KR20190134348A - Composition for preventing or treating bone disease comprising extracts of Ramalina litoralis or fractions thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition, a food composition, a quasi-drug composition, and a feed composition for preventing or treating bone diseases, containing a novel lichen Ramalina litoralis extract or a fraction thereof as an active component. Specifically, a novel lichen Ramalina litoralis extract or a fraction thereof of the present invention is derived from natural products and has low toxicity, and is excellent in inhibiting the formation or differentiation of osteoclasts, thereby being able to be usefully used for preventing or treating diseases induced by bone loss or inducing bone loss.

Description

Composition for preventing or treating bone disease comprising extracts of Ramalina litoralis or fractions

The present invention relates to a pharmaceutical composition, a food composition, a quasi-drug composition, and a feed composition for preventing or treating bone disease, including the novel lichen lavina ritoliris extract or a fraction thereof as an active ingredient.

Bone tissue of the human body is a dynamic organ in which bone remodeling, such as bone formation by osteoblasts and bone resorption by osteoclasts, is repeated continuously for life. Bone tissue is composed of cartilage and skeletal system, mechanical functions of supporting and muscle attachment, protecting the organs and bone marrow, and preserving them to maintain the homeostasis of calcium and phosphorus ions. Bone tissue that functions like this is composed of cell substrates such as collagen and glycoprotein, and various kinds of cells such as osteoblasts, osteoclasts and bone cells. Of these, osteoclasts are derived from hematopoietic stem cells and are responsible for uptake of aged bone. Osteoblasts are cells derived from bone marrow stromal cells and play a major role in bone formation.

Among these osteoclasts, RAW264.7 monocyte cells of the mouse are differentiated into multinucleated osteoclasts by the receptor activator of nuclear factor ĸB (RANK) ligand (RANKL). This differentiation process promotes the activation of mitogen-activated protein kinase (MAPK) by RANKL binding to RANK, which is associated with osteoclast differentiation by the transcription factor NF-ĸB entering the nucleus. It is possible by increasing the expression of tartrate-resistant acid phosphatase, matrix metalloproteinase-9, and c-Src tyrosine kinase, and multinucleated osteoclasts formed by this process produce mineralized bone. Absorbs. In addition, binding of RANKL to RANK promotes the activity of TRAF6 (tumor necrosis factor receptor-associated factor 6), thereby promoting the activity of transcription factors such as MARK or NF-ĸB, AP-1, NFATc1 (LEE ZH, KIM). HH.Signal transduction by receptor activator of nuclear factor kappa B in osteoclasts.Biochem Biophys Res Commun. 2003 May 30, 305, 211-4). Therefore, blocking of signaling pathways activated by RANKL is recognized as one of the therapeutic approaches for the treatment of bone diseases including osteoporosis.

Osteoclasts cause abnormal bone tissue destruction and absorption due to imbalance with osteoblasts in the bone, resulting in osteoporosis, which decreases bone mass and bone density, osteomalacia, in which Qudptj lime is depleted, and normal bone tissue. Fibrous dysplasia, which is replaced by fibrous connective tissue and immature bone ossein, periodontal disease in which the alveolar bone is lost, and rheumatoid arthritis, which causes joint destruction and deformation. Known.

Currently, bisphosphonate-based therapies such as Fosamax (component name: aledronate), Actonel (component name: risedronate), and Zometa (component name: zoledronate) are widely used to treat bone damage caused by osteoclasts such as osteoporosis. It is used. However, the number of adverse events such as osteonecrosis, severe atrial fibrillation, incapacitation of bones or joints, and musculoskeletal pain has been increasing each year in patients taking bisphosphonate-based drugs (Br J Cancer 98: 1736 -1740 (2008). Cancer cells that have metastasized to bones in breast cancer and prostate cancer are also promoted to form osteoclasts, resulting in serious bone diseases, and drugs to treat them have not been developed. Therefore, there is a need for the development of drugs that can supplement the disadvantages of the existing bisphosphonate-based drugs, less toxic and effectively inhibit the formation and differentiation of osteoclasts.

Under these backgrounds, the present inventors have sought to find a compound that is derived from natural products and is less toxic and capable of inhibiting the formation or differentiation of osteoclasts. As a result, the novel lichen Lamaritta ritoliris extract or a fraction thereof is used to form osteoclasts or The present invention was completed by recognizing that it can be usefully used for the prevention or treatment of diseases induced by bone loss or causing bone loss by remarkably inhibiting differentiation.

One object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of bone diseases, including the novel lichen Lamaritta Litoraris extract or a fraction thereof as an active ingredient.

Another object of the present invention to provide a food composition for the prevention or improvement of bone disease comprising a novel lichen marinari ritoliris extract or a fraction thereof as an active ingredient.

Another object of the present invention to provide a quasi-drug composition for the prevention or improvement of bone disease comprising a novel lichen marinari ritoliris extract or a fraction thereof as an active ingredient.

Another object of the present invention is to provide a feed composition for the prevention or improvement of bone diseases comprising a novel lichen marinari ritoliris extract or a fraction thereof as an active ingredient.

As one embodiment for achieving the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of bone disease, including a novel lichen Lamaritta Litoraris extract or a fraction thereof as an active ingredient.

In detail, the pharmaceutical composition for preventing or treating bone diseases of the present invention may include a novel lichen Lamarin ritoraris extract or a fraction thereof.

As used herein, the term “lichen lichen” is a symbiotic complex of fungi and algae or / and cyanobacteria and is classified into about 14,000 species. At one time, it has been proposed to use lichen secondary metabolites as antibacterial or anti-herbicides. Some lichen extracts have been used in various treatments in the private sector, and lichen metabolites include antibiotics, antiviral, analgesic, antipyretic, etc. Has a variety of biological activities.

In the present invention, the term "Lamarina Litoraris" is a dendritic ligament that grows in rocks, grows like a shrub, and grows as a non-limiting attachment, and the total length is about 5 cm. The Lamarina litoraris is full-bodied, with a width of 1 mm, a lamellar layer loose, cartilage discontinuous, no cracks, and is known to have a thickness of 30 to 100 μm. The Lamarina Litoraris is also called the Strand.

The present inventors have conducted various studies using substances showing excellent prevention or treatment of bone diseases from natural resources existing in nature, and found that the extract of Lamarina Litoraris has an effect of inhibiting osteoclast differentiation or bone resorption. Confirmed. Through this, it was found that Lamarina Litoraris extract can be used as a pharmaceutical composition for preventing or treating bone diseases. The pharmaceutical composition comprising the extract of Lamarina Litoraris, which has the effect of preventing or treating bone disease, is not known at all until now, and has great significance in that it was first developed by the present inventors.

As used herein, the term "extract" refers to extracting the new lichen limarin or litoraris, but is not limited thereto, and is obtained by drying an extract obtained by extraction, a dilution or concentrate of the extract, and an extract. It may include extracts of all formulations that can be formed using the extract itself and the extract, such as dried products, modifiers thereof, purified products or mixtures thereof.

The novel lichen Lamarin Litoraris extract of the present invention can be prepared using common extraction methods, separation and purification methods known in the art. The extraction method is not limited thereto, but methods such as dipping extraction, hot water extraction, cold extraction, reflux cooling extraction or ultrasonic extraction may be used.

The novel lichens can be extracted with the marinara litoliris water, alcohol having 1 (C ₁ ) to 6 carbon atoms (C ₆ ) or a mixed solvent thereof, but is not limited thereto. Since the degree of extraction and the degree of loss may vary, an appropriate organic solvent may be selected and used. Specifically, it can be extracted using water, an organic solvent or a mixed solvent thereof. More specifically, ethanol may be used, but is not limited thereto.

In addition, the solvent extract may further comprise the step of filtering the extract to remove the suspended solid particles. As an example, the particles may be filtered using cotton, nylon, or the like, or ultrafiltration, freezing, centrifugation, or the like may be used, but is not limited thereto.

Concentration of the extract may be used, such as concentrated under reduced pressure, reverse osmosis concentration.

The drying step after concentration may be a freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, foam drying, high frequency drying or infrared drying. In some cases, the method may further include grinding the final dried extract.

As used herein, the term "fraction" refers to the result obtained by performing fractionation to separate a specific component or a specific group of components from a mixture comprising several different components.

The fractionation method of obtaining the fraction in the present invention is not particularly limited as long as it can exhibit a bone disease prevention or treatment effect, it may be carried out according to a method commonly used in the art. For example, solvent fractionation is performed by treating various solvents, ultrafiltration fractionation is carried out through an ultrafiltration membrane having a constant molecular weight cut-off value, and separation according to various chromatography (size, charge, hydrophobicity or affinity) is performed. Chromatography), and combinations thereof.

The kind of the fractionation solvent used to obtain the fraction in the present invention is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the fractionation solvents include polar solvents such as water, distilled water and alcohols; Nonpolar solvents, such as hexane, ethyl acetate, chloroform, dichloromethane, etc. are mentioned. These may be used alone or in combination of two or more thereof. In the case of using the alcohol in the fractionation solvent, it is preferable to use an alcohol having 1 (C ₁ ) to 6 (C ₆ ) carbon atoms.

As used herein, the term "bone disease" includes all diseases induced by bone loss or decreased bone density, and specifically, osteoporosis, osteomalacia, osteoopenia, and bone atrophy. , Osteoarthritis, rheumatoid arthritis, periodontal disease, osteolysis, fibrous dysplasia, Paget's disease, osteogenic imperfect, hypercalcium Hypercalcemia, and the like, but is not limited thereto.

As used herein, the term "prevention" refers to any action that inhibits or delays the development of a bone disease by administration of a pharmaceutical composition according to the present invention, and "treatment" is suspected of bone disease by administration of the pharmaceutical composition. And any action in which the symptoms of the affected individual improve or beneficially change.

The composition of the present invention is characterized by inhibiting osteoclast differentiation or bone resorption. The composition of the present invention can be usefully used as a prophylactic or therapeutic agent for bone diseases by effectively inhibiting the expression of NFATc1, OSCAR, TRAP, DC-STAMP, which are known as markers for differentiation of osteoclasts.

In the present invention, the term "osteoclast" is a cell derived from a macrophage precursor (macrophage precursor), the osteoclast progenitor cells are macrophage colony stimulating factor (M-CSF), NF-ĸB of Differentiation into osteoclasts by receptor activator ligands (RANKL) and the like means the formation of multinucleated osteoclasts through fusion. Osteoclasts bind to bone through αvβ3 integrin, etc., create an acidic environment and secrete various collagenases and proteases to cause bone resorption. Osteoclasts do not proliferate into fully differentiated cells and cause apoptosis at the end of their lifespan for about two weeks.

In the embodiment of the present invention, after administering the composition of the present invention to the mouse monocytes differentiated into osteoclasts, the staining of TRAP (tarrate-resistant acid phosphate), an osteoclast differentiation marker, resulted in significant concentration-dependent TRAP activity. By inhibiting it, it was confirmed that the osteoclast formation was inhibited (FIG. 1). In addition, as a result of RT-PCR analysis to confirm the mRNA expression patterns of markers involved in osteoclast differentiation, the expression of osteoclast differentiation markers was remarkably increased when the shoe extract and fraction of the present invention were treated. It was confirmed that the decrease (Fig. 2).

As described above, since the composition of the present invention effectively inhibits the differentiation of osteoclasts through TRAP activity inhibition, it can be seen that it is possible to prevent or treat bone diseases such as osteoporosis by increasing the activity of osteoclasts.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent, which may comprise a non-naturally occuring carrier. Specifically, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, polycaprolactone, polylactic acid (Poly Lactic Acid, poly-L-lactic acid, Mineral oil and the like.

The pharmaceutical composition may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral dosage forms, external preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. May include various amorphous carriers, microspheres, nanofibers, and the like.

When formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may comprise at least one excipient such as starch, calcium carbonate, It may be prepared by mixing sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate and talc may also be used.

Liquid preparations for oral administration include suspensions, solutions, 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. have.

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories, and the like. 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.

The content of the novel lichen lamarin ritoraris extract or fractions thereof contained in the pharmaceutical composition of the present invention is not particularly limited, but is 0.01 to 100% by weight, 0.01 to 50% by weight, more specifically based on the total weight of the final composition It may be included in an amount of 0.01 to 20% by weight.

In another aspect, the present invention provides a method for preventing or treating bone disease, comprising administering the pharmaceutical composition to a subject.

Since the pharmaceutical composition of the present invention exhibits a prophylactic or therapeutic effect of bone disease, the method of the present invention comprising the step of administering it to a subject can be usefully used for the prevention or treatment of bone disease.

As used herein, the term "individual" means any animal, including humans, who may or may have a bone disease, such as monkeys, dogs, cats, rabbits, marmots, rats, mice, cattle, sheep, pigs, goats, Means birds, fish, etc., and may be, for example, a mammal including a human, but is not limited thereto.

As used herein, the term "administration" means introducing the composition into a subject in any suitable manner, and the route of administration may be administered through a variety of routes as long as the target tissue can be reached. In particular, the pharmaceutical compositions of the present invention may be administered orally, intravenously, subcutaneously, intradermal, nasal, intraperitoneal, intramuscular, transdermal, and the like, including suitable intravenous administration if necessary for local treatment. Administration by methods and routes. For example, it may be administered by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections, but is not limited thereto.

The therapeutic method of the present invention can be administered in a pharmaceutically effective amount of a pharmaceutical composition comprising Lamarina Litoraris.

As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, and an effective dose level refers to the severity of the disease, the activity of the drug, and the like. Including the age, body weight, health, sex of the patient, sensitivity to the drug of the patient, time of administration of the composition of the present invention used, route of administration and duration of release, treatment with the composition of the invention used or co-administered Factors, and factors well known in the medical arts. The pharmaceutical compositions of the present invention may be administered alone or in combination with known pterygium therapeutics. In consideration of all the above factors, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects.

In addition, the dosage of the pharmaceutical composition may be determined by those skilled in the art in consideration of the purpose of use, the degree of addiction of the disease, the age, weight, sex, history, or type of substance used as an active ingredient of the patient. For example, the pharmaceutical composition of the present invention can be administered at 1 mg / kg to 200 mg / kg, specifically 1 mg / kg to 100 mg / kg, more specifically 20 to 40 mg / kg per adult However, the frequency of administration of the composition of the present invention is not particularly limited, but may be administered once a day or multiple times in divided doses. The dosage does not limit the scope of the invention in any aspect.

As another aspect, the present invention provides a food composition for the prevention or improvement of bone diseases, including a novel lichen marinara ritoraris extract or a fraction thereof as an active ingredient.

Specifically, the novel lichen Lamarin ritoliris extract or fractions thereof of the present invention may be added to a food composition for preventing or improving bone diseases.

In the present invention, the term "improvement" is any action that improves or benefits the symptoms of suspected and onset individuals of bone diseases that are prevented or treated by using a composition comprising a novel lichen Lamarina ritoliris extract or a fraction thereof as an active ingredient. Say.

In the present invention, the term "food" is meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products, including ice cream, various soups, beverages, tea, drinks, alcoholic beverages It includes all foods in the usual sense, such as vitamin complexes, health functional foods, and the like, as long as it can include the novel lichen Lamaritta or Litoraris extract or fractions thereof. It may also include the form of pills, powders, granules, acupuncture, tablets, capsules or solutions.

In the present invention, the term "health functional food" refers to a food prepared and processed using raw materials or ingredients having a useful function to the human body according to Act No. 6767 of the Health Functional Food Act, "functional" is the structure of the human body And it means to obtain a useful effect for health use, such as regulating nutrients or physiological action for function. On the other hand, health food refers to foods that have active health maintenance or promotion effect compared to general foods, and health supplement food means foods for the purpose of health supplement, in some cases, the term functional health food, health food, health supplement food Can be used interchangeably. Health functional food of the present invention can be prepared by a method commonly used in the art. It can be prepared in various forms of formulation, unlike the general medicine has the advantage that there is no side effect that can occur when taking long-term use of the drug as a raw material and can be excellent in portability.

When preparing the food composition may be prepared by adding the raw materials and components commonly added in the art, the kind is not particularly limited. For example, various herbal extracts, food acceptable food additives, or natural carbohydrates may be included as additional ingredients, such as conventional foods, but are not limited thereto.

As another aspect, the present invention provides a quasi-drug composition for the prevention or improvement of bone diseases comprising a novel lichen marinari ritoliris extract or a fraction thereof as an active ingredient.

Specifically, the novel lichen marinara ritoliris extract or fractions thereof of the present invention may be added to the quasi-drug composition for the prevention or improvement of bone diseases.

As used herein, the term "quasi drug" refers to a fiber, a rubber product or the like used for the purpose of treating, alleviating, treating or preventing a disease of a human or animal, has a weak action on the human body or does not directly act on the human body, Or non-machinery and the like, as well as preparations for the use of disinfection, insecticides and similar applications for the prevention of infectious diseases, for the diagnosis of human or animal diseases. Other than articles used for the purpose of treatment, alleviation, treatment, or prevention, other than instruments, machines or devices, and not goods, machines or devices used for the purpose of pharmacologically affecting the structure and function of humans or animals Means the goods. In addition, the quasi-drug may include external skin preparations or personal hygiene products.

The external skin preparations are not particularly limited thereto, and in particular, may be prepared in the form of ointments, lotions, sprays, patches, creams, powders, suspensions, gels or gels, and in particular for the personal hygiene products Specific examples include, but are not limited to, soaps, cosmetics, wipes, tissue paper, shampoos, skin creams, face creams, toothpastes, lipsticks, perfumes, makeup, foundations, ball touch, mascara, eye shadows, sunscreen lotions, hair care products, Air freshener gel or cleaning gel. Further examples of quasi-drug compositions of the present invention include disinfectant cleaners, shower foams, gagrins, wet wipes, detergent soaps, hand washes, humidifier fillers, masks, ointments or filter fillers.

When using the novel lichen limari or ritoliris extract or fractions thereof of the present invention as an quasi-drug additive, the extract or fraction can be added as it is or used with other quasi-drugs or quasi-drug components, and can be appropriately used according to a conventional method. The amount of the active ingredient may be appropriately determined depending on the purpose of use.

As another aspect, the present invention provides a feed composition for the prevention or improvement of bone diseases comprising a novel lichen marinari ritoliris extract or a fraction thereof as an active ingredient.

Specifically, the novel lichen Lamarin ritoliris extract or fractions thereof of the present invention may be added to the feed composition for the prevention or improvement of bone diseases.

As used herein, the term "feed" means any natural or artificial diet, one meal, or the like or any ingredient of the one meal for the animal to eat, ingest, and digest.

The kind of the feed is not particularly limited, and may be used a feed commonly used in the art. Non-limiting examples of the feed include, but are not limited to, vegetable feeds such as cereals, fruits, food processing by-products, algae, fibres, pharmaceutical by-products, oils, starches, gourds or grain by-products; And animal feeds such as proteins, minerals, fats and oils, minerals, fats and oils, single cell proteins, zooplankton or foods. These may be used alone or in combination of two or more thereof.

The novel lichen Lamarina Litoraris extract or fractions thereof of the present invention are derived from natural products and have a low toxicity, and are excellent in inhibiting the formation or differentiation of osteoclasts, thereby preventing diseases induced by bone loss or causing bone loss. Or may be usefully used for treatment.

Figure 1 is a photograph showing the results of TRAP staining of differentiated osteoclasts according to the treatment concentration of the new lichen Lamarina Litoraris extract.
Figure 2 is a graph confirming the mRNA expression level of osteoclast differentiation markers (NFATc1, OSCAR, TRAP, DC-STAMP) according to the treatment of the new lichen Lamarina Litoraris extract by RT-PCR.
Figure 3 is a photograph confirming the protein expression level of osteoclast differentiation marker (NFATc1) according to the treatment of the new lichen Lamarina Litoraris extract by Western blot.
Figure 4 is a photograph confirming the RANKL-induced bone resorption pit according to the treatment of the novel lichen Lamarina Litoraris extract.
The quantitative results of the experiments are expressed as mean and standard deviation. Statistical differences were analyzed using Student's t-test and marked with an asterisk (*) considering that the p value was less than 0.05 (p values * <0.05, ** <0.01, *** < 0.001 versus control).

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Experimental material

Recombinant mouse receptor activator of nuclear factor-ĸB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) were purchased from the R & D System (MN), cell culture medium, fetal bovine serum (FBS), and penicillin / streptomycin. Was purchased from Inbitrogen Life Technologies (NY). The CCK-8 assay kit was purchased from Dojindo Molecular Technologies (ML), and all reagents used for reverse transcription and real-time PCR master mix are from Enzynomics. NFATc1 monoclonal antibody and actin polyclonal antibody were purchased from SantaCruz Biotechnology (CA, USA).

Example 1 Preparation of Lamarina Litoraris Extract and Fractions

After lichens were separated and dried at room temperature, lichen lichens were extracted with acetone and extracted with methanol at a shaking attachment for 24 hours at room temperature. Acetone and methanol extracts were filtered using Whatman filter paper No. 1 and concentrated in vacuo on a rotary concentrator. Concentrates were dissolved at different concentrations of acetone and methanol and stored frozen for later study.

Example 2: Culture and Differentiation of Osteoclasts

The femur and tibia of five-week-old mice were separated and bone marrow spaces were washed with water using a 1cc syringe to obtain bone marrow cells. The isolated bone marrow cells were cultured in α-MEM (α-minimum essential medium) medium containing 10% FBS, antibiotics, M-CSF (30ng / ml) for 3 days. After 3 days, the attached cells were used as bone marrow macrophage (BMM).

Phagocytes were cultured with the addition of RANKL (10 ng / ml), and the Lamarina Litoraris extract and fractions prepared in Example 1 were treated with concentrations (0.1, 0.3, 1, 3 and 10 μg / ml). After 4 days, the cultured cells were stained with tartarate resistance acid phosphatase (TRAP) solution (Sigma Aldrich, USA) and the cells stained in red were considered osteoclasts.

Experimental Example: Analysis of osteoclast formation inhibitory activity of Lamarina Litoraris extract and fractions

Experimental Example 1 TRAP Inhibitory Activity Assay

The multinucleated osteoclasts were fixed with marina Litoraris extract and fractions of the preparation for 10 minutes in 3.7% formalin and stained red with TRAP solution when the permeability of the cell membranes increased with 0.1% Triton X-100. The stained osteoclasts were recognized as only osteoclasts having three or more nuclei. TRAP activity was measured at 37 ° C by treating TRAP-stained osteoclasts with TRAP buffer containing 100 mM para-nitrophenyl phosphate (p-nitrophenyl phosphate, Sigma Aldrich, USA) (100 mM sodium citrate pH 5.0, 50 mM sodium tartrate). After reacting for 5 minutes and transferring the reacted supernatant to a new plate, 0.1N NaOH was added in the same amount to confirm absorbance at 405 nm.

As shown in FIG. 1, it was confirmed that the treatment of Ramarin Litoraris extract to red stained osteoclasts induced by RANKL significantly reduced the number of red stained osteoclasts while inhibiting TRAP activity in a concentration-dependent manner.

This suggests that Lamarina Litoraris extracts and fractions inhibit RANKL-induced osteoclast formation.

Experimental Example 2: Inhibitory activity of osteoclast differentiation related gene expression (RT-PCR)

RT-PCR analysis was performed to confirm the mRNA expression of key differentiation markers involved in osteoclast differentiation. Specifically, NFATc1, OSCAR, TRAP, and DC-STAMP were identified.

RNA was isolated from the cells cultured in Example 2 by TRIzol (Invitrogen) solution according to the manufacturer's method. 1μg of isolated RNA was synthesized by cDNA using oligo dT primer, dNTP, buffer, dithiothreitol, RNase inhibitor and Superscript II reverse transcriptase. The synthesized cDNA was obtained by real-time PCR amplification using SYBR green using a primer.

As shown in Figure 2, when the expression level of the marker in the cells treated nothing was 0, it was confirmed that the improvement of the expression of all markers during RANKL treatment (Vehicle) to induce differentiation of osteoclasts. On the other hand, the treatment of the extracts and fractions (10μg / ml) of the Lamarina Litoraris according to the present invention, it was confirmed that significantly reduced the expression of differentiation markers of all osteoclasts increased.

Experimental Example 3: Inhibitory activity of osteoclast differentiation related protein expression (Western blot)

Western blot analysis was performed to confirm the protein expression of NFATc1 among the key differentiation markers involved in osteoclast differentiation.

Specifically, the cells cultured in Example 2 using lysis buffer (50mM tris-Cl, 150mM NaCl, 5mM EDTA, 1% Triton X-100, 1mM sodium fluoride, 1mM sodium vanadate, 1% deoxycholate, and protease inhibitors) Were dissolved and centrifuged (14,000 rpm) to obtain pure protein. Proteins were quantified using a DC Protein assay kit (Bio-Rad, Hercules, CA, USA) and the same amount of protein was isolated from 10% SDS-polyacrilamide gel. The separated protein was transferred to PVDF membrane (Amersham Biosciences) and the degree of protein expression was confirmed using the antibody.

As a result, as shown in Figure 3, the protein expression of NFATc1 was increased by the treatment of RANKL, it was confirmed that the protein expression of NFATc1 markedly reduced as a result of the treatment of the extracts and fractions of Lamarina Litoraris.

This suggests that Lamarina Litoraris extract and fractions inhibit protein expression of NFATc1 and further inhibit osteoclast formation.

Experimental Example 4: Bone resorption inhibitory activity (Resorption Pit Assay)

Resorption Pit Assay was performed to confirm the inhibitory effect of RANKL-induced bone resorption activity.

Specifically, the osteoclasts were cultured on dentin disks, and the effects of Ramarin Litoraris extracts on various concentrations were confirmed for RANKL-induced bone resorption, and bone resorption activity of dentin disks was measured. .

As a result, as shown in Figure 4, the treatment of RANKL increased bone resorption activity, the pit was generated, and as a result of the treatment of the Ramarina Litoraris extract and fractions, the pit significantly decreased due to the decrease in bone resorption activity I could confirm that.

This suggests that Lamarina Litoraris extract and fractions inhibit bone resorption activity and further inhibit osteoclast formation.

From the above results, it can be seen that the Lamarina ritoraris extract and fraction according to the present invention has an inhibitory activity of osteoclast formation or differentiation.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (8)

Novel lichen Lamarina litoralis ( Ramalina litoralis ) pharmaceutical composition for the prevention or treatment of bone diseases comprising an extract or a fraction thereof as an active ingredient.
The pharmaceutical composition of claim 1, wherein the extract is extracted using water, alcohol having 1 (C ₁ ) to 6 carbon atoms (C ₆ ), or a mixed solvent thereof.
According to claim 1, wherein the bone disease (osteoporosis), osteomalacia (osteomalacia), osteopenia (osteopenia), bone atrophy (bone atrophy), osteoarthritis (rheumatoid arthritis), periodontal disease (periodontal disease) Pharmaceutical composition, which is at least one selected from the group consisting of osteolysis, fibrous dysplasia, Paget's disease, osteogenic imperfect, and hypercalcemia. .
The pharmaceutical composition of claim 1, wherein the composition inhibits the differentiation of osteoclasts.
The pharmaceutical composition of claim 1, wherein the composition inhibits expression of a protein involved in osteoclast differentiation.
Novel lichen Lamarina litoralis ( Ramalina litoralis ) food composition for the prevention or improvement of bone diseases comprising an extract or a fraction thereof as an active ingredient.
A novel quasi lichen Lamarina litoralis ( Ramalina litoralis ) extract or a quasi-drug composition for the prevention or improvement of bone diseases comprising the fraction as an active ingredient.
Feeding composition for the prevention or improvement of bone disease comprising a novel lichen Lamarina litoralis ( Ramalina litoralis ) extract or a fraction thereof as an active ingredient.

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