KR101756405B1 - Composition for preventing or improving postmenopausal osteoporosis comprising Scopolin - Google Patents

Abstract

The present invention relates to a composition for preventing or ameliorating postmenopausal osteoporosis comprising scopolin or a pharmaceutically acceptable salt thereof as an active ingredient. The scopoline of the present invention has a good efficacy for promoting osteoblast differentiation And it has an effect of effectively inhibiting the reduction of bone density after menopause, the lowering of the density of bone microstructure, and the reduction of blood osteogenesis markers. Therefore, it is expected that the scopolin of the present invention and the composition containing it as an active ingredient can be usefully used as a food composition for prevention and improvement of postmenopausal osteoporosis.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or improving postmenopausal osteoporosis comprising scopolin as an active ingredient,

The present invention relates to a composition for preventing, ameliorating or treating post-menopausal osteoporosis (type 1 osteoporosis, type 1) containing scopolin, a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

Estrogen, a typical hormone that symbolizes female, is an important female hormone that regulates the lifespan of women, from menstruation to pregnancy to menopause. Estrogens are known to be follicles, leprosy, and female hormones that are secreted mainly from the placenta of the female ovaries, and are collectively referred to as estrone (E1), estradiol (E2), and estriol (E3). Estrogens affect a wide range of tissues and organs, especially the uterus, urinary system, breast, skin, bones, and blood vessels that are needed to maintain flexibility and steady state. In particular, estrogen acts on osteoblasts involved in bone formation to maintain bone formation and inhibits bone resorption by acting on osteoclasts involved in bone removal And plays an important role in maintaining the homeostasis of bone (Fig. 1). Thus, postmenopausal osteoporosis due to estrogen deprivation due to menopause is one of the most severe postmenopausal women's diseases (Fig. 2).

Osteoporosis refers to a condition in which bone mass (bone mass) within a unit volume is abnormally reduced compared to the normal value of a normal person's sex, age, and race. It is characterized by fractures in the hip, wrist bone, vertebra bone, etc., with the bone being easily broken by a very minor impact such as bending the waist or sitting down. (Osteoporosis type 1), osteoporosis (type 2), and secondary osteoarthritis (osteoarthritis) due to other causes such as drugs (Fig. 3).

Postmenopausal osteoporosis (Type 1 osteoporosis, type 1) occurs in women when the postmenopausal estrogen hormone is deficient and the bone constituents are absorbed into the body tissues and calcium absorption through the intestines is reduced (Fig. 4). In addition, osteoclast differentiation and proliferation are inhibited by the decrease of estrogen hormone. As a result, osteogenesis is inhibited, osteoclast activation increases bone resorption more than osteogenesis, and bone loss is enhanced A decrease in bone mass occurs (FIG. 5). Depending on the person, it can proceed rapidly after menopause.

Geriatric osteoporosis (Type 2 osteoporosis, type 2) is caused by bone loss in both sexes with increasing age. Reduction of active vitamin D in the body is accompanied by a decrease in intestinal calcium absorption and a decrease in the number of osteoblasts that newly produce osteocytes.

Secondary osteoporosis is osteoporosis that is caused by various diseases or medications that affect functions related to bone cell production and maintenance of the human body. Hyperthyroidism, hyperparathyroidism, Cushing's syndrome, rheumatoid arthritis, hyperprolactinemia, etc. Steroid hormone preparations and thyroid hormone preparations may cause secondary osteoporosis.

When the menopause begins, the ovarian function is reduced and the female hormone secreted from the ovaries is reduced, resulting in a variety of symptoms. After menopause, osteoporosis may occur after menopause due to rapid decrease in bone density and decrease in bone mass. Postmenopausal osteoporosis is a disease that affects the quality of life because it can cause back pain or other bone related diseases and can easily become a fracture. In particular, women who have had early menopause or ovariectomy before age 50 may be more vulnerable to postmenopausal osteoporosis.

Therapeutic agents for postmenopausal osteoporosis include therapeutic agents such as Bonvivar Plus (a composition component: bisphosphonate series) and acclasta (ingredient: 5 mg injectable solution of zoledronic acid), which are compound modification drugs. However, due to side effects, there is a growing demand for therapeutic agents or health functional foods derived from natural products. Healthy functional foods such as calcium, vitamin D and isoflavones, which are good for bone health, are widely used but their effects are limited. Therefore, there is a need to develop a pharmaceutical formulation or health functional food for prevention, improvement, and treatment of postmenopausal osteoporosis more effectively.

Korean Patent No. 10-0981350 (published on September 30, 2010)

It is an object of the present invention to provide a composition for preventing, ameliorating or treating osteoporosis in postmenopausal women containing scopolin, a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient, And a composition for preventing, ameliorating, and treating osteoporosis after postmenopausal osteoporosis caused by decrease of estrogen hormone of osteoporosis.

The present invention provides a pharmaceutical composition for preventing or treating postmenopausal osteoporosis comprising scopolin, a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a health food composition for preventing or ameliorating postmenopausal osteoporosis comprising scopolin, a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention relates to a composition for preventing or ameliorating postmenopausal osteoporosis comprising scopolin or a pharmaceutically acceptable salt thereof as an active ingredient. The scopoline of the present invention has a good effect for promoting osteoblast differentiation , Reduction of bone mineral density after menopause, decrease in density of bone microstructure, and reduction of blood osteogenesis markers. Therefore, it is expected that the scopolin of the present invention and the composition containing it as an active ingredient can be usefully used as a food composition for prevention and improvement of postmenopausal osteoporosis.

1 is a schematic diagram showing a mechanism involved in bone formation and bone resorption of estrogen.
FIG. 2 shows the pathogenesis of postmenopausal osteoporosis that occurs when estrogen deficiency occurs after menopause.
FIG. 3 is a schematic diagram showing the types of osteoporosis, and postmenopausal osteoporosis is type 1 osteoporosis (type 1).
FIG. 4 shows the mechanism of onset of postmenopausal osteoporosis (type 1 osteoporosis, type 1) caused by a decrease in calcium absorption through the intestine and absorption of bone constituents into body tissues due to lack of estrogen hormone after menopause.
FIG. 5 is a schematic diagram showing the mechanism of osteoclast killing and inhibition and osteoclast activation due to postmenopausal estrogen hormone deficiency.
Figure 6 shows the chemical structure of Scopolin.
7 shows the results of cytotoxicity (Fig. 7A), ALP (osteoblast differentiation marker), alkaline phosphatase (Fig. 7A), and osteoblast differentiation markers after treating osteoclast MC3T3-E1 cells with scopolin at a concentration of 1 μg / ml, 5 μg / ml and 10 μg / , alkaline phosphatase activity measurement (FIG. 7b), ALP staining (FIG. 7c), and Alizarin red S staining of mineralization (FIG. 7d). Scopolin was not toxic to osteoblasts, Indicating that there is a significant effect on promoting differentiation.
FIG. 8 shows changes in bone mineral density (FIG. 8A) at 20 mg / kg / day and 40 mg / kg / day administration of scopolin in a postmenopausal mouse model (ovariectomized mouse) After taking a micro-CT photograph (FIG. 8B),% bone volume BV (FIG. 8C), trabecular thickness (Tb.Th) (FIG. 8D), trabecular number Tb.N) (FIG. 8E), trabecular spacing (Tb.Sp) (FIG. 8F), and scopoline significantly decreased the bone mineral density due to menopause and decreased bone microstructure densities Lt; / RTI >
FIG. 9 is a graph showing the relationship between OPG (osteoprotegerin) (FIG. 9A) and receptor activator (RANKL) (FIG. 9A) of bone metabolism by separating serum from mouse blood after administration of scopolin 20 mg / kg / day and 40 mg / (Fig. 9c) of the nuclear factor-kappaB ligand (Fig. 9b) and the ratio of OPG / RNAKL (Fig. 9c) The effect of inhibiting the increase is remarkably suppressed.

Accordingly, the inventors of the present invention have confirmed the in vitro and in vivo efficacy of scopolin, a single compound effective in reducing postmenopausal bone mineral density and lowering of bone microstructural densities, which are caused by estrogen hormone reduction in postmenopausal women, Completed.

The present invention provides a pharmaceutical composition for preventing or treating postmenopausal osteoporosis comprising scopolin, a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

Specifically, the female postmenopausal osteoporosis is caused by a decrease in estrogen secretion, and the estrogen secretion decrease is caused by a decrease in bone density, a decrease in bone microstructure densities, a decrease in bone formation markers and an increase in bone resorption markers Lt; / RTI >

More specifically, the bone formation marker is OPG (osteoprotegerin), and the bone resorption marker may be RANKL (receptor activator of nuclear factor-kappa B ligand), but is not limited thereto .

The scopolin of the present invention is represented by the formula (1), and the chemical formula thereof is C ₁₆ H ₁₈ O ₉ .

≪ Formula 1 >

In the present invention, the pharmaceutically acceptable salts include salts with organic acids selected from the group consisting of oxalic acid, maleic acid, fumaric acid, malic acid, tartaric acid, citric acid and benzoic acid, or inorganic acids selected from the group consisting of hydrochloric acid, sulfuric acid, phosphoric acid and hydrobromic acid May be a salt.

When the composition of the present invention is a pharmaceutical composition, the pharmaceutical composition may be formulated into a cream, a gel, a patch, a spray, an ointment, a warning agent, a lotion, a liniment, a pasta agent and a cataplasma agent. Meanwhile, the pharmaceutical composition may contain a pharmaceutically acceptable carrier in addition to the scopolin. Such pharmaceutically acceptable carriers are those conventionally used in pharmaceutical preparations, and include lactose, dextrose, water The present invention relates to a process for the preparation of a medicament for the treatment and / or prophylaxis of cancer, comprising administering a therapeutically effective amount of a compound selected from the group consisting of cross, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, Carboxymethylcellulose, carboxymethylcellulose, hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. In addition, the pharmaceutical composition may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. as an additive.

The pharmaceutical composition may be administered according to the severity of postmenopausal osteoporosis. Typically, topical administration is preferred. The dosage of the active ingredient in the pharmaceutical composition may vary depending on the route of administration, the severity of the disease, the age, sex, and weight of the patient, and may be administered once to several times per day.

The present invention also provides a health food composition for preventing or ameliorating postmenopausal osteoporosis comprising scopolin, a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

The health food composition may be provided in the form of powder, granule, tablet, capsule, syrup, beverage or ring, and the health food composition may be mixed with food or food additives other than scopolin according to the present invention, And can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose, for example, prevention, health or therapeutic treatment.

The effective dose of scopolin contained in the above health food composition can be used in accordance with the effective dose of the above pharmaceutical composition. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, Range, and it is clear that the active ingredient can be used in an amount of more than the above range because there is no problem in terms of safety.

There is no particular limitation on the type of the health food, and examples thereof include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Drinks, alcoholic beverages and vitamin complexes.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Scopolin's  Chemical structure

The single test compound used in the animal experiments in the present invention is Scopolin. The scopolin has a chemical structure of C ₁₆ H ₁₈ O ₉ and is a naturally occurring single compound that is abundantly contained in Lycii radicis cortex and the like (Fig. 6).

< Example  2> Scofolin  Promotes osteoblast differentiation promotion

Osteoblasts are derived from mesenchymal stem cells and are responsible for new bone formation. Since the role of osteoblasts is important after postmenopausal women whose estrogen is rapidly decreased, the effect of scopolin on osteoblast proliferation and differentiation was examined.

The cytotoxicity of scopolin was assessed by cell proliferation analysis using the EZ-Cytox Enhanced Cell Viability Assay Kit product using water-soluble Tetrazolium salt. Specifically, MC3T3-E1 cells (3 × 10 ³ cells / well), which are osteoblasts, were cultured in DMEM supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 100 units / L penicillin and 100 mg / L streptomycin After incubation at 37 ° C with 5% CO ₂ in essential medium (α-MEM) medium, scopollin was treated with 3 concentrations (1 μg / ml, 5 μg / ml, 10 μg / ml) And cultured for 2 days. Tetrazolium salt was added to each cultured cell and sample, incubated at 37 ° C for 2 hours, and absorbance was measured at 450 nm. Compared to the control (control) treated with physiological saline, in the experimental group treated with scopolin at a concentration of 1 μg / ml (Sco1), 5 μg / ml (Sco5) and 10 μg / And did not adversely affect cell proliferation (Fig. 7A).

The effect of scopolin on osteoblast differentiation was evaluated using MC3T3-E1 cells. MC3T3-E1 cells were treated with scopolin after induction of differentiation for 3 days by addition of ascorbic acid (50 μg / ml) and β-glycerophosphate (10 mM) to induce osteogenic differentiation. The scopolin was dissolved in an aqueous solution of 1% dimethyl sulfoxide (DMSO) and treated with MC3T3-E1 cells at a concentration of 1 μg / ml (Sco1), 5 μg / ml (Sco5) and 10 μg / The cells were cultured in the same amount of 1% DMSO solution without scopolin treatment as a control. Alkaline phosphatase (ALP) activity was measured in order to examine the differentiation effect of osteoblast. Specifically, after cells were washed with physiological saline, the cells dissolved in the cells were treated with p-nitrophenylphosphate, which is an ALP substrate, and cultured at 37 ° C for 1 hour. The absorbance was then measured at a wavelength of 405 nm after addition of 0.5N NaOH as a reaction stop solution. As a result, there was a statistically significant increase in osteoblast differentiation in the group treated with 5 μg / ml (Sco5) or 10 μg / ml (Sco10) of scopolin (FIG. 7b) . Negative control). In addition, osteoblast differentiation was evaluated by ALP staining, and ALP staining of cells treated with 5 μg / ml of scopolin was increased (FIG. 7c).

Mineralization of osteoblasts was assessed using Alizarlin red S staining method after 3 weeks of incubation in the control group and 5 μg / ml of scopolin-treated group. The mineralization of cells treated with scopolin was increased (Fig. 7d). From these results, it was confirmed that scopolin was effective in promoting osteoblast differentiation.

< Example  3> In ovariectomized menopausal mice model, scopollin of  Efficacy of osteoporosis prevention after menopause

A 10-week-old ovariectomized-mouse (OVX mouse) was used as a postmenopausal animal model to evaluate the in vivo efficacy of scopolin (additional feeding for 2 weeks for ovariectomy after ovariectomy at 8 weeks of age). OVX mouse group (negative control group) administered only with physiological saline to the ovariectomized mice, strontium chloride (a control group), which is an effective compound for improving bone density, in the ovariectomized mice, SrCl ₂₎ to 20 mg / kg / day dose by oral administration (oral injection) a SrCl ₂ mouse group is the bone mineral density improved efficacy test was used as a positive control. The experimental group was orally injected with scopoline at a dose of 20 mg / kg / day and 40 mg / kg / day. 10-week-old sham-operated and ovariectomized ddY female mice were purchased from the Central Laboratory Animals and transferred to a clean animal breeding area after a 1-week purification period in the laboratory animal quarantine room. The weight of each mouse was measured and group separation was performed so that there was no statistically significant weight difference between the experimental groups. The scopolin used in the experiment was dissolved in a 1% DMSO aqueous solution to prepare a test solution. To carry out the laboratory animal center, a sterilization operation was performed by irradiation with a gamma ray.

The initial bone mineral density (BMD) was measured with a PIXImus bone densitometer at the start of the animal experiment. The mice were anesthetized by injecting 50 μl of a mixed anesthetic of zolethyl and rumpun (1: 2 mixture in 2: 3 ratio with physiological saline), fixed in a bone density measuring frame, and measured for bone density . After 6 weeks and 12 weeks of the administration of scopoline, the bone mineral density of the mice was measured with a PIXImus bone densitometer. After completion of the 12 week experiment, blood samples were taken and the femoral bones were extracted and micro-CT was taken. The bone volume percentage (% bone volume, BV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular spacing (Tb.Sp).

A significant decrease in bone mineral density increase rate at 6 and 12 weeks after ovariectomy of ovariectomized OVX mice compared to normal ovariectomized Shame mice (Fig. 8a), and bone microstructure densities on micro-CT images (Fig. 8B). In addition, the bone volume ratio (FIG. 8C), cancellous bone sorbent thickness (FIG. 8D), and cancellous bone sorbent number (FIG. 8E) were significantly lower and cancellous bone sorbent space (FIG. However, in the experimental group in which scopollin was administered at a dose of 20 mg / kg / day and 40 mg / kg / day for 12 weeks, the decrease in bone density and decrease in bone microstructure density caused by menopause was suppressed (FIGS. 8a and 8b) The bone volume percentage, cancellous bone thickness, cancellous bone degeneration and cancellous bone degeneration were all suppressed (Figs. 8c, 8d, 8e, 8f). These results were similar to those of the positive control group SrCl ₂ mice. Statistical analysis also showed significant efficacy (*: p <0.05 vs. OVX negative control).

Next, the changes in the blood bone markers were examined. After 12 weeks of postmenopausal mouse modeling, serum samples were collected and analyzed for serum protein levels of OPG (osteoprotegerin), a bone formation marker, and RANKL (receptor activator of nuclear factor-kappa B ligand) Respectively. (Fig. 9A), increased RANKL (Fig. 9B) and decreased OPG / RANKL ratio (Fig. 9C) in ovariectomized OVX mice compared to normal ovariectomized Shame mice. However, in the experimental group where scopolin was administered at a dose of 20 mg / kg / day and 40 mg / kg / day for 12 weeks, there was a decrease in OPG, osteoclastogenesis marker, increase in RANKL and decrease in OPG / RANKL ratio (Figs. 9A, 9B, and 9C). These results were similar to those of the positive control group SrCl ₂ mice. Statistical analysis also showed significant efficacy (*: p <0.05 vs. OVX negative control).

Claims (8)

delete delete delete delete delete delete delete A method of promoting in vitro osteoblast differentiation comprising treating osteoblast cells with scopolin or a pharmaceutically acceptable salt thereof in vitro.

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