and RNA having a value in the range of 1.7 to 1.9 was used for the experiment. All experiments were done under RNase-free conditions.

Reference Example 3. RT-PCR

1 μg total RNA was added at 70 ° C. 5 min, 37 ° C. 5 min, 37 ° C. 60 min, with oligo (dT) ₁₈ primer, dNTP (0.5 μM), 1 unit RNase inhibitor, and M-MuLV reverse transcriptase (2U). The reaction was stopped by warming at 70 ° C. for 10 min. Polymerase Chain Reaction (PCR) was performed to amplify IL-6, COX-2, and β-Actin from the synthesized cDNA, 2 μl cDNA, 4 μM 5 'and 3' primers, 10 x buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.1% Triton X-100), 250 μM dNTP, 25 mM MgCl ₂ , 1 unit Taq polymerase (Promega, USA), mix to 25 μl with distilled water and then Perkin-Elmer Thermal Cycler PCR was performed. At this time, PCR conditions were 94 ℃ / 20 seconds, 55 ~ 60 ℃ / 30 seconds, 72 ℃ / 40 seconds, 30 times, the product produced by PCR is subjected to electrophoresis on 1.5% agarose gel and Et-Br ( Staining with ethidium bromide confirmed specific bands

Sequence Listing of Primers and Size of Gene Products Used in RT-PCR gene Sequence of primer Fragment size (bp) IL-6 Forward direction 5'-AATTCGGTACATCCTCGACG-3 ' 421 Reverse 5'-GCGCAGAATGAGATGAGTTG-3 ' COX-2 Forward direction 5'-TTCAAATGAGATTGTGGGAAAATTGCT-3 ' 305 Reverse 5'-AGATCATCTCTGGCCTGAGTATCTT-3 ' β-Actin Forward direction 5'-ATGGGTCAGAAGGATTCCTATG-3 ' 588 Reverse 5'-CAGCTCGTAGCTCTTCTCCA-3 '

Reference Example 4. Western blotting analysis

MG-63 cells at a concentration of 4 × 10 ⁵ cells / ml were precultured for 18 hours in a 5% CO ₂ incubator using DMEM medium. Thereafter, the medium was removed, and the test material prepared at 10-fold concentration (1 mg / ml) and fresh medium containing IL-1β (10 ng / ml) were simultaneously treated and cultured under the same conditions as the pre-culture. Cells were washed 2-3 times with PBS (phosphate buffered saline) and then 200 μl of lysis buffer was added, dissolved for 30 minutes to 1 hour, and then centrifuged at 15,000 rpm for 15 minutes to remove cell membrane components. Protein concentration was quantified using the Bio-Rad Protein Assay Kit by standardizing BSA (bovine serum albumin). 20-30 μg of lysate was denatured by 10% mini gel SDS-PAGE (Poly Acrylamide Gel Electrophoresis), which was transferred to PVDF membrane (BIO-RAD) at 200 mA for 2 hours. The blocking of the membrane was performed overnight in TTBS (TBS + 0.1% Tween 20) solution containing 5% skim milk (blocking reagent). Anti-goat COX-2 (Santa-Cruz) is 1: 1000 for examining the amount of COX-2 expression, and anti-goat COX-2 ( Santa-Cruz) was diluted 1: 1000 in a TTBS solution, reacted at room temperature for 2 hours, and washed three times with TTBS. As a secondary antibody, HRP (Horse Radish Peroxidase) conjugated anti-goat IgG (Amersham Co.) was diluted to 1: 5000, reacted at room temperature for 40 minutes, washed three times with TTBS and then ECL substrate (Amersham Cooxid). 1 minute after reaction with.) And photosensitive to the X-ray film.

Experimental Example 1. Measurement of the level of production of interleukin-6, a pro-inflammatory cytokine

To investigate the effect of the narrow leaf periphery and the solvent fraction of the leaf on the production of IL-6, mRNA expression of IL-6 in MG-63 cells was confirmed.

Since IL-1β is known to increase IL-6 levels in osteoblasts, 10 ng / ml IL-1β was used to induce the production of IL-6 from MG-63 cells. MG-63 cells, a human osteoblast cell line, were adjusted to 1 × 10 ⁶ cells / ml using DMEM medium, and then inoculated into a 24 well plate and cultured 18 hours before in a 5% CO ₂ incubator. Thereafter, the medium was removed, and 50 μl of the test substance prepared at 10-fold concentration (1 mg / ml) and a fresh medium containing 450 μl of the final concentration of IL-1β (10 ng / ml) were treated simultaneously. Incubated at. After 24 hours, the culture medium was centrifuged (12,000 rpm, 3 min) to measure the IL-6 content of the supernatant obtained (An., 2002). All samples were stored at −20 ° C. or below before quantification.

IL-6 ELISA was quantified; were quantified using the (Human enzyme-linked assay kit immnunosorbent Farmingen system, Inc, USA) r ² value of the standard curve for the standard value was 0.99 or more.

MG-63 cells (4 10 ⁵ cells / ml) were incubated 18 hours ago and treated with 10-fold concentrations of test drug and IL-1β (final concentration 10 ng / ml) incubated hourly, followed by IL- The inhibitory effect on IL-1β-induced cytokine mRNA expression was investigated by treating 1β, thin leaf paragon, and the sample together at a concentration of 100 μg / ml. As a result of treatment with 100 μg / ml of the narrow leaf periphery and the solvent fractions for IL-6 production inhibition, as shown in FIG. 2, 80% MeOH extract and Hexane, CHCl ₃ , EtOAc fraction showed high inhibitory effect. The 80% MeOH extract, hexane, CHCl ₃ and EtOAc fractions showed high inhibitory effect. In addition, by confirming the expression of the housekeeping gene β-Actin at the same time, the narrow leaf periphery and It has shown that it does not affect and decrease itself. As a result of treating the solvent-specific fractions by concentration, as shown in Figures 3 to 5, it was confirmed that the concentration-dependent inhibition of the expression of IL-6 not caused by cytotoxicity.

Experimental Example 2. Measurement of COX-2 Expression

Induction of COX-2 production using IL-1β (10 ng / ml) in MG-63 cells was followed by RT-PCR. Learned through. As shown in FIG. 6, COX-2 was significantly increased by IL-1β stimulation, and treatment of the narrow leaf perforation and fractions significantly inhibited mRNA expression in the Hexane and EtOAc fraction treatment groups.

In addition, COX-2 production was confirmed by Western blot protein amount quantitative analysis. As a result of treatment of the narrow leaf periphery and fractions, Hexane, EtOAc fraction showed a strong inhibitory effect compared to the IL-1β treatment group, even at the protein level as shown in Figure 7, it was confirmed that excellent COX-2 expression inhibition.

Experimental Example 3. Measurement of osteoclast formation

TRAP assay kit was used to investigate the inhibition of TRAP, a biochemical marker of osteoclasts. Induction of osteoclast formation using sRANKL (100 ng / ml) in RAW 264.7 cells was performed by TRAP staining to determine the degree of inhibition of osteoclast formation by narrow leaf striations and fractions.

RAW 264.7 cells were placed in a 24 well plate at 4.3 × 10 ⁴ cells / well, and then cultured for 4 days in a 37 ° C., 5% CO ₂ incubator with sRANKL 100ng / ml. A certain amount The medium was taken and centrifuged to quantify the amount of TRAP, an indicator of osteoclasts, with the supernatant and TRAP assay kit. After removing all the medium and washed once with PBS, fixed solution was added 50ul / well. After fixing the cells for 5 minutes, washed three times with deionized water, 50ul / well each of the substrate solution was added. It was left to stand at 37 ℃ for 1 hour while blocking the light. After washing twice with deionized water and drying, the number of mature osteoclasts was counted under a microscope.

As a result, the osteoclast formation was markedly increased by sRANKL, and it was confirmed that the osteoclast formation inhibitory effect was excellent in the narrow leaf periphery and the fraction treatment group hexane, CHCl ₃ and EtOAc fraction treatment group. 8 and 9 are shown by varying the treatment time of the narrow leaf zenith and the fraction, it was confirmed that the cytotoxicity is lower than when treated for 7 days when treated for 4 days at 100㎍ / ml concentration, Figure 9 and Comparing FIG. 10 and FIG. 11, in the 50 ㎍ / ml concentration treatment group compared to the 100 ㎍ / ml concentration treatment group, it was confirmed that the cytotoxicity by the narrow leaf periphery and fractions was significantly reduced in the 52 ㎍ / ml concentration treatment group. . In addition, as shown in Figure 12, it was confirmed that the osteoclast formation inhibitory effect and cytotoxicity of the CHCl ₃ fraction treatment group having the strongest cytotoxicity selectively inhibits the osteoclast formation without cytotoxicity up to 50㎍ / ml concentration Could.

The narrow leaf periphery of the present invention and extracts and fractions exhibit an inhibitory effect on the expression of IL-6 and COX-2 in the osteoporosis-induced osteoporosis-induced cells and the inhibition of TRAP levels, which are indicative of osteoclast formation. Excellent inhibitory effect on the involved INF-γ can be usefully used as a pharmaceutical composition and health supplement food for the prevention and treatment of osteoporosis.

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A pharmaceutical composition for the prevention and treatment of osteoporosis, containing narrow leaf zenith and methanol soluble extract or narrow leaf zenith and chloroform soluble extract as active ingredients. delete delete A dietary supplement for the prevention and improvement of osteoporosis, which contains a narrow leaf zenith and methanol soluble extract or a narrow leaf zenith and chloroform soluble extract as an active ingredient. delete

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