KR101132017B1 - A Composition Containing Hispidulin for Diseases Associated with Bones - Google Patents

Abstract

  The present invention exhibits the effect of inhibiting the differentiation of osteoclasts using hispidulin as an active ingredient, and relates to a composition for preventing and treating bone-related diseases such as rheumatoid and osteoporosis and a food comprising the same. Is a safe and less toxic to human body by extracting hispidulin, a kind of flavonoids histopilin screened from various plants, as an active ingredient, and hispidulin is ERK , JNK, p38 and the transcription factor c-Fos, NFATc1 through the signaling pathways to inhibit osteoclast differentiation, and the composition and foods containing the same can be expected to prevent and treat bone-related diseases such as rheumatoid arthritis and osteoporosis It is about.

Description

  A composition containing hispidulin for diseases associated with bones} as an active ingredient with hispiduline (Ｈｉｓｐｉｄｕｌｉｎ)

The present invention uses hispiduline as an active ingredient, through the inhibition of osteoclast differentiation through the activation of MAPK signaling (JNK, p38) and the signaling pathways of the transcription factors c-Fos and NFATc1, such as rheumatoid arthritis and osteoporosis. The present invention relates to a composition for preventing and treating bone-related diseases and a food comprising the same.

Hispidullin (5,7-Dihydroxy-2- (4-hydroxyphenyl) 6-methoxy-4H-1-benzopyran-4-one) is a compound having the following formula, and is an antioxidant (Hispidulin: Antioxidant properties and effect on mitochondrial energy metabolism. Free Radical Research, December 2005, pages 1305 -.. 1315) is known to have effects such as inhibition of platelet aggregation by increasing the nature and cAMP levels, but (Hispidulin, a natural flavone, inhibits human platelet aggregation by increasing cAMP levels Eur J . Pharmacol, 1988 147 (1) :. 1-6), Heather Lynn pidul c-Fos and NFATc1 me, so that the effect on related rheumatoid arthritis and bone-related diseases such as osteoporosis or described in any document implies the bar none.

Accordingly, the inventors of the present invention have repeatedly studied to develop a substance that inhibits the activation of the MAPK pathway (JNK, p38) and the protein expression of the transcription factors c-Fos and NFATc1, resulting in hispidu contained in Artemisia princeps pampan. Lin was found to inhibit osteoclast differentiation by activating the MAPK pathway (JNK, p38) and inhibiting signal transduction of the transcription factors c-Fos and NFATc1.

   The present invention is to provide a composition for the prevention and treatment of bone diseases related to the differentiation inhibitory effect of osteoclasts using hispiduline as an active ingredient.

The present invention also includes a health supplement for the prevention of bone-related diseases such as rheumatoid arthritis and osteoporosis associated with the differentiation inhibitory effect of osteoclasts comprising hispidulin as an active ingredient.

  The composition for preventing and treating bone-related diseases according to the present invention is characterized by osteoclasts having hispidullin as an active ingredient.

  In the composition for the prevention and treatment of bone-related diseases according to the present invention, the hispiduline is characterized in that extracted from Artemisia princeps Pampan.

  The composition for preventing and treating bone-related diseases according to the present invention is characterized in that the dose is 10 ~ 500mg / day.

  The dietary supplement comprising the composition according to the present invention is characterized in that any one of tea, sweets, beverages, capsules.

  In the present invention, by using hispidulin extracted from the wormwood as an active ingredient, it is safe and less toxic to the human body, and can control the formation of osteoclasts through the inhibition of c-Fos and NFATc1.

   The composition according to the present invention may further comprise a pharmaceutically acceptable carrier, in addition to hispidulin as an active ingredient, such carriers include excipients, binders, lubricants, disintegrants, coating emulsifiers, suspensions, solvents, And at least one carrier selected from the group consisting of stabilizers, absorption aids, water for injection, and isotonic agents.

   The pharmaceutically acceptable carriers described above may be employed by the skilled person in the selection, and their type and amount will vary depending on the formulation of the composition, and it is obvious that the present invention is not limited thereto.

   The compositions according to the invention can be formulated in a variety of oral or injectables, preferably in oral formulations. Examples of such oral preparations include granules, tablets, capsules, liquids, and the like.

   The dosage of the composition according to the present invention will vary depending on the age, sex, etc. of the patient, but preferably 10 to 500 mg / day, more preferably 500 mg / day, based on the weight of hispidullin, Depending on the dosage over. Depending on the particular mode of administration chosen and the requirements of the patient, it may be selected based primarily on the volume, viscosity, weight, etc. of the fluid, and it may be desirable to examine the patient to optimize dosage and frequency.

   The composition according to the present invention leads to the differentiation of osteoclasts and breaks the balance between osteoclasts and osteoblasts. As osteoclasts increase, bone density decreases, which can be used for the prevention and treatment of osteoporosis and bone-related diseases such as rheumatism, as well as for the treatment of warm-blooded animals such as mice, dogs, rabbits, cats, and chickens.

   In general, hispiduline can be extracted by known methods. Specifically, hispiduline is a kind of flavonoids that is separated from Artemisia princeps Pampan, and hispidulin (hispi Extraction using the process of making and extracting doulin).

In addition, there is provided a health supplement comprising the composition of the present invention, non-limiting examples of such health supplements include health supplements such as tea, confectionery, beverages, capsules.

As described above, according to the present invention, the osteoclasts are inhibited through the inhibition of signaling pathways of c-Fos or NFATc1, which are JNK, p38 and transcription factors, and are differentiation master genes of osteoclasts. Since it can be, the prevention and treatment of bone-related diseases such as rheumatoid arthritis, osteoporosis may be possible according to the present invention.

1A is a chemical structural formula of hispiduline.
1B is a degree of inhibition of the differentiation of hispidullin osteoclasts by a concentration dependent method.
Figure 1 shows hispidulin is an inhibitory measure on the number of TAP-positive multinucleated cells (MNC).
Figure 1 D. was examined the effect of hispidullin on cell growth, cell growth was analyzed by MTT assay, hispidullin was treated at various concentrations for 24 hours. Hispidullin is a diagram showing no effect on cell growth rate in low 264.7 cells.
Figure 2 is a diagram inhibiting the induction of hispidulin in the bone marrow macrophages (RANKL)-osteoclasts.
Figure 2A shows hispidulin inhibits the formation of TAP-positive multinucleated cells (MNC) in a concentration dependent manner.
Figure 2b is a diagram showing the reduction for Trap-positive multinucleated cells (MNC).
3 is a diagram showing the inhibition of the expression of specific genes in osteoclasts in a concentration-dependent manner of hispidullin.
4 is a diagram showing that hispidullin inhibited the activities of JNK and p38 but did not affect the activity of ERK.
Figure 4 B. shows that phosphorylation of icapabi (IB) and p65 is significantly inhibited by hispiduline.
FIG. 5 shows that the increased induction of c-fos mRNA levels is significantly reduced by hispiduline.
6A. RANKL-induced increase in NFATc1 mRNA is markedly inhibited by 10 μM hispiduline.
FIG. 6B is a diagram showing that 10 μM hispidullin significantly inhibits RANKL-induced NFATc1 transcriptional activity.

   Hereinafter, the present invention will be described in more detail with reference to formulation examples and examples, but the scope of the present invention is not limited only to the following examples.

Preparation Example 1 Preparation of Pharmaceutical Composition

Tablets, capsules, powders and injections were prepared according to the following compositions, respectively.

1) tablets

Hispidullin 500.0 mg

Lactose 500.0 mg

Talc 5.0 mg

Magnesium Stearate 1.0mg

2) Capsule

   Capsules were prepared by sieving hispidulin and mixing with excipients, then filling into gelatin capsules according to the following composition.

Hispidullin 500.0 mg

Starch 1500 10.0 mg

Magnesium stearate 100.0 mg

3) powder

   The following components were mixed in a conventional powder preparation method, put into a bag, and sealed, and powder was prepared.

Hispidullin 500.0 mg

Lactose 100.0 mg

Talc 5.0 mg

4) Injection

   The following components were filled into 2.0 ml ampoules by injection method and sterilized to prepare injections.

Hispidullin 50.0 mg

Antioxidant 1.0 mg

Tween 80 1.0mg

Distilled water for injection ≤2.0 mg

Preparation Example 2 Preparation of Health Supplement Food

    The following ingredients were mixed with water to prepare a beverage in a conventional manner, but after adjusting to a total of 100 ml, sterilization and cooling for 15 seconds at 95 ℃ to make a drink.

Hispidulin 500.0 mg

Citric Acid 10.0 mg

Gum arabic 5.0 mg

5.0 mg of sugar

Example 1 Cell Culture and Osteoclast Induction

Mouse murine monocyte / macrophage RAW 264.7 cell line was obtained from the American American Type Culture Collection (Manasas, VA, USA). Cells were incubated with 10% FBS, 2 mM glutamine, 100 U / ml penicillin G and 100 μg / ml streptomycin sulfate (growth medium) contained in DMEM at 37 ° C. with 5% CO ₂ moist air.

For differentiation of osteoclasts, raw 264.7 cells contained 10% FBS, 2 mM L-glutamate, 100 U / ml penicillin and 100 mg / ml streptomycin a-MEM. It was then seeded in 3 × 10 ³ cells / well in 96-well culture dishes and incubated with 50 ng / ml soluble RANKL for 6 days.

Example 2 Isolation of Bone Marrow-derived Macrophages

  Six-week-old Institute of Cancer Research (ICR) mice were purchased from Damool Science (Daejeon, Korea). Cells were obtained from tibia and femoral bone marrow and in a-MEM with 10% fetal bovine serum (FBS), including 30 ng / ml macrophage M-csf (M-CSF) at 37 ° C. in a CO 2 incubator overnight. Incubated.

Non-adherent cells were cultured in a 100 mm petri dish and in 30 ng of M-CSF for 3 days. Adherent cells are washed and used as bone marrow-derived macrophages (BMMs) and cultured until the cells differentiated into osteoclasts until the proximity of M-CSF and RANKL, the cells are cytochemically osteoclasts, tartrate resistant acid Staining was performed for phosphatase (TRAP). Trap positive multinuclear cells (MNCs) with more than three nuclei were counted.

Example 3 Cell Proliferation Assay

Rat macrophage lines were added to 96-well plates by dispensing 5 × 10 ³ cells / well, and then treated with hispidulin 0.1, 1, and 10 μM for 24 hours, followed by washing with physiological saline. After treatment with MTT 100mg / ml to the medium, (4,5-dimethylthiazolyl-2) -2,5-diphenyltetrazolium bromide was incubated for 2 hours at 37 ℃. After removing the medium, washed with physiological saline, and incubated for 10 minutes by adding 200 μL of DMSO (DMSO), it was measured at the absorbance of 540nm.

Example 4 Tartrate-resistant acid phosphatase staining (TRAP staining)

Tartrate-resistant acid phosphate (TRAP) staining was performed according to the protocol of Sigma's TRAP staining kit, and trap positive multinucleated cells containing three or more nuclei were counted on an optical microscope.

Example 5 Effects on the Regulation of Osteolytic Genes in RANKL-Stimulated RAW 264.7 Cells

  Differentiation of osteoclasts is associated with increased regulation of specific genes such as TRAP, RANK, and cathepsin K in response to RANKL. Total RNA was prepared and analyzed by Alti-Phisal (RT-PCR) to examine the inhibitory potency of hispidulline for the expression of osteoclast specific genes. Ranke (50 ng / ml) significantly induced trap, rank and cathepsin K in RAW 264.7 cells. Hispidullin was found to inhibit the expression of osteoclast specific genes in a concentration-dependent manner (FIG. 3). Hispidullin did not affect the expression of housekeeping gene GAPDH. This data shows that hispidullin affects some gene regulation induced during the differentiation of osteoclasts.

   Total RNA was isolated in accordance with the instructions of Trizol (invitrogen) manufacturer. 2 μg of total RNA was reverse transcribed using the Super Script ™ First-Strand Synthetic System (Invitrogen) and the reaction product was amplified with specific primers. A series of primers for each gene was designed from Bioneer (Daejeon, Korea), and primer sequences are listed in Table 1 below.

Table 1 Primer Sequence

PCR products were separated on 1.2-2.0% agarose gel and stained with ethidium bromide. Concentration measurements were performed using Phosphoimager and Quantity One software (Version 4.3.1) (Bio-Rad, Hercules, CA, USA). As shown in the results, when the compound of the above formula was added in culture, it was found that the expression of c-Fos and NFATc1, which are increased genes when RANKL was added, was significantly suppressed. In other words, the compounds of the above formula was found to inhibit the osteoclast differentiation-related master genes c-Fos and NFATc1 excellently.

Example 6 Western blot

  Add lysis buffer (lysis buffer, 20 mM Tris-HC1, pH 7.5, 137 mM NaCl, 10% glycerol, 1% Trixon X-100, 1 mM Na3V04, and 1 mM PMSF) to the cells and collect them for 15 minutes at 16,000 × g. Centrifuged at 4 ° C. The supernatant was used as whole cell extract, and the whole cell extract or nuclear extract of hispidulin was electrophoresed with 10% SDS-PAGE, and then the protein separated by polyvinylidene difluoride membrane (PVDF) membrane was transferred. . PVDF membranes were incubated for 1 hour at room temperature with 5% skim milk powder dissolved in TTBS (tris buffered saline solution containing 0.5% Tween-20), followed by rabbit antibody (1000-fold diluted with TTBS 1: 5% skim milk powder). Incubation was for 16 hours at ℃. After washing the membrane with TTBS, dilution of horseradish-conjugated antibody (Horseradish peroxidase-conjugated anti-rabbit or mouse antibodies) with TTBS 1: 5,000 ~ 1: 10,000) times with 5% skim milk powder as a secondary antibody. After incubation for 1 hour at room temperature, the amount of protein bound to the antibody is quantified by an enhanced chemi luminescence (ECL) reaction.

   It has been shown that MAPKs (ERK, JNK and p38) are activated in response to RANKL in osteoclast precursor cells (Lee et al., 2002), and the activity of MAPKs involved in the RANKL-signaling pathway, cells of hispidullin. RAW 264.7 to determine my mechanism was studied in RAW 264.7 cells. 200ng of RANKL induced activation of all 3 MAPKs in RAW264.7 cells after treatment. Hispidulin inhibited the phosphorylation and p38 phosphorylation of JNK, but did not inhibit phosphorylation of ERK (FIG. 4A).

Activation of NF-κB transcription factors is a fundamental step for the differentiation of osteoclasts (Franzoso et al., 1997; Kotake S, 1999; Vaira et al., 2008). Phosphorylation of p65, a subunit of NF-κB and phosphorylation of IκB, a protein that inhibits NF-κB, is known to activate NF-κB (Viatour et al., 2005). Hispidullin significantly inhibited the phosphorylation of IkB and p65 induced by RANKL (Fig. 4b).

Example 7 Transfection and Luciferse Reporter Activity Evaluation

Seed RAW264.7 cells 5 x 10 ⁴ cells / well in a 24-well dish and use serum-containing medium to grow 90-95%. Luciferase reporter plasmid constructs per well were co-transfected into cells with Lipofectamine 2000 (Invitrogen) with 1.0 μg of NFATc1 binding and 0.5 μg of pCMV-β-galactosidase control vector according to the manufacturer's instructions.

   After 12 hours of transfection, the medium was replaced and the cells were treated with RANKL and hispidullin. Cells were washed with PBS and lysed in 1 x Reporter Lysis Buffer (Promega). The activity of firefly luciferase in cellular extracts was measured using a luciferase reporting assay system according to the manufacturer's instructions (Promega).

Comparative luciferase activity was obtained by normalizing firefly luminescent enzyme activity against β-galactosidase activity.

Example 8 Statistical Analysis

All experimental data shown are represented as meaning ± SEM and repeated at least 3 hours. Statistical analysis was performed and one-way analysis of variance (ANOVA) was followed by students' t-tests, with p-values of 0.05 or less considered important.

Claims (5)

Pharmaceutical composition for the prevention and treatment of rheumatoid arthritis and osteoporosis using hispidullin as an active ingredient.
The pharmaceutical composition for preventing and treating rheumatoid arthritis and osteoporosis according to claim 1, wherein the hispidullin is extracted from Artemisia princeps Pampan.
The pharmaceutical composition for preventing and treating rheumatoid arthritis and osteoporosis according to claim 1 or 2, wherein the composition has a dosage of 10-500 mg / day.
The health supplement for preventing rheumatoid arthritis and osteoporosis according to claim 1 or 2, comprising the composition.
    The health supplement for preventing rheumatoid arthritis and osteoporosis according to claim 4, wherein the health supplement is any one of tea, sweets, beverages, and capsules.

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