WO2024154990A1 - Pharmaceutical composition comprising neorhodomela munita extract for preventing or treating bone disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating bone disease, comprising a Neorhodomela munita extract as an active ingredient. The Neorhodomela munita extract has the effects of inhibiting the differentiation of macrophages into osteoclasts and inhibiting the activity of osteoclasts, and thus can be effective for the described uses.

Description

Pharmaceutical composition for preventing or treating bone disease comprising extract of Red-brown Blackthorn

The present invention relates to a pharmaceutical composition for the prevention or treatment of bone disease containing an extract of Neorhodomela munita as an active ingredient.

Human bones are composed of osteoblasts, osteocytes, and osteoclasts. Among them, osteoblasts play the role of forming bone tissue through the proliferation phase, bone matrix formation phase, and calcification phase. Additionally, osteoclasts play a role in bone resorption. In adult bones that have completed growth, a bone remodeling process occurs through continuous regeneration and repeated bone resorption, in which old bone is removed by osteoclasts and replaced by new bone by osteoblasts.

When the balance between bone absorption and production is lost, osteoporosis occurs. Osteoporosis is a disease in which the balance between bone formation and bone resorption is broken, resulting in a decrease in bone mass and the risk of fractures continues to increase due to deterioration of the microstructure of bone tissue. It is a condition in which the minerals (especially calcium) and matrix that make up bone are reduced. The gap between the bone microstructures widens and the microstructures become thinner, increasing the risk of the bone being easily fractured by even a small impact. Known causes of osteoporosis include old age, lack of exercise, low body weight, smoking, low-calcium diet, menopause, and ovarian resection. In particular, when women reach menopause, bone loss rapidly progresses due to hormonal changes.

To treat such osteoporosis, hormones, alendronate, calcitonin, raloxifene, Na-F, calcitriol, or bisphosphonate preparations are administered. However, treatment of osteoporosis requires a long period of time, and long-term use of the drug increases the possibility of side effects such as urinary stones, endometrial cancer, and breast cancer.

Accordingly, recent research has been active on natural products that can promote bone formation while minimizing bone loss without other side effects.

In the above-described situation, the present inventors made efforts to discover natural products that can improve bone-related diseases such as osteoporosis, and as a result, it was confirmed that the Siberian red-black extract had an effect of suppressing osteoclast differentiation and activity, thereby completing the present invention. .

Accordingly, the purpose of the present invention is to provide a pharmaceutical composition capable of improving/treating bone diseases such as osteoporosis, which contains an extract of Siberian sagebrush as an active ingredient.

In order to achieve the above object, the present inventors discovered the extract of the red snail as a substance that has a therapeutic effect on bone-related diseases, for example, diseases such as osteoporosis. Specifically, when macrophages were treated with RANKL (receptor activator of NK-κB ligand), an osteoclast differentiation inducer, to induce differentiation into osteoclasts, when the macrophages were treated with the above-mentioned red-brown extract, differentiation of macrophages into osteoclasts occurred. It was found that it was suppressed (Figures 1 and 2). In addition, the expression of osteoclast differentiation markers and the activity of osteoclasts were also inhibited by the red-brown extract (Figures 3 to 5).

Accordingly, one aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of bone disease, comprising an extract of Siberian sagebrush as an active ingredient.

Neorhodomela munita ( Neorhodomela munita or Neorhodomela aculeata (Perestenko) Masuda 1982) is a type of seaweed belonging to the phylum Rhodophyta , Florideophyceae , Rhodomelaceae , and genus Neorhodomela , found on the east coast of Korea. , lives in the intertidal zone of the west and south coasts. The thallus is dark brown, almost black, and grows in a cartilaginous column shape, producing small branches irregularly. The small branches have many thorn-like protrusions.

The extract of the red snail has anti-inflammatory and antibacterial effects and is listed in the International Cosmetic Ingredients Directory published by the American Cosmetic Association. However, its effectiveness in preventing or treating bone diseases is not yet known.

The term "extract" used in the present invention refers to a preparation obtained by squeezing the extraction target into an appropriate leachate and concentrating or filtering the leachate, but is not limited to, an extract obtained by extraction treatment, a dilution or concentrate of the extract, It may be a dried product obtained by drying the extract, a crude purified product, or a purified product thereof.

The above-mentioned red-brown extract can be prepared using general extraction, separation, and purification methods known in the art. The extraction method is not limited to this, but methods such as hot water extraction, hot water extraction, cold needle extraction, reflux cooling extraction, or ultrasonic extraction can be used.

In the present invention, the extraction solvent may be selected from the group consisting of water, alcohol with 1 to 6 carbon atoms, dichloromethane, acetone, and mixed solvents thereof, and is preferably ethanol.

In one embodiment of the present invention, an extract prepared by immersing the entire dried bright red snail in ethanol, repeating ultrasonic treatment, and drying under reduced pressure was used (Example).

On the other hand, the extract of the red snail may be included in an amount of 0.0001 to 90% by weight based on the total weight of the composition, but is not limited thereto. For example, the black-brown extract may be included in an amount of 0.0001 to 80% by weight, 0.001 to 70% by weight, or 0.001 to 50% by weight, based on the total weight of the composition.

In the present invention, the bone disease includes osteoporosis, periodontal disease, osteomalacia, osteopenia, bone defect, bone atrophy, osteonecrosis, fracture, and osteolysis ( It may be selected from the group consisting of osteolysis, osteoarthritis, and osteogenesis imperfecta, and is preferably osteoporosis.

As used herein, the term “osteoporosis” refers to a condition in which the microstructure of bone tissue deteriorates due to a decrease in bone minerals (especially calcium) and matrix, and as a result, the risk of fracture continues to increase. When osteoclastic activity is greater than osteoblastic activity, it occurs when the balance of bone remodeling is broken. Causes include impaired calcium absorption, vitamin D deficiency, drug side effects, lack of exercise, excessive drinking, menopause, and depression. In particular, in the case of women, it is known that when estrogen secretion decreases after menopause, free radicals accumulate in cells, triggering bone loss.

The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the desired method, and when formulated, commonly used fillers, extenders, It is manufactured using diluents or excipients such as binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc. These solid preparations are made by adding at least one excipient, such as starch, calcium carbonate, It is manufactured by mixing sucrose, lactose, or gelatin. Additionally, in addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, or syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. You can.

Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, etc. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerol, gelatin, etc. can be used.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, 'pharmaceutically effective amount' means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, activity of the drug, and the type and severity of the patient's disease. It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the field of medicine. For example, the pharmaceutical composition of the present invention can be administered once a day in a dose of 0.1 mg/kg to 200 mg/kg, preferably 1 mg/kg to 50 mg/kg, or divided into three times a day. there is. The above dosage does not limit the scope of the present invention in any way. However, since it may increase or decrease depending on the route of administration, severity of obesity, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

Another aspect of the present invention provides a food composition for preventing or ameliorating bone disease, comprising the above-described black-black extract as an active ingredient.

Since the food composition uses the same ingredients as the pharmaceutical composition for preventing or treating bone diseases, overlapping content between the two is omitted to avoid excessive description in the specification.

In the present invention, the food composition may be provided in the form of powder, granules, tablets, capsules, syrup, beverage, or pills, and is used with other foods or food additives in addition to the active ingredient, the red sage extract, according to a conventional method. It can be used appropriately. The mixing amount of the active ingredient can be appropriately determined depending on its purpose of use, for example, prevention, health, or therapeutic treatment.

The effective dose of the active ingredient contained in the food composition can be used in accordance with the effective dose of the pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or health control, it may be below the above range, Since the active ingredient poses no safety issues, it is certain that it can be used in amounts exceeding the above range.

The food composition includes ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. Examples of the above-mentioned carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a flavoring agent, natural flavoring agents and synthetic flavoring agents can be used. For example, when the food composition of the present invention is manufactured as a drink, citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, etc. may be additionally included in addition to the active ingredients of the present invention.

Another aspect of the present invention provides a composition for inhibiting osteoclast differentiation in vitro using the above-mentioned red-brown extract.

In one embodiment of the present invention, the extract of the red snail effectively inhibits the differentiation of macrophages into osteoclasts induced by RANKL treatment, and therefore can be used for the purpose of inhibiting osteoclast differentiation.

Another aspect of the present invention provides a method for improving or treating bone disease, comprising administering the pharmaceutical composition to a subject in need of treatment.

The red snail extract according to the present invention has the effect of inhibiting the differentiation of macrophages into osteoclasts and inhibiting the activity of osteoclasts, so it can be usefully used for the prevention or treatment of bone diseases.

Figure 1 shows the results of TRAP (tartrate-resistant acid phosphate) staining after differentiating macrophages into osteoclasts and treating them with a red-brown extract.

Figure 2a shows the results of confirming the ratio of TRAP+ osteoclasts with three or more nuclei in one cell after differentiating macrophages into osteoclasts and treating them with a red-black extract.

Figure 2b shows the results of confirming the ratio of TRAP+ osteoclasts with more than 10 nuclei per cell after differentiating macrophages into osteoclasts and treating them with a red-black extract.

Figure 3 shows osteoclast differentiation and activation markers DC STAMP (dendritic cell specific transmembrane protein), OSCAR (osteoclast-associated receptor), and TRAP after treating macrophages with an osteoclast differentiation inducer (RANKL) and a black lily extract. This is the result of confirming the mRNA level of NFATc1 (Nuclear Factor Of Activated T Cells 1).

FIG. 4A graphically displays the results of DC STAMP in FIG. 3.

Figure 4b graphically displays the results of OSCAR in Figure 3.

Figure 4c graphically displays the results of TRAP in Figure 3.

Figure 4d graphically displays the results of NFATc1 in Figure 3.

Figure 5a shows the results of confirming the decomposition area formed when osteoclasts decompose a bone-mimicking mattress after treating macrophages with an extract of red-black while differentiating them into osteoclasts.

Figure 5b graphically displays the results of Figure 5a.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more embodiments and the scope of the present invention is not limited to these examples.

Example: Extract Preparation

The red-lipped blacktail extract (MABIK NP30210062) was distributed through the Marine Extract Bank of the National Marine Biological Resources Institute. The extract was prepared as follows. Neorhodomela munita (Perestenko) Masuda, collected in Taean-gun, Chungcheongnam-do in 2021, was washed three times and freeze-dried. 30 g of dried red black powder was added to 400 ml of 70% ethanol, and extracted three times with an ultrasonic extractor (40 KHz, 60 minutes) at room temperature. The obtained extract was filtered and concentrated to obtain a final extract of 800 mg. In the following experiment, the powder was dissolved in DMSO and used.

Experimental Example 1: Inhibition of osteoclast activation

2-1. TRAP staining

RAW264.7 cells, a mouse macrophage cell line, were distributed in a 48-well plate at 3 x ¹⁰ cells per well, and differentiation was induced by treating RANKL (receptor activator of NK-κB ligand), an osteoclast differentiation inducer, at a concentration of 40 ng/ml. did. Afterwards, the culture was cultured for a total of 4 days, with the medium changed every 2 days, and when the medium was replaced, the bright red blackhead extract was treated together.

After 4 days, the differentiated cells were fixed with fixative at room temperature for 5 minutes and washed several times with distilled water. A substrate solution of sodium tartrate and acid phosphatase was mixed, and cells were treated with the prepared mixture to stain tartrate-resistant acid phosphate (TRAP), an osteoclast differentiation marker. Staining was performed at 37°C for 15 to 45 minutes after covering the plate with a lid to prevent drying. Afterwards, the dye was discarded and the reaction was stopped by washing three times with distilled water. After observing under a microscope, photographs were taken and the number of stained cells was calculated using the Image J program.

As a result of the experiment, it was found that the differentiation of osteoclasts was significantly suppressed in the group treated with the red blackhead extract compared to the control group treated only with RANKL (Figure 1).

In addition, since osteoclasts are characterized by having multiple nuclei, the ratio of TRAP ⁺ osteoclasts with more than 3 (N≥3) or more than 10 (N≥10) nuclei per cell was calculated. As a result, compared to the control group treated only with RANKL, the proportion of TRAP ⁺ osteoclasts with more than 3 (N≥3) or more than 10 (N≥10) nuclei was significantly reduced in the group treated with the Black Red Negro extract. This was confirmed (Figure 2).

2-2. Identification of osteoclast differentiation markers

RAW264.7 cells were distributed in a 6-well plate at 3x10 ⁴ cells per well, and pretreated with 10 or 20 ㎍/㎖ concentration of RAW264.7 cells for 1 hour. Afterwards, each well was treated with RANKL at a concentration of 40 ng/ml, cultured for a total of 4 days with medium replaced every 2 days, and cells were recovered with trizol. mRNA was isolated from the recovered cells using the Trizol-chloroform method and quantified using nano-drop. Afterwards, cDNA was synthesized using 500 ng of the isolated mRNA as a template using a cDNA synthesis kit (oligo dT primer), and RT-PCR was performed using the synthesized cDNA as a template. RT-PCR was performed a total of 35 cycles of annealing at 56°C for 30 seconds and extension at 72°C for 1 minute. The primer sequences used for RT-PCR are as follows:

DC-STAMP F (SEQ ID NO: 1): TTGAACCGAGCTGCATTCCT;

DC-STAMP R (SEQ ID NO: 2): GCACTACCTTGGCCTTACCT;

OSCAR F (SEQ ID NO: 3): CCCAGTCTGTCTTGCGGTAG

OSCAR R (SEQ ID NO: 4): TCTGGGTTGGAGGGTCCTAA

TRAP F (SEQ ID NO: 5): CTCCTGCCTGTTCTCTTCCCA

TRAP R (SEQ ID NO: 6): AAGAGAGAAAGTCAAGGGAGTGGC

NFATc1 F (SEQ ID NO: 7): GGAGAGTCCGAGAATCGAGAT

NFATc1R (SEQ ID NO: 8): TTGCAGCTAGGAAGTACGTCT

GAPDH F (SEQ ID NO: 9): CAAGGTCATCCATGGACAACTTTG

GAPDH R (SEQ ID NO: 10): GTCCACCACCCTGTTGCTGTAG

As a result, the mRNA levels of DC STAMP (dendritic cell specific transmembrane protein), OSCAR (osteoclast-associated receptor), TRAP, and NFATc1 (Nuclear Factor Of Activated T Cells 1), which are markers of osteoclast differentiation and activation, were significantly higher in the group treated with the falciparum extract. It was confirmed that there was a decrease in (Figures 3 and 4).

2-3. Pit assay

Bone resorption assay kit (CSR-BRA-48KIT, COSMOBIO) was used to confirm whether the RAW264.7 cells can inhibit the activity of RAW264.7 cells differentiated into osteoclasts by RANKL treatment. Specifically, RAW264.7 cells were spread on a culture dish coated with a bone-mimicking mattress containing a calcium-phosphate layer, and differentiation was induced by treatment with RANKL, an osteoclast differentiation inducer, at a concentration of 40 ng/ml. Afterwards, the culture was cultured for a total of 4 days, with the medium changed every 2 days, and when the medium was replaced, the bright red blackhead extract was treated together. After 6 days, osteoclasts attached to the culture dish were removed by treatment with 5% sodium hypochlorite for 5 minutes. The culture dish was additionally washed with water and dried, a picture was taken of each well, and the pit area formed when osteoclasts decomposed the bone-mimicking mattress was measured using Image J. The decomposition area is proportional to the activity of osteoclasts.

As a result of the measurement, it was confirmed that compared to the control group treated only with RANKL, the group treated with the Black Red Negro extract showed a significant decrease in the decomposition area starting from the treatment concentration of 10 ㎍/㎖ (FIG. 5).

Claims (9)

A pharmaceutical composition for the prevention or treatment of bone disease containing an extract of the red sagebrush as an active ingredient, The bone disease is selected from the group consisting of osteoporosis, periodontal disease, osteomalacia, osteopenia, bone defect, bone atrophy, osteonecrosis, fracture, osteolysis, osteoarthritis, and osteogenesis imperfecta. A pharmaceutical composition for preventing or treating bone disease. The method of claim 1, wherein the fine red blackhead extract is extracted with a solvent selected from the group consisting of water, alcohol with 1 to 6 carbon atoms, dichloromethane, acetone, and mixed solvents thereof, for the prevention or treatment of bone disease. Pharmaceutical composition. The pharmaceutical composition for the prevention or treatment of bone disease according to claim 2, wherein the black lily extract is an ethanol extract. The pharmaceutical composition for preventing or treating bone diseases according to claim 1, wherein the pharmaceutical composition inhibits the differentiation of macrophages into osteoclasts or the activity of osteoclasts. The method of claim 1, wherein the pharmaceutical composition promotes osteoclast differentiation selected from the group consisting of DC STAMP (dendritic cell specific transmembrane protein), OSCAR (osteoclast-associated receptor), TRAP, and NFATc1 (Nuclear Factor Of Activated T Cells 1). A pharmaceutical composition for preventing or treating bone disease, which inhibits the expression of a marker. A food composition for the prevention or improvement of bone disease containing the extract of the red sagebrush as an active ingredient, The bone disease is selected from the group consisting of osteoporosis, periodontal disease, osteomalacia, osteopenia, bone defect, bone atrophy, osteonecrosis, fracture, osteolysis, osteoarthritis, and osteogenesis imperfecta. A food composition for preventing or improving bone disease. The method of claim 6, wherein the fine red blackhead extract is extracted with a solvent selected from the group consisting of water, alcohol with 1 to 6 carbon atoms, dichloromethane, acetone, and mixed solvents thereof, for the prevention or improvement of bone disease. Food composition. The food composition for preventing or ameliorating bone disease according to claim 7, wherein the black-brown extract is an ethanol extract. A method for improving or treating bone disease comprising administering the pharmaceutical composition according to claim 1 to an individual in need of treatment, The bone disease is selected from the group consisting of osteoporosis, periodontal disease, osteomalacia, osteopenia, bone defect, bone atrophy, osteonecrosis, fracture, osteolysis, osteoarthritis, and osteogenesis imperfecta.

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