KR100766431B1 - Bone tissue regeneration promoter comprising oleanane compounds - Google Patents

Abstract

The present invention relates to a bone tissue regeneration accelerator using an oleanane compound as an active ingredient, specifically hederagenin 3-O- [2-O-acetyl-alpha-L-arabinofyranoside] or hederagenin A bone tissue regeneration accelerator comprising 3-O-alpha-L-arabinofyranoside as an active ingredient.

Oleanane compound according to the present invention promotes bone mineralization process of osteoblasts by increasing the cell proliferation of human fetal osteoblasts, increase the activity of basic phosphatase and protein expression of osteocalcin and BSP, bone defects By forming new bone in the part, it can be usefully used for bone regeneration, such as bone tissue, or fracture and bone damage, destroyed by periodontal disease.

Description

Bone tissue regeneration promoter comprising oleanane compounds as an active ingredient             

1 is a view showing a process for extracting, separating and purifying an oleanane compound from the fast-acting according to the present invention.

Figure 2 is a diagram showing the effect of the oleonan-based compound according to the present invention on cell proliferation for human fetal osteoblasts.

Figure 3 is a diagram showing the basic phosphatase activity against human fetal osteoblasts of the oleonan-based compound according to the present invention.

Figure 4 is a diagram showing the expression of osteocalcin and BSP in human fetal osteoblasts of the oleonan-based compound according to the present invention.

Figure 5 is a diagram showing the new bone regeneration for the skull defect of the oleonan-based compound according to the present invention.

The present invention relates to a bone tissue regeneration accelerator using the oleanane compound as an active ingredient.

Hard tissue in the human body (hard tissue) is largely classified into bones and teeth, a disease that occurs due to the problem of the periodontal disease. Periodontal disease is a disease that forms periodontal pockets due to chronic inflammation and causes periodontal tissue destruction such as alveolar bone. Therefore, periodontal disease, which is the cause of periodontal disease, removes plaque and periodontal disease, and the periodontal disease can be treated if the periodontal tissue is newly formed structurally and functionally while the alveolar bone is regenerated. In order to regenerate lost alveolar bone, bone transplantation using various bone materials should be performed in addition to general periodontal treatment. In addition, it is necessary to administer biological bone regeneration accelerators such as growth factors that can enhance bone induction. .

If the material used for bone graft is classified according to the source, it can be divided into autologous bone, allogeneic bone, xenograft bone, synthetic bone, and the like.

First, autologous bone graft is a bone graft taken from the patient's own body, which may be the most ideal implant, but the limit of the amount of bone graft, additional surgery for bone graft, damage to the donor site at the time of bone extraction, and postoperative procedure There are disadvantages such as the possibility of complications.

Second, allogeneic bone is donated by organs (bones) from brain lions and made by tissue banks, which compensates for the disadvantages of autologous bones, but the possibility of disease transmission from the donor and immune rejection may be a problem.

Third, xenografts and synthetic bones are similar in structure and composition to human bones and can compensate for the disadvantages of autologous and allogeneic bones, but they have the least bone formation ability because they do not have bone formation promoting factors and are only fillers. It only serves as.

In recent years, much attention has been directed to some growth factors that can control the proliferation and differentiation of cells in the method of treating bone regeneration or healing of periodontal tissue. (Terranova VP., Wikesjo UM .: Extracellular matrices and polypeptide growth factors as mediators of functions of cells of the periodontium.A Review. J Periodontol 58 (6): 371-380, 1987.). Growth factors are polypeptide molecules secreted from the inflammatory site that regulate biological mechanisms that occur during wound healing, regulate the migration of connective tissue cells, the growth and synthesis of proteins, and the extracellular matrix material. Platelet-derived growth factor (PDGF) among growth factors is known to promote cell proliferation of human bone cells (Piche JE., Graves DT .: Study of the growth factor requirements of human bone-derived cells: a comparison with human fibroblasts.Bone 10 (2): 131-138, 1989.), insulin-like growth factor (IGF) is known to promote DNA synthesis of osteoblasts (Canalis E., Pash). J., Gabbitas B., Rydziel S., Varghese S .: Growth factors regulate the synthesis of insulin-like growth factor-I in bone cell cultures.Endocrinology 133 (1): 33-38, 1993.). In addition, bone morphogenic protein (BMP) is known to increase bone formation in jaw defects, and it is known that the addition of bone morphogenetic protein to proteoblasts isolated from rats results in higher basic phosphatase activity. (Yamaguchi A., Katagiri T., Ikeda T., Wozney JM., Rosen V., Wang EA., Kahn AJ., Suda T., Yoshiki S .: Recombinant human bone morphogenetic protein-2 stimulates osteoblastic maturation and inhibits myogenic differentiation in vitro. J Cell Biol 113 (3): 681-687, 1991.).

On the other hand, in addition to such growth factors, research has been made on substances that regenerate periodontal tissue and bone in oriental medicine. The interest in periodontal disease in oriental medicine started a long time ago, periodontal disease has been known as the first symptom listed in the emperor bore (경). In recent years, attention has been drawn to the fact that the drugs extracted from herbal medicines have fewer side effects and can be used for a long time.As a treatment for periodontal disease, the anti-inflammatory and antimicrobial effects on periodontal disease bacteria and the effects on the periodontal tissue regeneration ability Approaches and analyzes have been attempted, and typical herbal preparations include safflower seed, hubac, and sokdan.

Safflower seed is known to be effective in strengthening fractures, bone fractures, clavicles, as well as various bone diseases and fragile bones. Safflower seed extract has been reported to increase osteoproliferation and basic phosphatase activity of osteoblasts and to promote the formation and maturation of new bone, and thus have an excellent effect on bone formation, regeneration and healing (Yun Dong-hwan, Lee Seung-cheol, Kim Myung-eun) , Kim, Eun-Chul, Hyung-Geun Kim, Yun-Chul Kim, and Hyung-Chul Shin: Effect of Safflower Seed Extract on Osteoblast-like Cell Activity and Bone Regeneration.Journal of Korean Periodontology 28 (4): 769-786, 1998.) It has been reported to be effective in wound healing and new bone formation by topical administration of extracts (Dukgyu Kyu, Sungwoo Woo, Kyungtae Yoo, Jaejin Kim, Heungsik Kim, Hyung-Geun Shin, New Type: Effect of topical administration of safflower seed extract on the regeneration of skull defects in rats. Korean Journal of Periodontology 29 (2): 297-313, 1999. In addition, Korean Patent Application Nos. 10-1993-27375 and 10-1993-60028 describe that safflower seeds are effective in treating damaged bones.

Thick pepper extract is described in the Republic of Korea Patent Application No. 10-1992-16516 has a useful effect in the prevention and treatment of periodontal disease, and the Republic of Korea Patent Registration No. 10-204165 discloses the damage of bones, such as osteotomy, osteoctomy, collarbone It is said to have a beneficial effect on the treatment of damaged bone by promoting regeneration.

On the other hand, Phlomidis Radix is a perennial herb belonging to the Lamiaceae family and its medicinal parts are roots and rhizomes. Sokdan is an herbal medicine that has been widely used in clinical practice for improving liver and kidney function, smoothing blood flow, and connecting muscle and bone. It is described as golgol (속), sokdan (천), cheonshikdan (gawa), etc., and can be said to have been named in the sense of connecting most broken bones. In addition, it is known that anti-inflammatory analgesic, drainage, hemostasis, tissue regeneration, strengthening heart activity, inhibits platelet aggregation.

The effects of extracts obtained from the heat-filtered mixture of Sokdan with distilled water on the basic phosphatase activity of osteoblasts were reported to be significantly increased compared to the control group (Kim Dae-gyeom, Kim Tak, Pee Sung-hee, Kim Hyun-a, Choi Kwang-soo). , Hyoung-Geun Kim, New Type: Effects of several herbal preparations on the synthesis of basic phosphatase in MC3T3-E1 cells.Journal of Korean Journal of Dental Medicine 29 (4): 751-765, 1999.). Later, laboratory studies evaluating the effect of dichloromethane fractions on the induction of bone formation in human fetal osteoblasts have been reported to increase cell activity and basic phosphatase activity and to have an effective effect on the formation of bone nodules. , Dichloromethane fractions of fasting may affect the activity and differentiation of bone cells and suggest the possibility of using them as a regenerant of broken bone tissue (Lee Young-jun, Hee-in Choi, Yuncheol Kim, New-type, Hyung-Geun Lee: Effect on the induction of bone formation in blasts, Korean Journal of Periodontal Science 33 (2): 259-269, 2003.).

Therefore, the inventors of the present invention, while searching for a substance that can be used for a long period of time as a regeneration agent of bone tissue from a herbal drug, the olean-based compound isolated from the rapid increase the cell proliferation of human fetal osteoblasts, and basic phosphatase activity Increasing and increasing the expression of osteoproteins, such as osteocalcin and BSP, by regenerating new bone in bone defects, confirming that it is useful for bone tissue damaged by periodontal disease, or bone defects such as fracture and bone damage and the present invention Completed.

The present invention is to provide a bone tissue regeneration accelerator using the oleanane compound as an active ingredient.

The present invention provides a bone tissue regeneration accelerator using the oleanane compound as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a bone tissue regeneration accelerator using the oleanane compound represented by the following formula (1) as an active ingredient.

(In Formula 1, R is hydrogen or an acetyl group.)

The oleanane-based compound as an active ingredient in the composition of the present invention may be used in the form of a pharmaceutically acceptable salt, and includes all salts, hydrates, and solvates prepared by conventional methods. As salts, acid addition salts formed with pharmaceutically acceptable free acids are useful, and inorganic acids and organic acids can be used as the free acid, and hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc. can be used as the inorganic acid. Organic acids include citric acid, acetic acid, lactic acid, maleic acid, umarin acid, gluconic acid, methanesulfonic acid, glyconic acid, succinic acid, 4-toluenesulfonic acid, trifluoroacetic acid, galluxuronic acid, embon acid, glutamic acid, or as Partic acid and the like can be used.

In the present invention, the oleanane-based compound was extracted, separated and purified from the fast-acting compound, which can be obtained by all conventional methods, and commercially available reagents can be used.

Extraction, separation and purification methods of the oleanane compound from the fast-acting according to the present invention are as follows.

Clean the inside quickly, dry and cut it. The fine fastener was repeatedly extracted with methanol for 2 hours, filtered, and the filtrate was concentrated under reduced pressure to obtain a methanol extract. The obtained methanol extract was dissolved in 60% aqueous methanol solution and partitioned sequentially using n-hexane and dichloromethane. The 60% methanol aqueous solution fraction was concentrated under reduced pressure again, and the obtained residue was dissolved in distilled water and then partitioned using n-butanol. Each solvent fraction is removed using a reduced pressure concentrator to obtain n-hexane, dichloromethane and n-butanol fractions.

The dichloromethane fraction showing the significant effect in the bone formation induction test of human fetal osteoblasts from the solvent fraction is separated by the following method.

The dichloromethane fractions obtained above were subjected to solvent fractionation using n-hexane: ethyl acetate: methanol: water (1: 1: 1: 1 (v / v), 2L), and fraction 1 (upper) and fraction 2 (lower). Get)

Fraction 1 was subjected to silica gel column chromatography using chloroform: methanol (12: 1 ⇒ 8: 1) as the effluent solvent and separated into eight small fractions (fractions 11 to 18) according to the spot pattern on TLC. Fraction 14 of the subfraction was separated into four fractions (fractions 141 to 144) by Sephadex LH-20 column chromatography (eluent: chloroform). Fraction 141 was subjected to reverse phase C-18 column chromatography (eluent: 80% methanol) to give compound 1 (hederagenin 3-O- [2-O-acetyl-alpha-L-arabinofyranoside] Get)

Fraction 2 was subjected to silica gel column chromatography using chloroform: methanol (9: 1 ⇒ 3: 2) as the effluent solvent and separated into five small fractions (fractions 21 to 25) according to the spot pattern on TLC. Fraction 23 of the subfraction was repeated with silica gel column chromatography using chloroform: methanol: water (9: 1: 0.1) as an elution solvent to obtain Compound 2 (hederagenin 3-O-alpha-L-arabinofi). Lanoside).

Of the oleanane compounds of the formula 1 according to the present invention, the number of cells in the group treated with Compound 1 is significantly increased compared to the negative control group at 10 ^-6 , 10 ^-7 M in the group cultured for 5 days, Compound 2 The number of cells in the group treated with 10 ^-5 , 10 ^-6 M showed a significant increase compared to the negative control group (p <0.05).

In addition, in the oleanane compound of Formula 1 according to the present invention, the basic phosphatase activity in the group treated with Compound 1 is significantly increased compared to the negative control at 10 ^-7 to 10 ^-11 M, and treated with Compound 2 One group showed a significant increase from 10 ^-6 to 10 ^-12 M compared with the negative control (p <0.05).

In addition, among the oleanane compounds of Formula 1 according to the present invention, the group treated with Compound 1 and Compound 2 in Western blot analysis is not expressed at 5 days, 7 days, and 10 days at the beginning of the mineralization process, In one, the expression of osteocalcin and BSP is increased.

In addition, when the oleanane-based compound of Formula 1 according to the present invention is directly administered after forming a bone defect in the white skull, the compound 1 administration group significantly increased new bone formation at 1, 2, and 4 weeks compared to the control group. At week 4, most of the defects were replaced with new bone (p <0.05). Compound 2 administration group significantly increased new bone formation at 2 and 4 weeks compared to the control group (p <0.05).

Therefore, the oleonan-based compound of Formula 1 according to the present invention increases the cell proliferation of human fetal osteoblasts, increases the activity of basic phosphatase, and increases the protein expression of osteocalcin and BSP to undergo bone mineralization of osteoblasts. By accelerating and forming new bone in the bone defect, it can be usefully used for bone defect regeneration such as bone tissue destroyed by periodontal disease or fracture and bone damage.

The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in addition to the oleanane compound.

The composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers include polymeric compounds such as polylactic acid and polyglycolic acid, collagen, hyaluronic acid, saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and these components One or more components can be mixed and used, and other conventional additives, such as an antioxidant, a buffer solution, and bacteriostatic agents, can be added as needed. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

The compositions of the present invention may be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method, and the dosage may be based on the weight, age, sex, health status, The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease. The daily dosage is about 0.2 to 30 mg / kg, preferably 5 to 20 mg / kg, of the oleanane-based compound of formula 1, and more preferably administered once to several times a day.

The composition of the present invention can be used alone or in combination with methods using surgery, hormone therapy, drug treatment and biological response modifiers for the regeneration of bone tissue or bone defects destroyed by periodontal disease.

Hereinafter, preferred examples and experimental examples are presented to help understand the present invention. However, the following Examples and Experimental Examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited by the Examples.

Example  From fast Oleanan system  Extraction of compounds, Separation and Purification

1. Extraction from Rapid

Sokdan, purchased from Iksan, Korea, was washed, dried, and cleaned. 1.2 kg of fine fasteners were repeatedly extracted with 5 L of methanol for 2 hours, filtered, and the filtrate was concentrated under reduced pressure to obtain 196 g of methanol extract. The resulting methanol extract was dissolved in 60% aqueous methanol solution and partitioned sequentially using n-hexane and dichloromethane. The 60% methanol aqueous solution fraction was concentrated under reduced pressure again, and the obtained residue was dissolved in distilled water and then partitioned using n-butanol. For each solvent fraction, the solvent was removed using a vacuum concentrator, and n-hexane, dichloromethane and n-butanol fractions were obtained in 4.5g, 6.7g and 56.0g, respectively.

As a result of testing the bone formation induction effect of human fetal osteoblasts in the solvent fraction, it showed a significant effect in the dichloromethane fraction.

2. Dichloromethane From fractions Oleanan system  Isolation and Purification of the Compound

The dichloromethane fraction obtained in 1 was subjected to solvent fractionation using n-hexane: ethyl acetate: methanol: water (1: 1: 1: 1 (v / v), 2L), and fraction 1 (upper layer, 2.7g) and Fraction 2 (lower layer, 3.1 g) was obtained.

Fraction 1 was subjected to silica gel column chromatography using chloroform: methanol (12: 1 ⇒ 8: 1) as an effluent solvent and eight small fractions (fractions 11 to 18) according to the spot pattern on TLC (thin layer chromatography). Separated. Fraction 14 (794.5 mg) in the small fraction was separated into four fractions (fractions 141 to 144) by Sephadex LH-20 column chromatography (eluent: chloroform). Fraction 141 (360 mg) was subjected to reverse phase C-18 column chromatography (solvent: 80% methanol) to obtain compound 1 (66 mg).

Fraction 2 was subjected to silica gel column chromatography using chloroform: methanol (9: 1 ⇒ 3: 2) as the effluent solvent and separated into five subfractions (fractions 21 to 25) according to the spot pattern on TLC. Fraction 23 (885 mg) in the small fraction was subjected to repeated silica gel column chromatography using chloroform: methanol: water (9: 1: 0.1) as an elution solvent to obtain compound 2 (63.2 mg).

3. Oleanan system  Structural Analysis of Compounds (Compounds 1 and 2)

The material obtained through the above example, ¹ H-NMR, ¹³ C-NMR, DEPT (Distortionless Enhancement by Polarization Transfer), HMQC (1H-Detected Heteronuclear Multiple-Quantum) through nuclear magnetic resonance (NMR) analysis (JEOL 500) Spectra such as Coherence) and Heteronuclear Multiple-Bond Coherence (HMBC) were obtained and the molecular weight was determined using a mass spectrometer (Electron Spray Ionization mode) to determine molecular weight and molecular structure.

The measurement results are as follows. As a result of comparative analysis with the published literature, Compound 1 of Formula 1 is hederagenin 3-O- [2-O-acetyl-alpha-L-arabinofyranoside] (Woo WS, Choi JS, Seligmann O., Wagner H., Sterol and triterpenoid glycosides from the roots of Patrinia scabiosaefolia . Phytochemistry , 22 (4), 1045-1047 (1983)), compound 2 is hederagenin 3-O-alpha-L-arabinofyranoside (Jung KY, Son KH, Do JC, Triterpenoids from the roots of Dipsacus asper . Arch. Pharm. Res., 16 (1), 32-35 (1993)).

[Compound 1]: Hederagenin  3-O- [2-O- Acetyl Alpha-L Arabinopyranoside ]

Hederagenin  3-O- [2-O- acetyl -a-L- arabinopyranoside ]

1) Properties: Colorless needle crystal

2) Molecular Weight: 646

3) Molecular formula: C ₃₇ H ₅₈ O ₉

4) (-)-ESI-MS m / z (rel. Int.): 645 [MH] ^- (100)

5) ¹ H-NMR (500 MHz, pyridine-d ₅ ) δ 0.91, 0.92, 0.93, 0.99, 1.02, 1.22 (3H each, s, CH ₃ ), 2.10 (3H, s, C H ₃ COO-), 4.90 (1H, doublet, J = 7.1 μs, anomeric H), 5.47 (1H, br.d, 12-H)

6) ¹³ C-NMR (125 MHz, pyridine-d ₅ ) δ 38.5 (C-1), 26.0 (C-2), 80.9 (C-3), 43.2 (C-4), 46.9 (C-5) , 17.9 (C-6), 32.6 (C-7), 39.6 (C-8), 48.0 (C-9), 36.6 (C-10), 23.5 (C-11), 122.4 (C-12), 144.6 (C-13), 41.8 (C-14), 28.2 (C-15), 23.7 (C-16), 46.5 (C-17), 42.0 (C-18), 46.2 (C-19), 30.8 (C-20), 34.0 (C-21), 33.0 (C-22), 13.4 (C-23), 63.4 (C-24), 15.8 (C-25), 17.3 (C-26), 26.0 ( C-27), 180.0 (C-28), 23.6 (C-29), 33.1 (C-30), 103.8 (C-1 '), 74.2 (C-2'), 72.3 (C-3 '), 69.6 (C-4 '), 67.0 (C-5'), 21.1 ( C H ₃ COO-), 169.9 (CH ₃ C OO-)

[Compound 2]: Hederagenin  3-O-alpha-L- Arabinopyranoside

Hederagenin  3-O-α-L- arabinopyranoside

1) Properties: Colorless needle crystal

2) Molecular Weight: 604

3) Molecular formula: C ₃₅ H ₅₆ O ₈

4) (-)-ESI-MS m / z (rel. Int.): 603 [MH] ^- (100)

5) ¹ H-NMR (500 MHz, pyridine-d ₅ ) δ 0.92, 0.94, 0.95, 1.00, 1.02, 1.25 (3H each, s, CH ₃ ), 4.98 (1H, d, J = 7.1 ㎐, anomeric H ), 5.47 (1H, broad doublet, 12-H)

6) ¹³ C-NMR (125 MHz, pyridine-d ₅ ) δ 38.6 (C-1), 26.0 (C-2), 81.7 (C-3), 43.3 (C-4), 47.4 (C-5) , 18.0 (C-6), 32.7 (C-7), 39.6 (C-8), 48.0 (C-9), 36.8 (C-10), 23.7 (C-11), 122.4 (C-12), 144.6 (C-13), 41.8 (C-14), 28.2 (C-15), 23.7 (C-16), 46.5 (C-17), 42.0 (C-18), 46.2 (C-19), 30.8 (C-20), 34.0 (C-21), 33.0 (C-22), 13.3 (C-23), 64.3 (C-24), 15.9 (C-25), 17.3 (C-26), 26.0 ( C-27), 180.0 (C-28), 23.6 (C-29), 33.1 (C-30), 106.5 (C-1 '), 73.0 (C-2'), 74.6 (C-3 '), 69.5 (C-4 '), 66.8 (C-5')

Experimental Example  One  : Cell proliferation

In order to determine the effect of the oleonan-based compound according to the present invention on bone formation of human fetal osteoblasts according to cell proliferation, the following experiment was performed.

1. Cell Culture

Human fetal osteoblastic cell line (hFOB _1.19 , Catalog No. CRL-11372; American Type Culture Collection, Manassas, VA) was treated with 10% FBS (fetal bovine serum, GibcoBRL, Grand island, NY, USA). 6-well petri dish containing 2 ml of a mixture of DMEM: F-12 HAM (1: 1) (Sigma, St. Louis, MO, USA) with 0.03 mg / ml G-418 (Duchefa, Netherlands) Titration cells (5 × 10 ⁴ cells / well) were dispensed. This was incubated with a continuous supply of 95% air and 5% CO ₂ at 34 ℃ temperature and 100% humidity conditions. Cultures were exchanged every two days until sufficient proliferation of cells occurred and subcultures were performed at a ratio of 1: 3.

2. Cell count  Measure

The monolayer cells that reached denaturation in the petri dish were separated with 0.25% trypsin / EDTA, suspended in culture and aliquoted into 6-well plates to 1 × 10 ⁴ cells. After 24 hours, the culture solution was removed, and the experimental group contained Compound 1 or Compound 2 isolated in the above Examples 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 , 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 , 10 ^-11 , 10 ^-12 M each was added at 10 concentrations, distilled water was added to the control group, and 10 ^-7 M dexamethasone (dexamethasone) was added to the positive control group. After 3 days and 5 days of incubation, trypsin-blue was stained with trypan-blue, and the number of living cells was measured using a hemocytometer.

For the analysis of the experimental results, the mean and standard deviation were calculated using SPSS WIN version 10.0. For the statistical significance, the pretest was processed by one-way ANOVA and the post test was performed by tukey test. (P <0.05).

The results are shown in FIG.

As shown in Figure 2, the number of cells in the group treated with Compound 1 was not significantly different between the control group and the experimental group in the group cultured for 3 days, 10 ^-6 , 10 ^{-7 in the} group cultured for 5 days M increased significantly compared to the negative control (p <0.05).

In addition, the number of cells in the group treated with Compound 2 was significantly increased in the group cultured for 3 days at 10 ⁻⁵ and 10 ⁻⁶ M compared with the negative control group (p <0.05), and the group cultured for 5 days ^Was significantly increased at 10 ^-5 , 10 ^-6 , 10 ^-7 , and 10 ^-8 M compared with the negative control (p <0.05).

Therefore, it can be seen that the oleanane compound according to the present invention has a slight cell proliferation effect at a certain concentration.

Experimental Example  2  : Basic phosphatase activity

In order to determine the effect of the oleonan-based compound according to the present invention on the bone formation of human fetal osteoblasts, the following experiment was performed.

Fetal osteoblasts were dispensed to 1 × 10 ⁵ cells / well in a 6-well culture dish, and then until a single dense layer was formed in a DMEM / F-12 (1: 1) mixture with 10% FBS. Incubated in C, 100% humidity, 5% CO ₂ air mixed incubator. After a single dense layer was formed, the medium was removed and washed twice with a DMEM / F-12 (1: 1) mixture, followed by 10% FBS, G-418 antibiotic, 50 μl / ml ascorbic acid, 10 mM To the DMEM / F-12 (1: 1) mixture with sodium beta-glycerophosphate added 10 ⁻⁷ M of dexamethasone to the positive control, the experimental group was isolated in the above example. 10 kinds of Compound 1 or Compound 2 each 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 , 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 , 10 ^-11 , 10 ^-12 M Concentrated and further incubated for 5 days. After a certain incubation time, the medium was removed, cells were separated by trypsin-EDTA and centrifuged for 6 minutes at 15,000 rpm. The supernatant was removed and suspended in an ultrasonic grinder with the addition of 0.2 ml of sterile distilled water. 0.1 ml of 0.1 M glycine NaOH buffer (pH 10.4), 15 ml of p-nitrophenyl phosphate (pNPP; Sigma, USA) in 0.1 ml of each cell suspension, 0.1% Triton X- 0.1 ml of 100 / salt and 0.1 ml of sterile distilled water were mixed well and the reaction was incubated at 37 ° C. for 30 minutes. These reactions were stopped by adding 0.6 mL of 0.1 N sodium hydroxide. The cultured cells were transferred to a 96-well culture dish, and hydrolysis of p-NPP was indicated by the difference in absorbance in the ELISA reader at 410 nm wavelength, and p-nitrophenol (p-NP; Sigma, USA) The reference value was used. Protein concentration was measured using a BCA protein assay reagent (Pierce, USA), basic phosphatase activity was expressed as nmol / 30 min / mg.

Basic phosphatase is an osteoblast marker of 160 kDa and is expressed in osteoblasts, promyeloblasts, osteoblasts, osteosarcoma cells and the like. The enzyme converts proteins into phosphoproteins by locally increasing the concentration of phosphate ions, which are known to play a key role in the mineralization of bone minerals with calcium binding properties. On the other hand, it was reported that the activity was low in cells without bone formation ability, suggesting that this enzyme is useful as a marker for cells with bone formation ability (Wlodarski KH, Reddi AH .: Alkaline phosphatase as a marker of osteoinductive cells. Calcif Tissue int 39: 382-385, 1986.).

For the analysis of the experimental results, the mean and standard deviation were calculated using SPSS WIN version 10.0. For the statistical significance, the pretest was processed by one-way ANOVA and the post test was performed by tukey test. (P <0.05).

The results are shown in FIG.

As shown in FIG. 3, the group treated with Compound 1 showed a significant increase in 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 , 10 ^-11 M compared with the negative control group (p <0.05) , Especially at 10 ^-8 , 10 ^-9 M, showing the highest activity, these two concentrations were used for Western blot experiments.

In addition, the group treated with Compound 2 showed a significant increase in the negative control group at 10 ^-6 , 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 , 10 ^-11 , 10 ^-12 M (p) <0.05), especially at 10 ⁻⁶ , 10 ⁻⁷ M, showing the highest activity, these two concentrations were used in the Western blot experiment.

Therefore, it can be seen that the oleanane compound according to the present invention increases the activity of the basic phosphatase expressed early in the bone mineralization process, thereby promoting the bone mineralization process of osteoblasts.

Experimental Example  3  Western Blot  analysis

In order to examine the effect of the oleonan-based compound according to the present invention on the osteoblast induction of human fetal osteoblasts, the expression of osteocalcin and BSP was observed.

Cells cultured at each concentration for a certain period of time were washed twice with phosphate buffer solution (1 × PBS), followed by extraction of cellular proteins with lysis buffer and copper in BCA solution (Bicinchoninic acid, Sigma Co, USA). Protein concentration was measured by mixing (II) sulfate (Copper (II) sulfate; Sigma Co, USA) at 50: 1. Each 100 ㎍ extracted protein was used to perform 15% SDS-polyacrylamide gel electrophoresis (SDS-polyacrylamide gel electrophoresis) and then transferred to PVDF (immobilon-P membrane, Milipore, USA).

To prevent binding of nonspecific antibodies, membranes were treated with each blocking solution (Zymed, USA) for 1 hour at room temperature. Thereafter, the reaction was performed for 90 minutes using the primary antibody osteocalcin (Bioogenesis, UK) and BSP (bone sialoprotein, Chemicon International. CA., USA). Osteocalcin is a vitamin K-dependent protein that is specifically distributed in bone and dentin, accounts for 20% of bone's comparative protein, and is a peptide of 46 amino acids and a molecular weight of 4.9 kDa, a gamma-carboxyrutamic acid. (Υ-carboxyhlutamic acid; Gla) appears in the mineral deposits involved in bone and cartilage formation with residues, and is most clinically useful among bone formation markers, also called bone Gla protein. BSP is a comparative protein found in bone and chalky chalk. It contains high concentrations of sialic acid, phosphoserine and sulphotyrosin, and the presence of glutamic acid residues and cell attachments. Helps bone cells adhere to mineralized substrates. Osteocalcin and BSP are both characterized by late stages of bone mineralization.

After reacting with the primary antibody, washed twice with phosphate buffer solution (1 × PBS), and the secondary antibody anti-mouse and anti-rabbit IgG (with basic phosphatase) Santacruze Biotechnology, USA) was reacted at room temperature for 60 minutes, washed twice with phosphate buffer (1 × PBS) for 7 minutes, and then reacted for 1 minute with ECL kit for Hyperfilm-MP (Amersham, UK). Exposed to.

The results are shown in FIG.

As shown in Figure 4, the group treated with Compound 1 and Compound 2 was not expressed at the beginning of the mineralization process 5 days, 7 days, 10 days, but the expression of osteocalcin and BSP increased in the late 14 days, experimental group There was no big difference between the two.

Therefore, it can be seen that the oleonan-based compound according to the present invention increases the protein expression of osteocalcin and BSP involved in the late stage of bone mineralization, thereby promoting bone mineralization of osteoblasts.

Experimental Example  4  : Bone formation  In vivo

In order to determine the effect of the oleanane compound according to the present invention on bone formation, the following experiment was performed.

1. Experimental Animal

The experimental animals were divided into three groups of 24 Sprague-Dawly white papers (Daemul Science, Korea) of 3 months old and weighing about 250 g. In the control group, the defects were washed with physiological saline and then sutured without any treatment. The experimental group was divided into two groups, and the compound 1 prepared in the above example was added to the first group, and the compound 2 prepared in the above example was 0.2 to the second group. Mg doses were administered.

2. Formation of experimental site

Intravenous anesthesia was performed by intramuscular injection of 0.5 ml of ketamine hydrochloride (Ketamine HCl; Ketalar, Yuhan Co., Korea). 2% lidocaine hydrochloride (lidocaine HCl) was injected into the liquor for local anesthesia and hemostatic effect. After the incision was made in the median of the white skull, the periosteum was detached and the valve was elevated. Then, a bone defect was formed using a 4 mm trephine bur (3i, FL, USA) and pressure-bleeding with gauze.

3. Compound Administration

In the first group of the experimental group, 0.2 mg of Compound 1 prepared in the above Examples and the second group of Compound 2, respectively, were administered. After each compound was administered, layered sutures were performed closely with 4-0 absorbent sutures (Vicryl, Ethicon, England). In the control group, the bone defects were sufficiently washed with physiological saline to compare normal bone healing, and the surgical site was closed. In order to prevent postoperative infection, 0.2 ml muscle injection of gentamycin (Korea Microbiological Research Institute, Korea) was conducted to all experimental animals.

4. Histological Examination

For histological examination, the animals were sacrificed into 1 week, 2 weeks, and 4 weeks after the experiment, and the bones of the implants including the surrounding tissues were collected and fixed in 10% neutral formalin solution for 2 days. Each sample was decalcified by the formic acid-sodium citrate method, then embedded in paraffin, tissue sections were made to a thickness of 4 μm, and hematoxylin-eosin (Hematoxylin-). Eosin staining and Modified Masson Trichrome (Goldner's method) staining were performed and observed with an optical microscope.

The results are shown in FIG.

As shown in FIG. 5, in the 1-week group, bleeding and granulation tissues were observed in the defects in the control group, and regeneration of new bone was observed in the margins of the defects, but no osteoclast activity was observed (1A). In the group treated with Compound 1, necrosis of the fibrous and bone tissues occurred slightly in some of the defects, but the formation of new bone was significantly increased compared to the control group (1C, 1C-1). In the group administered with Compound 2, granulation tissue and osteoclasts were observed, but formation of new bone was observed to a degree similar to that of the control group (1B). Overall, similar findings were observed in the 1 week group in the formation of new bone, but the formation of new bone was significantly increased in the compound 1 group compared to the control group.

In the two-week group, the bleeding and inflammation were decreased in the control group compared to the one-week group, the densities of the fibrous connective tissues were increased, and the amount of new bone formation was increased compared to the one week in the control group (2A). In the group treated with Compound 1, inflammatory cell infiltration was reduced compared to 1 week, the highest amount of osteoblasts were observed compared to the other experimental groups, and new bone was migrated from the margins rather than the center of the defect to fill the defect. Compared with the experimental group, it showed the most new bone formation (2C, 2C-1). In the group administered with Compound 2, inflammatory cell infiltration was observed more than the control group, but new bone formation was significantly increased (2B).

In the 4 week group, the new bone generated at the margin of the defect matured in the control group (3A). In the group treated with Compound 1, new bone grew in the bone cutting area and filled most of the defects, leading to the findings in the center of the defect, showing the most osteogenic findings compared to the other experimental groups (3C, 3C-1). In the group to which the compound 2 was administered, the finding that the new bone filled about 1/3 of the bone defect was observed (3B).

5. Histometric Analysis

A total of 96 microscopic images from 24 tissue samples stained with Masson's trichrome were measured for the amount of new bone using an Image Pro II image analyzer (Media Cybermetrics, USA).

The mean and standard deviation were calculated using SPSS WIN version 10.0. The preliminary test was one-way ANOVA and the post-test was tested using tukey test for statistical significance. (P <0.05).

The results are shown in Table 1.

group 1 week 2 weeks 4 Weeks Control 9.85 ± 0.77 17.30 ± 2.57 23.72 ± 1.12 Compound 1 administration group 25.82 ± 0.86 * 36.87 ± 3.22 * 43.83 ± 3.64 * Compound 2 administration group 10.62 ± 2.36 26.77 ± 1.67 * 30.58 ± 1.80 *

※ Significantly increased compared to the control group (p <0.05)

As shown in Table 1, Compound 1 and Compound 2 administration group according to the present invention significantly increased compared to the control group. Therefore, it can be seen that Compound 1 and Compound 2 according to the present invention stimulate the bone cells to promote bone formation.

Examples of preparations for the compositions of the present invention are illustrated below.

Formulation example  Pharmaceutical Formulation  Produce

1. Preparation of powder

Oleanane compound of Formula 1 2g

1g lactose

The above ingredients were mixed and filled in airtight cloth to prepare a powder.

2. Preparation of Tablets

100 mg of oleanane compound of Formula 1

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

3. Preparation of Capsule

100 mg of oleanane compound of Formula 1

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

4. Preparation of Injection Solution

10 μg / ml of oleanane compound of Formula 1

Dilute hydrochloric acid BP until pH 3.5

Injectable sodium chloride BP up to 1 ml

Dissolve the oleanane compound of Formula 1 above in an appropriate volume of sodium chloride BP for injection, adjust the pH of the resulting solution to pH 3.5 using dilute hydrochloric acid BP, and adjust the volume with sodium chloride BP for injection. Mix well. The solution was filled into a 5 ml Type I ampoule made of clear glass, encapsulated under an upper grid of air by dissolving the glass, and sterilized by autoclaving at 120 ° C. for at least 15 minutes to prepare an injection solution.

Oleanane compound according to the present invention promotes bone mineralization process of osteoblasts by increasing the cell proliferation of human fetal osteoblasts, increase the activity of basic phosphatase and protein expression of osteocalcin and BSP, bone defects By forming new bone in the part, it can be usefully used for bone regeneration, such as bone tissue, or fracture and bone damage, destroyed by periodontal disease.

Claims (5)

Bone tissue destroyed by periodontal disease using the oleanane compound represented by the formula (1) as an active ingredient, or bone tissue regeneration accelerator due to fracture or bone damage. <Formula 1>

(In Formula 1, R is hydrogen or an acetyl group.) According to claim 1, wherein the oleonan compound is bone tissue regeneration promoter, characterized in that hederagenin 3-O- [2-O-acetyl-alpha-L- arabinofyranoside]. [Claim 2] The bone tissue regeneration accelerator according to claim 1, wherein the oleanane-based compound is hederagenin 3-O-alpha-L-arabinofyranoside. The bone tissue regeneration accelerator according to any one of claims 1 to 3, wherein the oleanane-based compound is extracted, separated, and purified from the inner ear. delete

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