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WO2009028839A1 - Composition pharmaceutique comprenant de la licochalcone a destinée à prévenir ou à traiter des maladies osseuses - Google Patents

Composition pharmaceutique comprenant de la licochalcone a destinée à prévenir ou à traiter des maladies osseuses Download PDF

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WO2009028839A1
WO2009028839A1 PCT/KR2008/004912 KR2008004912W WO2009028839A1 WO 2009028839 A1 WO2009028839 A1 WO 2009028839A1 KR 2008004912 W KR2008004912 W KR 2008004912W WO 2009028839 A1 WO2009028839 A1 WO 2009028839A1
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licochalcone
cells
treated
bone
differentiation
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Seong Hwan Kim
Yong Ki Min
Soon Nam Kim
Myung Hee Kim
Su-Ui Lee
Cheol-Hee Kim
Seung Hyun Jung
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Korea Research Institute of Chemical Technology KRICT
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Korea Research Institute of Chemical Technology KRICT
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones

Definitions

  • the present invention relates to a pharmaceutical composition for preventing or treating bone diseases such as osteoporosis, fracture, periodontal disease and bone growth disorder, which comprises licochalcone A as an active ingredient.
  • Bone homeostasis is maintained by the bone remodeling process which controls the balance between osteoclastic bone resorption and osteoblastic bone formation, and increased osteoclastic activity or decreased osteoblastic activity can induce an imbalance in the bone remodeling, which results in metabolic bone diseases, e.g., osteoporosis. Therefore, there have been many studies to keep the bone remodeling process balanced by inhibiting the osteoclastic bone resorption, or enhancing the osteoblastic bone formation.
  • Osteoclasts originate from monocytes of hematopoietic progenitor cells, and mouse monocyte/macrophages RAW264.7 are differentiated into multinucleated osteoclasts upon RANKL (receptor activator of nuclear factor KB (RANK) ligand) stimulation (Boyle et al., Nature, 423(6937), 337-342, 2003).
  • RANKL receptor activator of nuclear factor KB (RANK) ligand
  • This differentiation is enhanced by the interaction of RANKL and RANK activating MAPK (mitogen-activated protein kinase), which results from the elevated expression of osteoclast differentiation-related molecules such as TRAP (tartrate-resistant acid phosphatase), MMP-9 (matrix metalloproteinase- 9) and c-Src tyrosine kinase by the action of NF- ⁇ B transcription factor, and then multinucleated cells can resorb mineralized bone.
  • MAPK mitogen-activated protein kinase
  • TRAF6 tumor necrosis factor receptor-associated factor 6
  • osteoclast differentiation-specific molecules including AP-I and NFAT can be regulated by ERK and NF- ⁇ B known as signaling molecules which play important roles in the survival and bone resorption of osteoclasts (Takayanagi et al., Dev Cell, 3(6), 889-901, 2002; and Asagiri et al., J Exp Med, 202(9), 1261-1269, 2005).
  • osteoporosis Rosen et al., J Clin Endocrinol Metab., 86(3), 957-64, 2001; and Garces C et al., Maturitas, 54(1), 47-54, 2006
  • active ingredients derived from natural substances for the treatment of osteoporosis without adverse effects Wang et al., Ann Pharmacother, 40(5), 836-49, 2006; and Putnam et al., Phytother Res., 21(2), 99-112, 2007.
  • Osteoblasts arise from mesenchymal stem cells, and the differentiation of osteoblasts contributes to mineralization such as calcium deposition, thereby preserving the bone strength and the homeostasis of calcium and hormone metabolisms. It has been demonstrated that the calcium deposition triggered by osteoblast differentiation is regulated by vitamin D or parathyroid hormone, and the bone formation induced by osteoblast diffentiation has been reported to be mediated by the synthesis of alkaline phosphatase (ALP) via cross-talk of variable signaling pathways involved in bone morphogenetic protein (BMP),
  • ALP alkaline phosphatase
  • BMP bone morphogenetic protein
  • Anti-resorptives inhibiting osteoclast activity and anabolic agents stimulating osteoblast activity have been employed for preventing or treating osteoporosis, fracture and periodontal disease (Zhang et al., Shanghai Kou Qiang Yi Xue, 7(2), 99-103, 1998; and Cahill et al., Biol Blood Marrow Transplant, 10(10), 709-717, 2004), and also for the treatment of bone growth disorders that occur after burn injury (Klein et al., Osteoporos Int., 16(6), 631- 635, 2005). These compounds are however, generally administered to patients over a long period, and thus, there is a need for developing an improved drug for such diseases.
  • licochalcone A has been known to have anti-inflammatory, whitening and anti-virus activities (Kolbe L et al., Arch Dermatol Res., 298(1), 23-30, 2006; Fu B et al., Agric Food Chem, 53(19), 7408-7414, 2005; and Friis- Moller et al., Planta Med, 68(5), 416-9, 2002), but its use for inhibiting bone resorption and enhancing bone formation has not yet been disclosed.
  • licochalcone A can be advantageously used for the treatment of bone diseases with minimal side effects.
  • bone diseases including osteoporosis, fracture, periodontal diseases involved in the bone mineral density, and bone growth disorders caused by lowered osteoclastic bone resorption and enhanced osteoblastic bone formation that occurs after burn injury.
  • a pharmaceutical composition for preventing or treating a bone disease selected from the group consisting of osteoporosis, fracture, periodontal disease and bone growth disorder comprising licochalcone A of formula (I) as an active ingredient:
  • FIG. Ia the cell viabilities of mouse monocytes RAW264.7 treated with various concentrations of licochalcone A during osteoclastic differentiation;
  • FIG. Ib the inhibitory effect of licochalcone A against the osteoclastic differentiation of mouse RAW264.7 cells
  • FIG. Ic the inhibitory effect of licochalcone A against the activity of TRAP, a marker for the osteoclastic differentiation
  • FIG. 2a the inhibitory effect of licochalcone A against the MAPK activity
  • FIG. 2b the inhibitory effect of licochalcone A against NF- ⁇ B
  • FIG. 3 the inhibitory effect of licochalcone A against the bone resorption of differentiated osteoclasts
  • FIG. 4a the inhibitory effect of licochalcone A against the osteoclastic differentiation of mouse hematopoietic progenitor cells
  • FIG. 4b the inhibitory effect of licochalcone A against .the activity of TRAP during the osteoclastic differentiation of mouse hematopoietic progenitor cells;
  • FIG. 4c the inhibitory effect of licochalcone A against the ERK and NF- KB p65 activities
  • FIG. 4d the inhibitory effect of licochalcone A against the bone resorption of osteoclasts differentiated from mouse hematopoietic progenitor cells
  • FIG. 5 the viability of osteoblasts treated with licochalcone A
  • FIGs. 6a and 6b the agonistic effects of licochalcone A on the activity of ALP, a marker for the osteoblastic differentiation;
  • FIG. 7 the calcium density deposited by licochalcone A
  • FIGs. 8a and 8b the agonistic effects of licochalcone A on the calcium density and mineralization of osteoblasts
  • FIG. 9 the effect of licochalcone A on the bone formation of Zebra fish.
  • the inventive composition for preventing and treating bone diseases comprises licochalcone A of formula (I) as an active ingredient.
  • a of the present invention is commercially available or it may be prepared by a conventional method described in Wang et al., J Chromatogr A., 1048(1), 51-57, 2004.
  • An effective dose of licochalcone A inhibits osteoclastic bone resorption and enhances osteoblastic bone formation when administered to a mammal.
  • mouse monocytes RAW264.7 are treated with licochalcone A at a concentration of less than 5 ⁇ M, 1) TRAP activity, osteoclastic differentiation, RANKL- mediated ERK activity and phosphorylation of IKB inducing nuclear localization of NF -KB are suppressed, 2) RANKL-mediated expressions of Fra-2 (a member of AP-I protein complex) and NFATcI transcription factor are inhibited, and 3) osteoclasts differentiated from the treated RAW264.7 monocytes exhibit a reduced degree of bone resorption as compared to not-treated osteoclasts, without any change in the cell viability.
  • licochalcone A enhances the differentiation and the activity of osteoblasts.
  • mouse osteoblasts i.e., MC3T3-E1 subclone 4 cells
  • licochalcone A when mouse osteoblasts, i.e., MC3T3-E1 subclone 4 cells, are treated with licochalcone A at a concentration of less than 20 ⁇ M, the ALP activity which is known to be involved in the osteoblastic differentiation, mineralization including calcium deposition and the related gene expressions thereof, and the activities of MAPK (mitogen-activated protein kinase) and transcription factors are enhanced, which results in increased bone formation, without showing any drug toxicity.
  • MAPK mitogen-activated protein kinase
  • inventive pharmaceutical composition comprising licochalcone A can be advantageously used for preventing or treating bone diseases including osteoporosis, fracture, periodontal diseases involved in the bone mineral density, and bone growth disorders that occur after burn injury.
  • licochalcone A may be employed in an amount ranging from 0.0001 to 50 % by weight, preferably from 0.01 to 1 % by weight based on the total weight of the inventive composition.
  • composition of the present invention may be formulated for oral or parenteral administration via various routes together with pharmaceutically acceptable additives such as carriers, diluents and excipients.
  • pharmaceutically acceptable additives such as carriers, diluents and excipients.
  • the pharmaceutically acceptable additives which can be used may include excipients such as starch, sugar and mannitol; fillers and bulking agents such as calcium phosphate and silicic acid derivatives; binding agents such as cellulose derivatives (e.g., carboxymethylcellulose and hydroxypropylcellulose), gelatin, alginic acid and polyvinylpirrolidone; lubricants such as talc, calcium or magnesium stearate, hydrogenated castor oil and solid-phase polyethylene glycol; disintergents such as povidone, croscarmellose sodium and crospovidone; and surfactants such as polysorbate, cetyl alcohol and glycerol monostearate.
  • a proper daily dosage of licochalcone A in the present invention for mammals including human ranges from about 1 to 150 mg/kg, preferably from 1 to 15 mg/kg, which may be administered once daily or by dividing into several times a day.
  • the amount of the active ingredient actually administered should be determined in light of various relevant factors including the condition to be treated, the chosen route of administration, the age and weight of the individual patient, and the severity of the patient's symptoms; and therefore, the dosage suggested above should not be construed to limit the scope of the invention in any way.
  • the following Examples are intended to further illustrate the present invention without limiting its scope.
  • Example 1 Inhibitory effect of licochalcone A against osteoclastic differentiation
  • Step 1) Monocyte culture and cytotoxicity test Mouse monocytes RAW264.7 cells (ATTC TIB-71) were cultured in DMEM supplemented with 10% FBS, while changing the medium every 2 days.
  • the reagents used for the culture were purchased from Hyclone Co.. When the cells were 70% confluent, the cultured cells were harvested, seeded at a concentration of 1 x 10 3 cells/well on a 96-well plate, and cultured in ⁇ -MEM containing 100 ng/ml of RANKL (R&D systems) and 10% FBS for 24 hrs. Thereafter, licochalcone A (Merck, Cat No.
  • the viability of the cells was not affected when the concentration of licochalcone A was less than 5 ⁇ M.
  • Step 2) TRAP staining and activity analysis
  • TRAP staining was performed for the cells obtained in Step 1) using a leukocyte acid phosphatase kit (Sigma).
  • the stained cells were fixed in 10% formalin for 10 min, dehydrated using 95% ethanol, and dried at room temperature. Then, 100 fd of citrate buffer (50 mM, pH 4.6) containing 10 mM sodium tartrate and 5 mM p- nitrophenylphosphate was added to each well, and the plate was incubated for 1 hr. Thereafter, the reaction mixtures of each well was placed in a new 96-well plate, 90 ⁇ JL of 0.1 N NaOH was added to each well to terminate the enzyme reaction, and the absorbance of each well was measured at 405 nm. The TRAP activity was determined by calculating the percentage based on that of the control, and the value thus determined was tested using t-test (a, p ⁇ 0.01 ; b, p ⁇
  • FIGs. Ib and Ic The results are shown in FIGs. Ib and Ic.
  • licochalcone A inhibits the multinucleated osteoclastic differentiation of RAW264.7 monocytes
  • FIG. Ic licochalcone A reduces the RANKL-mediated TRAP activity in a dose- dependent manner.
  • licochalcone A exhibits significant inhibitory activity against osteoclastic differentiation without showing cytotoxicity at a concentration of less than 5 ⁇ M.
  • Example 2 Inhibitory effect of licochalcone A against osteoclastic differentiation-related gene expressions
  • RAW264.7 cells were treated with RANKL according to the same method as described in Step 1) of Example 1, and treated with licochalcone A at a concentration of 1 or 5 ⁇ M for 2 hrs.
  • RNA isolation and cDNA synthesis from each well were conducted using TRIzol reagent (Life Technologies) and Omniscript RT kit (Qiagen).
  • the synthesized cDNAs were each diluted 50 times, and subjected to PCR with Brilliant SYBR Green master mix (Stratagene) and 20 pmole of primer using Stratagene Mx3000P system.
  • the employed primers for TRAP gene had sequences of SEQ ID NOs: 1 and 2; MMP-9 gene, SEQ ID NOs: 3 and 4; and c-Src gene, SEQ ID NOs: 5 and 6. Further, the PCR condition consisted of an initial denaturation reaction for 10 min at 94 ° C ; 40 cycles of amplification reaction for 40 sec at 94 0 C (denaturation), 40 sec at 60 °C (annealing) and 1 min at 72 ° C (extension); and an final amplification cycle for 1 min at 95 °C (denaturation), 30 sec at 55 ° C (annealing) and 30 sec at 95 ° C (extension) for generating the melting curve of each PCR product.
  • the calculated amounts of the amplified products were normalized with those of GAPDH (glyceraldehyde 3 -phosphate dehydrogenase) (using primers having nucleotide sequences of SEQ ID NOs: 7 and 8) by 2 'AACT method, which were each tested using t-test (a, p ⁇ 0.001 (RANKL treated or not-treated); b, p ⁇ 0.001 (1 or 5 ⁇ M licochalcone A treated in the presence of RANKL). The results are shown in Table 1.
  • Example 3 Inhibitory effect of licochalcone A against MAPK and NF- ⁇ B activities
  • RAW264.7 cells were precultured according to the same method as described in Step 1) of Example 1, and treated with 1 or 5 ⁇ M licochalcone A for 2 hrs and 100 ng/mi of RANKL for 30 min.
  • the cultured cells were each lysed in RIPA buffer to obtain a cell lysate, and each protein content of the cell lysates were determined by BCA kit (Bio-Rad). 10 ⁇ g of the total protein from each cell lysate was resolved by 12% SDS-PAGE, and transferred to a nitrocellulose membrane.
  • the transferred blots were blocked with 10% skim milk solution, and incubated with an antibody for ERK (externally regulated kinases), p-ERK (phosphorylated externally regulated kinases), JNK (jun N-terminal kinase), pJNK (phosphorylated jun N-terminal kinase), p38 or p-p38.
  • ERK externally regulated kinases
  • p-ERK phosphorylated externally regulated kinases
  • JNK jun N-terminal kinase
  • pJNK phosphorylated jun N-terminal kinase
  • p38 or p-p38 p-p38.
  • cytoplasmic an nuclear fractions from the total proteins of each cell lysate were each isolated with NucBuster protein Extraction kit (Novagen), and incubated with an antibody for NF- ⁇ B p65 or p-I ⁇ B. The results are shown in FIG. 2b.
  • Example 4 Inhibitory effect of licochalcone A against AP-I and NFATcI transcription factors
  • Example 2 The procedure of Example 2 was repeated except for using primers for c-Fos gene had sequences of SEQ ID NOs: 9 and 10; Fra-1 gene, SEQ ID NOs: 11 and 12; Fra-2 gene, SEQ ID NOs: 13 and 14; and NFATcI gene, SEQ ID NOs: 15 and 16.
  • the results were each tested using t-test (a, p ⁇ 0.01; b, p ⁇ 0.001 (RANKL treated or not-treated); c, p ⁇ 0.01 (1 or 5 ⁇ M licochalcone A treated in the presence of RANKL), which are shown in Table 2.
  • Example 5 Inhibitory effect of licochalcone A against bone resorption of osteoclast
  • RAW264.7 monocytes were differentiated with RANKL, and treated with licochalcone A at a concentration of 0, 2 and 5 ⁇ M at the beginning of the osteoclastic formation. After culturing for 15 days, the cells were treated with
  • the licochalcone A-treated cells exhibited a dramatically reduced bone resorption as compared with the not-treated control cells.
  • Example 6 Inhibitory effect of licochalcone A against osteoclastic differentiation and bone resorption of mouse hematopoietic progenitor cells
  • Step 1) Isolation and osteoclastic differentiation of mouse hematopoietic progenitor cells
  • a 6- or 7-week-old DDY male mouse was sacrificed with nitrogen and sterilized with 70% ethanol, and a thigh bone was harvested from a thigh (a hind leg) of the mouse and dipped in PBS containing antibiotics. After washing the thigh bone with PBS to remove the remained tissues, the both epiphyses were cut away, followed by pushing out the bone marrows using a syringe filled with a culture medium. The bone marrows were collected in a Petri dish and pipetted to obtain single cells. After treating with 10 ng/ml of M-CSF for 1 day, the cells were harvested and used as hematopoietic progenitor cells.
  • the hematopoietic progenitor cells were seeded at a concentration of 3 x 10 5 cells/well on a 96- well plate, and M-CSF 30 ng/ml and RANKL 100 ng/ml were added thereto to differentiate into osteoclasts.
  • Step 2) TRAP staining and activity analysis
  • the cells obtained in Step 1) were treated with licochalcone A at a concentration of 1, 2, 5 or 10 ⁇ M, and subjected to TRAP staining and activity analysis described in Step 2) of Example 1. The results are shown in FIGs. 4a and 4b.
  • licochalcone A significantly inhibits the osteoclastic differentiation of hematopoietic progenitor cells and reduces the
  • RANKL-mediated TRAP activity in a dose-dependent manner.
  • Step 1) After incubating the cells obtained in Step 1) with M-CSF for 3 days, the medium was changed, and the cells were treated with licochalcone A for 2 hrs, and then, with RANKL 100 ng/ml for 30 min. The cells were lysed with RIPA buffer and the total proteins were harvested. After each determining protein contents by BCA kit (Bio-Rad), 10 ⁇ g of the total protein from each cell lysate was resolved by 12% SDS-PAGE, and transferred to nitrocellulose membrane (BioRad). The transferred blots were blocked with 10% skim milk solution, and incubated with an antibody for ERK, p-ERK and NF- ⁇ B p65. The results are shown in FIG. 4c. As shown in FIG. 4c, the treatment of licochalcone A remarkably reduces the p-ERK and NF- ⁇ B p65 activities, which are known to be increased by RANKL during the osteoclastic differentiation.
  • licochalcone A can potently inhibits the osteoclastic differentiation of mouse hematopoietic progenitor cells by effectively suppressing ERK signal pathway known to be activated during the osteoclastic differentiation.
  • Step 4) Inhibitory effect of licochalcone A against bone resorption
  • the seeded hematopoietic progenitor cells on a 96-well plate according to the procedure of Step 1) was incubated with M-CSF for 3 days, the medium was changed, and the cells were treated with 100 ng/ml of RANKL, followed by treating with licochalcone A at a concentration of 0, 2.5, 5 or 10 ⁇ M at the beginning of the osteoclast formation. After culturing for 15 days, the cells were treated with 100 ⁇ Z of 6% NaOCl for 5 min and washed three times with distilled water, followed by observing the bone resorption level using a microscopy (using BD BioCoat Osteologic MultiTest slide). The results are shown in FIG. 4d.
  • the licochalcone A-treated cells exhibited a dramatically reduced bone resorption as compared with the not-treated control cells.
  • Example 7 Analysis for osteoblast differentiation and alkaline phosphatase (ALP) activity
  • Step 1) Osteoblast culture and cytotoxicity test Cytotoxicity of licochalcone A was analysed using MC3T3-E1 subclone 4(ATCC CRL-2593) as osteoblasts, and the employed medium and the reagents were purchased from Hyclone Co..
  • the purchased osteoblasts were cultured in a differentiation inducing medium ( ⁇ MEM, 10% FBS, 50 ⁇ g/m# ascorbic acid and 10 mM ⁇ -glycerophosphate) while changing the medium every 3 days.
  • ⁇ MEM differentiation inducing medium
  • the cells were treated with licochalcone A at a concentration of 0, 1, 2, 5, 10 or 20 ⁇ M, and incubated for 3 days.
  • the absorbance of each well was measured using CCK-8 kit (Dojindo, JP) on day 1 and 3 after licochalcone A treatment began, and conversed into cell number to determine the cell viability.
  • the cells were seeded at a concentration of 1 x 10 3 cells/well on a 96-well plate and the absorbance thereof were measured 12 hrs before the above measurement. The results are shown in FIG. 5. As shown in FIG.
  • the cytotoxicity of licochalcone A was not observed when the concentration of licochalcone A was less than 20 ⁇ M, while the cells treated with licochalcone A at a concentration of less than 10 ⁇ M exhibited a little lower proliferation as compared with not-treated control cells.
  • the cultured cells were harvested using trypsin and seeded at a concentration of 1 x 10 4 cells/well on a 48-well plate.
  • the cells were cultured in a differentiation-inducing medium ( ⁇ MEM, 10% FBS, 50 ⁇ glml of ascorbic acid and 10 mM ⁇ -glycerophosphate) containing 2.5, 5 or 10 ⁇ M licochalcone A to induce differentiation, while changing the medium every 3 days.
  • ⁇ MEM differentiation-inducing medium
  • the licochalcone A-treated cells according to the same method described in Step 2) were lysed with a cell lysis buffer (10 mM Tris-HCl, pH 7.5,
  • licochalcone A significantly enhances the ALP activity, a marker for early osteoblastic differentiation as compared with not- treated control cells, especially when administered in an amount of 2.5 ⁇ M and 5 ⁇ M.
  • the licochalcone A-treated cells according to the same method described in Step 2) were washed three times with PBS, were fixed in 10% formalin for 30 sec and washed again with water. Thereafter, the cells were stained an alkaline solution for 1 hr with shading the light and washed again with water.
  • the employed alkaline solution was prepared by dissolving Fast blue RR 1 capsule (Sigma, cat. no. 85Ll- 1 kit) in 48 ml of water, and adding 2
  • Example 8 Effects of licochalcone A for the calcium deposition and mineralization of osteoblasts
  • the osteoblasts treated with licochalcone A at a concentration of 2.5 or 5 ⁇ M exhibited remarkably increased calcium depositions from 15 days after the treatment, as compared with not-treated control cells.
  • the osteoblasts treated with licochalcone A at a concentration of 2.5 or 5 ⁇ M exhibited remarkably increased calcium depositions as compared with not-treated control cells.
  • the cells were subjected to von Kossa staining as follows, in order to analysis the mineralization of osteoblasts. Particularly, the cells was washed with PBS, and fixed in 2.5% glutaraldehyde/PBS for 30 sec. The fixed cells was washed twice with distilled water and treated with 5% silver nitrate, followed by irradiating with ultraviolet ray and observing the mineralized part (black). The results are shown in FIG. 8b.
  • licochalcone A-treated osteoblasts exhibit an increased mineralization as compared with not-treated control cells, which suggests that licochalcone A promotes the osteoblastic differentiation, resulting in increased calcium deposition and mineralization.
  • Example 9 Effects of licochalcone A for the gene expressions related to the osteoblastic differentiation and mineralization
  • Biomarkers for the osteoblastic differentiation can be divided according to the appearance stage thereof during the differentiation. It has been known that at the early stage of the differentiation, ALP is expressed, and during the osteoblastic mineralization, the expressions of osteopontin (OP), osteocalcin (OC) and type I collagen (Col) are increased.
  • OP osteopontin
  • OC osteocalcin
  • Col type I collagen
  • the licochalcone A-treated cells according to the same method described in Step 2) of Example 7 were incubated for 18 days, and subjected to real time RT-PCR according to the same method as described in Example 2, except for using primers for ALP gene had sequences of SEQ ID NOs: 17 and 18; OP gene, SEQ ID NOs: 19 and 20; OC gene, SEQ ID NOs: 21 and 22; and Col gene, SEQ ID NOs: 23 and 24.
  • the results are shown in Table 3.
  • the osteoblasts treated with licochalcone A at a concentration of 2.5 and 5 ⁇ M exhibit significantly increased expressions of OP and OC genes, which are known to be involved in the mineralization, which suggests that licochalcone A can elevate the osteoblastic differentiation- promoting gene expressions.
  • Example 10 Effect of licochalcone A for the bone formation of osteoblasts
  • Embryos of a zebra fish which is a zebra-patterned aquarium fish, were placed into a 24-well plate (7-10 embryos per well) 5 days after fertilization, 1 ml of buffered medium (sea salt, 0.06 mg/lL) containing licochalcone A at a concentration of 2.5, 5 or 10 ⁇ M was added to each well, and the plate was maintained until 7 days post fertilization, while changing the medium every day.
  • the embryos were fixed in 4% paraformaldehyde and washed three times with PBS containing 0.1% tween 20 (PBST) at 10-min intervals, followed by treating the embryos with a bleaching solution (PBST containing 1% KOH and 3% H 2 O 2 ) at room temperature until pigment cells were removed (for about 30 min to 1 hr).
  • PBST PBS containing 0.1% tween 20
  • PBST containing 1% KOH and 3% H 2 O 2
  • the embryos was washed 3 times with PBST at 10-min intervals, treated with 1 ml of alizarin red S staining buffer (pH 4.2) to stain the formed bone.
  • the zebra fish embryos 8 days after fertilization exhibited significantly enhanced bone formations in Meckel's cartilage (MC), which can be developed into temporomandibular joint; palatoquadrate (pq); opercle (op) and vertebrae (vb) when treated with licochalcone A, especially in an amount of 5 ⁇ M, which corresponds to the results from the above cell examination.
  • MC Meckel's cartilage
  • pq palatoquadrate
  • opercle op
  • vb vertebrae
  • Formulation Example 1 Preparation of a pharmaceutical formulation comprising licochalcone A
  • a tablet comprising licochalcone A was prepared by mixing 120 mg of licochalcone A with 33 mg of microcrystalline cellulose, 33 mg of calcium hydrogen phosphate, 12 mg of sodium starch glycolate and 2 mg of magnesium stearate, and the resulting mixture was formulated into a tablet.

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Abstract

La licochalcone A de la présente invention inhibe puissamment la différenciation ostéoclastique et la résorption osseuse tout en améliorant la différenciation ostéoblastique, la minéralisation et la formation osseuse. Par conséquent, la composition pharmaceutique de l'invention comprenant de la licochalcone A peut être avantageusement utilisée afin de prévenir ou de traiter des maladies osseuses telles que l'ostéoporose, les fractures, les parodontopathies et les troubles de la croissance osseuse.
PCT/KR2008/004912 2007-08-31 2008-08-22 Composition pharmaceutique comprenant de la licochalcone a destinée à prévenir ou à traiter des maladies osseuses Ceased WO2009028839A1 (fr)

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WO2018079715A1 (fr) * 2016-10-27 2018-05-03 サントリーホールディングス株式会社 Composition pour inhibition d'activité ｆｏｘｏ１
CN112898146A (zh) * 2021-01-22 2021-06-04 中国科学院华南植物园 一种制备甘草查尔酮a的方法

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JPWO2018079715A1 (ja) * 2016-10-27 2019-09-19 サントリーホールディングス株式会社 Ｆｏｘｏ１活性阻害用組成物
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