KR19990038984A - Pharmaceutical composition for preventing and treating osteoporosis - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of osteoporosis, more specifically, to prevent osteoporosis because it contains a glabol, licorice (Glycyrrhiza glabra) extract or Glycyrrhiza glabra fraction represented by the following formula (1) as an active ingredient And pharmaceutical compositions useful for treatment.

Description

Pharmaceutical composition for preventing and treating osteoporosis

The present invention relates to a pharmaceutical composition for the prevention and treatment of osteoporosis, more specifically, to prevent osteoporosis because it contains a glabol, licorice (Glycyrrhiza glabra) extract or Glycyrrhiza glabra fraction represented by the following formula (1) as an active ingredient And pharmaceutical compositions useful for treatment.

Formula 1

Osteoporosis has been studied in the past mainly on bone minerals, namely, calcium and phosphorus metabolic abnormalities, and no progress has been made in identifying its pathogenesis. However, recently, the importance of research on metabolism of bone matrix proteins as well as minerals important for bone formation is emerging. Calcium adjuvant, widely used as an agent for treating osteoporosis, has recently been reported to be insignificant. In addition, the effects of hormone therapy using estrogen or calcitonin are not significant. It is reported that it is likely to cause.

Therefore, there is an urgent need for the development of new substances effective in the prevention and treatment of osteoporosis due to less toxicity and side effects with new actions and skeletal structures. In addition, attempts to create new drugs from natural products, especially herbal medicines, are actively underway since these new materials are very likely and found in natural products that have been used for a long time as folk remedies.

Osteoporosis is a type of osteoporosis that is more easily caused by bone weakness than itself, especially femoral fractures or vertebral fractures, which limit long-term activity and lead to a healthy life, resulting in 15% of elderly deaths. There is a problem.

Bone tissue is a dynamic tissue that is constantly formed by osteoblasts and destroyed and absorbed by osteoclasts. There are many uncertainties in the mechanism of bone absorption and formation, but osteoporosis is a disease caused by a weak balance between bone absorption and bone formation.

Licorice (Glycyrrhiza glabra) is used as a food, and its components are very diverse, but the main component is a flavonoid compound, and these components have various physiological and pharmacological effects such as melanin suppression, immune suppression, and anti-inflammatory agents. It is known, and it is a traditional medicine that is listed in various prescriptions as a sweetener in folk medicine and a representative antidote in herbal medicine.

However, the physiological activity of osteoporosis of various flavonoids contained in a large amount of licorice has not been revealed to date.

Therefore, the inventors of the present invention while studying the physiological activity of various flavonoids of licorice, methanol extract, hexane extract and ethyl acetate extract of licorice and hexane fraction and ethyl acetate fraction for each extract inhibits osteoporosis-induced action This potency was discovered, and as a result of tracking the active substance by bioassay-directed fractionation using a series of solvent extraction methods and column chromatography, the osteoporosis-induced inhibitory activity was represented by Chemical Formula 1, which is a strong flavonoid. The displayed glabrol was isolated.

Glabrol represented by Chemical Formula 1 is a substance known as a flavanone compound [Chem. Pharm. Bull. 24 (4) 752 to 755 (1976). Glabrol is known to be effective as an antifungal, antifungal, anti-tuberculant, and antimutagenic and also effective for hyperlipidemia and atherosclerosis, but glabrol has not yet been found to have physiological activity against osteoporosis.

An object of the present invention is to contain a pharmaceutical composition useful for the prevention and treatment of osteoporosis, because it contains a glabol, licorice (Glycyrrhiza glabra) extract or Glycyrrhiza glabra fraction represented by the formula (1) having an osteoporosis-inducing inhibitory activity as an active ingredient To provide.

FIG. 1 is a diagram showing the determined structure of glabrol represented by the formula (1).

FIG. 2 is a diagram showing C-H correlations obtained from HMQC and HMBC spectra of glabrole represented by Chemical Formula 1. FIG.

The present invention is characterized in that the pharmaceutical composition containing glabrolol, licorice (Glycyrrhiza glabra) extract or Glycyrrhiza glabra fraction represented by the following formula (1) as an active ingredient.

In another aspect, the present invention is characterized by a method of extracting and concentrating a licorice (Glycyrrhiza glabra), and then, solvent fractionation and column chromatography to separate and recover the glabrol represented by Chemical Formula 1.

Referring to the present invention in more detail as follows.

Licorice is obtained by using water, an organic solvent or a mixed solvent thereof as an extraction solvent. In this case, water, methanol, ethyl acetate or hexane is preferably used as the extraction solvent.

In addition, the licorice extract is fractionated with hexane and ethyl acetate to obtain a licorice fraction. In this case, various organic solvents may be used as the fractionation solvent, but the fractionation ratio when fractionation with hexane and ethyl acetate is the best.

In addition, the concentrate obtained by concentrating the licorice extract is subjected to solvent fractionation, and then subjected to column chromatography and recrystallized to obtain the glabrol represented by the formula (1). In other words, the concentrated solution is solvent fractioned with hexane, ethyl acetate, butanol and water, and then the active fraction is purified by column chromatography using silica gel, Sephadex, RP-18, etc. Chromatography) and VCC (Vacuum Column Chromatography) can be used to obtain high-purity glabrol.

The globulol, licorice extract or licorice fraction represented by the formula (1) obtained by the above method is combined with a conventional pharmaceutically acceptable carrier in the form of each or mixed with each other tablets, capsules, troches, liquids, etc. Preparations for oral administration of; Injectable preparations, such as ready-to-use dry powder for injection, which can be prepared and used as an injectable liquid or distilled water for injection at the time of injection; It can be formulated into a conventional formulation in the pharmaceutical field, such as formulations for topical administration such as aerosols and solutions.

Carriers that can be used in the present invention include carriers commonly used in the pharmaceutical field, such as binders, suspending agents, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspensions in the case of oral preparations. Topical agents, pigments and flavorings; In the case of topical administration agents, there are bases, excipients, lubricants and preservatives. In addition, during oral administration, in order to prevent the drug from being degraded by gastric acid, an antacid may be used in combination, or may be administered by formulating an oral administration solid preparation such as a tablet coated with an enteric coating.

The pharmaceutical preparations thus prepared may be administered orally or parenterally, intravenously, subcutaneously, intranasally or intraperitoneally.

The human dose of the active ingredient according to the present invention is appropriately selected depending on the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, the severity of the disease to be treated, but generally 1 It is preferable to administer about 15 to 1500 mg per day, but about 300 to 1000 mg for extracts or fractions, and about 200 to 800 mg for glabrol represented by the formula (1). Therefore, when the licorice extract of the present invention is prepared as a unit dosage form, each unit dosage form is prepared to have an amount of 5 to 500 mg of the active substance of the present invention in consideration of the above-mentioned effective dose range. Preferably it is formulated to contain 75 to 350 mg for extracts or fractions, and 50 to 300 mg for glabrol represented by the formula (1). The formulated unit dosage form may be administered several times at regular time intervals or by using a specialized dosage method according to the judgment of an expert who monitors or observes the administration of the drug as needed and the needs of the individual. It may be administered once to three times a day.

In addition, in the present invention, to determine the degree of inhibition of bone resorption of each of the globulol, licorice extract, licorice fraction represented by the formula (1) to establish the bone resorption inhibitory ability test of osteoclasts to investigate the osteoporosis inhibitory activity + It was used for the search. The test method is to measure the bone resorption capacity of osteoclasts using dentin fragments. The dentin fragments and osteoclast-like multinucleated cells are cultured together to form a pit in the dentin fragments and to measure the area of the pit. At this time, the area of the pit is reduced as much as bone absorption is suppressed, and thus can be distinguished from the control.

As a result of experiments on the bone absorption capacity of each of the globulol, licorice extract and licorice fraction represented by the formula (1), it was found that the activity of the glabrol represented by the formula (1) is the most excellent.

The present invention will be described in detail with reference to the following examples, but the present invention is not limited thereto.

Example 1 Separation and Purification of Active Material from Licorice

Plant taxonomically tested Glycyrrhiza glabra of legumes (Leguminosae) was obtained commercially. Finely chopped and dried 2 kg of licorice root was extracted with a solvent using an extractor with a cooler at least five times. The methanol extract was filtered at on and concentrated almost completely at 40 캜 or lower using a vacuum concentrator. The concentrate was suspended in water and the suspension was then solvent fractionated sequentially using hexane, ethyl acetate and butanol. Since the activity was shown in the hexane and ethyl acetate fractions of the fractions, each fraction was subjected to column chromatography using silica gel, Sephadex and RP-18.

Hexane fractions were fractionated into ten by column chromatography using silica gel. The eluting solvent was carried out in a concentration gradient using hexane and ethyl acetate (hexane / ethyl acetate = 10/0 → 0/10). Part 4 of the fraction was purified by separating the active material using MPLC (Medium Pressure Liquid Chromatography) and VCC (Vacuum Column Chromatography).

In addition, fractions 8 and 9 were attempted to separate and purify the active substance using MPLC, VCC, and prep HPLC to obtain a highly active substance, but the structure thereof was not determined.

The ethyl acetate fractions were fractionated into ten by column chromatography using silica gel. The elution solvent was carried out in a concentration gradient using methylene chloride and methanol (methylene chloride / methanol = 100/0 → 50/50). As a result of attempting to separate and purify the active substance using MPLC and VCC, fraction 1 was obtained with a highly active globulin represented by the formula (1). In addition, fraction 2 was used to separate and purify the active substance using MPLC and VCC to obtain a highly active substance, but its structure was not determined.

Example 2: Structural Determination

In order to identify the structure of the globulol obtained in Example 1 was carried out as follows.

The photoluminescence was measured at 22 ° C by dissolving 12 mg of the glabrol obtained in Example 1 in 3.5 ml of methanol. In the present invention, the photosensitivity of the glabrol obtained as a pale yellow amorphous solid is as follows.

[α] _D ²² = -48.0 (MeOH)

In addition, ¹ H-NMR (400 MHz) and ¹³ C-NMR (100 MHz) spectra of the glabrolol obtained in Example 1 were measured, and the chemical shift of each peak was measured using the residual solvent of methanol. It is shown as a relative value to the chemical shift value (3.3 ppm, 49.8 ppm). ¹ H-NMR and ¹³ C-NMR spectral data are shown in Tables 1 and 2 below.

¹ H signal assignment and ¹ H- ¹ H correlation H Chemical shift of H (δ) COZY 2 5.31 H3 3 2.70, 3.01 H2, H3 5 7.57 H6 6 6.49 H5 2' 7.19 5 ' 6.76 H6 ' 6 ' 7.12 H5 ' One" 3.29 H2 " 2" 5.18 H1 " 4" 1.62 5 " 1.59 One'" 3.30 H2 '" 2'" 5.33 H1 '" 4'" 1.72 5 '" 1.70

¹³ C Signaling and ¹³ C- ¹ H Correlation C Chemical shift of C (δ) DEPT HMQC HMBC 2 80.87 CH H2 H3, H2 ', H6' 3 44.79 CH ₂ H3 4 194.22 C H3, H5 5 126.67 CH H5 6 110.89 CH H6 7 163.03 C H7 H5, H1 " 8 117.06 C H6, H1 " 4a 114.90 C H6 8a 164.33 C H5, H1 " One' 131.47 C H2, H3, H5 ' 2' 128.82 CH H2 ' H6 ', H1' " 3 ' 129.40 C H5 ', H1' " 4' 156.40 C H2 ', H5', H6 ', H1' " 5 ' 115.59 CH H5 ' 6 ' 125.94 CH H6 ' H2 ' One" 22.96 CH ₂ H1 " 2" 123.36 CH H2 " H1 ", H4", H5 " 3 " 132.01 C H1 ", H4", H5 " 4" 25.94 CH ₃ H4 " H2 ", H5" 5 " 17.85 CH ₃ H5 " H2 ", H4" One'" 29.25 CH ₂ H1 '" H2 ' 2'" 123.78 CH H2 '" H1 '", H4'", H5 '" 3 '" 133.12 C H1 '", H4'", H5 '" 4'" 17.97 CH ₃ H4 '" H2 '", H5'" 5 '" 25.98 CH ₃ H5 '" H2 '", H5'"

The overall structure of the glabrol obtained in Example 1 is known from known glabrol [Tamotsu Saitoh, et al., Chem. Pharm. Bull, 24 (4), 1976, 752 to 755] was determined by ¹ H-NMR and ¹³ C-NMR, DEPT, and two-dimensional NMR COSY, HMQC, HMBC experiments. ^{In 1} H-NMR, methyl peaks at 1.59 ppm, 1.62 ppm, 1.70 ppm, and 1.72 ppm appeared as singlets, and the overlapping peaks of 3.29 ppm and 3.30 ppm were confirmed by HMBC and HMQC experiments.

The structure of the glabrolol obtained in Example 1 by the above method was determined and shown in FIG. 1, which is in good agreement with all the results observed in HMQC and HMBC. The correlated C-H results obtained from the HMBC spectrum are shown in FIG.

Example 3: Measurement of Bone Uptake Inhibition Activity

1. Preparation of Cells

end. Bone Marrow Cell Culture

Tibia was extracted from 6 to 9 week old male ddY mice, and bone marrow cells were recovered from the extracted tibia using a syringe to give α MEM medium (Gibco BRL, sodium bicarbonate 2.0 g / l, streptomycin 100 mg / l, penicillin). 100,000 units / ml was added and then filtered and sterilized in 5 ml to determine nucleated cell numbers. Inoculate 48well plate (Falcon) to 5 × 10 ⁵ cells / 0.25ml / well using α MEM medium added with 10% FCS (Hyclone), and add bone resorption factors and incubate for 6 to 8 days. The cell number was then confirmed [T. Akatsu et al., J. Bone. Miner. Res., 7, 1297-1306, 1992].

I. Osteoblast-like Cell Culture

Skulls were removed from newborn ddY mice aged 1 to 2 days, suspended in 10 ml of enzyme solution (0.1% collagenase and 0.2% testpase) and allowed to react at 37 ° C for 5 minutes. The cell suspension supernatant was recovered, and 10 ml of fresh enzyme solution was added, followed by reaction at 37 ° C for 10 minutes. This operation was repeated four times to recover the cell suspension supernatant. The cell suspension supernatant was inoculated into a culture dish using centrifugation (250 × g, 5 min) and then cultured to 3 to 5 × 10 ⁴ cells / 10 ml / 100 mm using αMEM medium (10% FCS addition). After incubation for 3 to 4 days, the cells were recovered using Tripsin-EDTA solution, and the cells were diluted to a concentration of 1 × 10 ⁶ cells / 1ml and stored at -70 ° C.

All. Coexistence Culture

Co-culture was performed using collagen gel (cell matrix Type I-A).

1) Preparation of Collagen Gel Solution

Collagen (cell matrix Type IA), 5-fold concentrated αMEM medium, 0.05M NaOH buffer solution (containing 2.2% NaHCO ₃ or 200mM HEPES, pH 7.4) at 7: 2: 1 at low temperature and then stored at low temperature, 100 mm 4 ml of the solution was added to the culture dish and then evenly applied and left at 37 ° C. for 5 minutes.

2) Coexistence Culture

15-20 ml of αMEM medium was added onto the applied collagen gel, and the osteoblast-like cells (about 1 × 10 ⁵ cell / 100 mm plate) and the bone marrow cells (about 1 × 10 ⁷ cell / 100 mm plate) were inoculated. Co-culture was performed for 6-8 days in the presence of vitamin D ( ^10-8 M) and PGE ₂ ( ^10-8 M). The culture solution was removed, 4 ml of 0.2% collagenase solution was added, and the adherent cells were separated by shaking culture at 37 ° C. for 20 minutes. After suspension and recovery of cells, the degree of differentiation of osteoclasts was evaluated and used for analysis.

la. Assessment of osteoclast differentiation

1) Preparation of Tartaric Acid Resistance Alkaline Phosphatase (TRAP) Reaction

5 mg of substrate naftol AS-MS phosphate (sigma N-4875) and 25 mg of fast red violet LB salt were dissolved in N, N-dimethylformamide (about 0.5 mL). 50 ml of 0.1 N NaHCO ₃ buffer (pH 5.0) with 50 mM tartaric acid was added. Refrigerated and used as a dye solution.

2) dyeing

After culturing the cells, the culture solution was removed, washed once with PBS, and the cells were fixed in PBS containing 10% formalin for 2 to 5 minutes. After fixing again in ethanol / acetone (1/1) for about 1 minute and dried. After treatment with TRAP staining solution for 15 minutes, washed with water and dried under a microscope to measure the number of TRAP positive cells of co-cultured cells to determine the degree of differentiation into osteoclasts.

2. Measurement of bone resorption capacity

end. Preparation of Hematoxylin Dye

After dissolving 1 g of hematoxylin in 500 ml of purified water, 500 ml of purified water and 0.2 g of sodium iodide were added and stirred for 15 minutes. 50 g of ammonium alum and 7.5 ml of acetic acid (glacial acetic acid) were added thereto, and the resultant was sufficiently stirred and filtered to use a dye.

I. reaction

After the addition of αMEM medium (10% FCS) to the 96 well microplate 100 μl / well, dentin sections (derived from ivory, thickness of about 0.2 mm, diameter of about 4 mm). The osteoclast-type multinucleated cells differentiated by co-culture previously were turbid in 10 ml of αMEM medium (10% FCS added), added 100 μl per well, and left in the incubator for 1-2 hours. The cells were incubated for 24 hours in a 24-well or 48-well microplate containing FCS) (1 dentin / 0.5-1.0 ml / well). At this time, the control group without adding the sample and the well with the addition of the sample for checking the pit formation inhibition were incubated together.

All. Measure

The culture solution was removed, 1 ml of 1 M ammonia water was added, and cells were completely removed from the sections using an ultrasonic cell mill. The sections were collected and stained with hematoxylin stain for about 1 minute. The excess dye solution was removed and the staining state of the pits formed on the sections was observed under a microscope, and the sections were washed with water and dried. The area of the pit was measured by an image analyzer. The inhibitory activity was confirmed by comparing the area of the control group and the pit area in the well to which the sample was added.

la. result

The pit formation inhibitory activity of each of the globulol, licorice organic solvent extract and licorice organic solvent fraction represented by the formula (1) is shown in Table 3 below. As a comparative sample, commercially available prefriflavone (Deoborn ^⑧ ) was used.

Sample (concentration) Inhibitory Activity (%) Licorice Methanol Extract (50µg / mL) 70 Licorice ethyl acetate extract (50µg / ml) 70 Licorice Hexane Extract (50µg / mL) 73 Licorice ethyl acetate fraction (10 µg / ml) 65 Licorice hexane fraction (10 μg / ml) 65 Glabrol (1 μg / ml) 72.6 Ifriflavones (1 μg / ml) 32

In the case of licorice extract and licorice fraction, there was a difference in concentration, but generally showed about 2 times higher activity than ifriflavone, and the purified glabulol represented by Formula 1 showed more than 2 times activity at the same concentration.

In addition, toxicity experiments were performed for each of the glabrol, licorice extract and licorice fraction represented by the formula (1). Each test substance was suspended in 0.5% Na-CMC and administered to mice (5 mice per group) and observed for 14 days to determine mortality. At this time, glabrol was administered in five stages of 1 g / kg, 500 mg / kg, 250 mg / kg, 125 mg / kg and 62.5 mg / kg, and licorice extract and licorice fraction were 2 g / kg and 1 g, respectively. / Kg, 500 mg / kg, 250 mg / kg, 125 mg / kg in five steps. As a result, posts 50% lethal dose (LD ₅₀₎ of the roll Rab is 1 g / ㎏ was 50% of licorice extract and licorice fraction lethal dose (LD ₅₀₎ was 2 g / ㎏.

As described above, the glabrol, licorice (Glycyrrhiza glabra) extract or the licorice (Glycyrrhiza glabra) fraction represented by Formula 1 according to the present invention is excellent in bone resorption ability, so as an active ingredient of the pharmaceutical composition for preventing and treating osteoporosis useful.

Claims (7)

The pharmaceutical composition for osteoporosis prevention and treatment, characterized in that the glabrol represented by the following formula (1) is contained as an active ingredient. Formula 1 A pharmaceutical composition for preventing and treating osteoporosis, characterized in that the licorice (Glycyrrhiza glabra) extract is contained as an active ingredient. A pharmaceutical composition for preventing and treating osteoporosis, characterized in that the licorice (Glycyrrhiza glabra) fraction is contained as an active ingredient. Licorice (Glycyrrhiza glabra) solvent extraction and concentration, and then separated and purified by performing solvent fractionation and column chromatography separation and recovery method of the glabrolol represented by the formula (1). Formula 1 The method of claim 4, wherein the extraction solvent is methanol, hexane or ethyl acetate. The method of claim 4, wherein the fractional solvent is hexane and ethyl acetate. The method of claim 5, wherein the column chromatography is performed by using silica gel, Sephadex or RP-18.