JP2006070003A - Cell proliferation promoting composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a wound treating composition, by finding out a medicine having cell proliferation action and applying the medicine to treatment of the wound, and to provide a medicinal composition capable of efficaciously treating the wound, by scarcely causing an adverse effect. <P>SOLUTION: The cell proliferation promoting medicine contains Centaurein or Jacein as an active ingredient. The wound treating composition contains the promoting medicine. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a fibroblast proliferation promoter and a wound treatment composition containing Centaurein or Jacein.

Wound healing is the bleeding / coagulation phase in which red blood cells and platelets appear, the inflammatory phase in which neutrophils and macrophages migrate, the proliferative phase mainly consisting of granulation and neovascularization by vascular endothelial cells and fibroblasts, and epidermal cells It progresses through a process called the remodeling phase in which migration and connective tissue proliferation occur. In the proliferative phase, angiogenesis by the proliferation of vascular endothelial cells precedes the migration of fibroblasts to the wound surface, which is essential for wound healing and is the most important reaction. In addition, fibroblasts migrate and proliferate from tissues around the wound site to promote extracellular matrix synthesis, which is the action of various growth factors such as FGF, PDGF, and TGF-β secreted from macrophages. it is conceivable that. As mentioned above, in the wound healing process, in addition to angiogenesis, fibroblast proliferation is important for granulation formation, so compounds that can enhance these processes may promote wound healing. There is.
On the other hand, Bidens pilosa, a plant belonging to the genus Sendangusa, has been reported to promote wound healing in vivo (see Patent Document 1).

JP2004-91434

  An object of the present invention is to find a drug having a cell proliferating action and to provide a wound treatment composition in which such a drug is applied to wound treatment. Another object of the present invention is to provide a pharmaceutical composition that can effectively treat a wound with few side effects.

上記細胞増殖促進剤において、繊維芽細胞が、ヒト皮膚繊維芽細胞であることが好ましい。
また、本発明は、上記繊維芽細胞増殖促進剤を含む、創傷治療組成物、に関する。 In the wound healing process, the present inventors have been searching for a drug having a cell proliferating action that can be used as a wound treatment composition, since the proliferation of fibroblasts is important for granulation formation in addition to angiogenesis. As a result, it was found that Centaurein and Jacein have an action, and the present invention was completed.
That is, the present invention is a fibroblast proliferation promoter comprising Centaurein (R ₁ = H, R ₂ = CH ₃ ) or Jacein (R ₁ = CH ₃ , R ₂ = H) represented by the following formula as an active ingredient: , Regarding.

In the cell growth promoter, the fibroblast is preferably a human skin fibroblast.
Moreover, this invention relates to the wound treatment composition containing the said fibroblast proliferation promoter.

  The wound healing in animals can be promoted or treated with the fibroblast proliferation promoter of the present invention and the wound treatment composition containing the same.

Centaurein(quercetqagetin 3,6,4'-trimethyl ether 7-O-β-D-glucoside)及びJacein（quercetqagetin 3,6,3'-trimethyl ether 7-O-β-D-glucoside)は、C3またはC6にメトキシ基を有し、C7に糖を有する点に特徴を有する。Centaurein及びJaceinは、水和物、溶媒和物、各種塩形態であってもよい。
Centaurein及びJaceinは繊維芽細胞に対して、強い増殖促進作用を示す。繊維芽細胞は、特にヒト皮膚繊維芽細胞において効果が著しい。 Hereinafter, the fibroblast proliferation promoter of the present invention and a wound treatment composition containing the same will be described.
[Fibroblast proliferation promoter]
The fibroblast proliferation promoter of the present invention is Centaurein (R ₁ = H, R ₂ = CH ₃ ) or Jacein (R ₁ = CH ₃ , R ₂ = H) represented by the following formula, and fibroblast proliferation It is included as a component having promoting activity. In the chemical formula, “glc” means glucose.

Centaurein (quercetqagetin 3,6,4'-trimethyl ether 7-O-β-D-glucoside) and Jacein (quercetqagetin 3,6,3'-trimethyl ether 7-O-β-D-glucoside) are C3 or C6 Is characterized by having a methoxy group at C7 and a sugar at C7. Centaurein and Jacein may be hydrates, solvates, and various salt forms.
Centaurein and Jacein have a strong growth promoting effect on fibroblasts. Fibroblasts are particularly effective in human skin fibroblasts.

The cell growth promoter of the present invention can be used as an active ingredient such as a wound treatment composition.
The cell growth promoter of the present invention may be in any form, and may be in the form of a solution such as an aqueous solution, or a powder or tablet. In the case of molding into tablets or the like, conventionally known pharmaceutically acceptable carriers, triturating agents, disintegrating agents, lubricants and the like can be used. Any pharmaceutical form can be appropriately prepared by those skilled in the art based on the art in the pharmaceutical field.
The route of administration of the composition can be various. When used for treating wounds such as skin, including scratches, cuts, rough skin, and the like, it is effective and preferably used as an external preparation.

  The content of Centaurein and Jacein in the cell growth promoter of the present invention is preferably included in the range of about 5 to 100% by mass as the dry solid content weight depending on the purpose, dosage form and the like.

[Wound treatment composition]
The wound treatment composition containing the cell growth promoter of the present invention can be used for various wound treatments of animals including humans. In this specification, a wound means the state which produced the defect | deletion in the surface of organ tissue, and includes a trauma, an abrasion in a skin, a cut, what is called rough skin, etc.
The wound treatment composition of the present invention may be in any form, and may be in the form of a solution such as an aqueous solution, or a powder or tablet. In the case of molding into tablets or the like, conventionally known pharmaceutically acceptable carriers, triturating agents, disintegrating agents, lubricants and the like can be used. Any pharmaceutical form can be appropriately prepared by those skilled in the art based on the art in the pharmaceutical field.
The route of administration of the composition can be various. When used for treating wounds such as skin, including scratches, cuts, rough skin, and the like, it is effective and preferably used as an external preparation.

(Centreurein and Jacein cell growth promoting mechanism)
1. Cell proliferation signaling pathways involving protein kinases When receptors bound to ligands transmit signals into cells by activating specific enzymes, many of them activate or inactivate protein kinases by phosphorylating substrate proteins The signal is transmitted to the next (Toru Akiyama, et al., “I understand the signal transduction”, Shepherd, pp.18-62 (2001)).
In order to investigate what kind of intracellular signal transduction pathways such as Centaurein and Jacein express such cell growth promoting action, especially for Centaurein, inhibitors against various protein kinases involved in signal transduction pathways The signal transduction pathway was examined using cell proliferation and protein expression changes of protein kinase as indices.
Details of the experiment will be described later. When human skin fibroblasts (HSF) were treated with Centaurein by adding Calphostin C, an inhibitor of protein kinase C (PKC), at a 50% inhibitory concentration (IC50). The cell growth promoting action shown by treatment with Centaurein alone was inhibited (FIG. 5B). Furthermore, phosphorylation of protein kinase C was observed in the treatment with Centaurein, and this activation was suppressed by calphostin C (FIG. 6B).

2. About influence of MAPK signaling pathway
Many growth stimuli from inside and outside the cell transmit a growth signal from the cytoplasm into the nucleus through the MAP kinase cascade while regulating protein phosphorylation.
Therefore, MAP kinase involved in cell proliferation and differentiation, ERK1 / 2 (p44 / 42), was investigated using Centaurein treatment on cell proliferation and protein expression using an inhibitor of its activation enzyme MEK (PD98059). .
Proliferation of human fibroblasts was promoted by treatment with Centaurein, but was suppressed by simultaneous treatment with PD98059, an inhibitor of activating enzyme MEK (FIG. 7B). Moreover, phosphorylation of ERK1 / 2 by Centaurein treatment was inhibited by calphostin C and PD98059, respectively (FIG. 8B).
From these facts, it is considered that the activity of protein kinase C and ERK is involved in the cell growth promoting action of Centaurein and Jacein.
In addition, many flavonoids represented by Quercetin inhibit enzyme activities such as tyrosine kinase (PTK), protein kinase C (PKC), and MAP kinase, and suppress cell proliferation, migration, angiogenesis, and cell cycle. There have been reports, and the present inventors have also confirmed the suppression of proliferation of human skin fibroblasts by Quercetin (data not shown). Centaurein and Jacein have the same skeleton as Quercetin, but are structurally different such as having a methoxy group at C3 or C6 and a sugar at C7. It is surprising that the difference in structure shows a completely different action such as cell growth inhibition / promotion.

(Effects of Centaurein and Jacein on wound healing models)
In an in vitro wound healing model, cell migration to the cell defect site by Centaurein and cell proliferation around the wound were observed (FIG. 9B). In addition, the cell migration and cell proliferation actions were inhibited by calphostin C and PD98059 (FIG. 9C).

(Manufacture of Centaurein & Jacein)
In the present invention, Centaurein and Jacein are described as being extracted from Bidens pilosa, a plant belonging to the genus Sendangsa, but may be extracted from any natural product, purified product, or synthetic product. It is natural.
In the present invention, the evidence pillow includes the following.

Bidens pilosa L. (Kosendangusa, Koshirono Sendangusa, Sakai Toyokusa)
Bidens pilosa L. var. Minor (Blume) Sherff (Shirobanasendangusa, Shironosendangusa, Koshironosendangsa, Kosendangsa, Sengyogusa)
Bidens pilosa L. var. Bisetosa Ohtani et S. Suzuki
Bidens pilosa L. f. Decumbens Scherff (high millet snow Bidens)
Bidens pilosa L. var. Radiata Scherff (may also include Tachiawayukisendangusa, Hiawayukisendangusa)
Bidens pilosa L. var. Radiata Schultz Bipontinus (Shironosendangusa)
Bidens biternata Lour. Merrill et Sherff (Sendangsa)
Bidens bipinnata L. (Kobano Sendangsa, Sendangsa)
Bidens cernua L.
Bidens frondosa L. (American Sendangsa, Seitakatakogi)
Bidens maximowicziana Oett
Bidens parviflora Willd
Bidens radiata Thuill. Var. Pinnatifida ( Turcz.) Kitamura ( Ezonotaukogi)
Bidens tripartita L.

The above-mentioned Sendangsa plant usually uses a raw plant obtained by drying in the sun or hot air (for example, 70 to 80 ° C.), or steaming, for example, steaming for about 1 hour to 1 and a half hours, and then drying the plant. Also good.
The use part of the above-mentioned Sendangsa plant may be any part of the root, the above-ground part (stem, leaf, flower, etc.) or the whole plant. In particular, the use of leaf and stem portions is preferred in terms of efficacy.

[Other additives]
Moreover, you may add another component or a plant to the cell growth promoter of this invention or the composition containing this further for the purpose of antioxidant.
[Method for extracting Sendangsa plants]
One embodiment of the method for obtaining the extract of the genus Sendanga contained in the composition of the present invention is as follows. As described above, the extract of the genus Sendangsa is dried or dried after steaming with steam. Alternatively, it is steamed with steam and dried, and extracted by adding a solvent at room temperature or under heating. As an extraction method, for example, it is possible to obtain an extract by immersing and standing, or using an extraction device such as a Soxhlet extractor.

Examples of solvents used for extraction include water, methanol, ethanol, propanol, butanol, ethylene glycol, propanediol, butanediol, glycerin and other alcohols, and their hydrates, acetone, ethyl methyl ketone, chloroform, Methylene chloride and ethyl acetate and their hydrates may be used. Moreover, the mixture containing 2 or more types of the said solvent may be sufficient. The amount of the solvent added can be, for example, about 1 L to 100 L with respect to 1 kg of the total dry weight of the plant used.
The temperature at the time of extraction can be normally performed at room temperature to about the boiling point. The extraction time depends on the temperature and the solvent, but when extraction is performed at room temperature to about boiling point, it can be performed over a range of about 1 to 300 hours.

  The extract may be concentrated or solid (dry product) by distilling off the solvent if necessary. When used as a concentrate, it may be used as it is after the concentration is adjusted. Further, the extract may be used after being subjected to treatment such as activated carbon treatment, resin treatment such as HP20, low-temperature standing, filtration, etc. for decolorization and removal of unnecessary substances. Further, the extract can be used by fractionating a highly active fraction by an appropriate separation means such as a gel filtration method, a silica gel column chromatography method, or a reversed phase or normal phase high performance liquid chromatography method. In the present invention, the Sendangusa plant extract includes such a fraction. The obtained extract may be used as it is, or as a concentrate or a solid (eg, powder). Further, other components may be mixed according to the purpose of use.

・ Reagent Genistein (CALBIOCHEM)
KT5720 (CALBIOCHEM)
KT5823 (CALBIOCHEM)
Calphostin C (CALBIOCHEM)
PD 98059 (CALBIOCHEM)
Dulbecco's modified eagle medium (low glucose) (GIBCO)
Fetal Bovine Serum (GIBCO)
Dialyzed fetal bovine serum (GIBCO)
0.25% Trypsin-0.02% EDTA-PBS (GIBCO)
L-Glutamine (GIBCO)
Penicillin / Streptomycin (GIBCO)
MEM Non-Essential-Amino-Acid Solution 10 mM (GIBCO)
Tissue culture dish (35 mm, 100 mm, 96 well) (FALCON)
Cell Counting Kit-8 (Dojindo Laboratories)

・ Reagents and instruments for Western blotting Acrylamide (BIO-RAD)
Ammonium persulfate (GIBCO)
Nitrocellulose transfer membrane (Schleicher & Schuell)
Skimmed milk (snow mark)
Tween-20 (Kanto Chemical)
TEMED (Sigma)
Bovine serum albumin fraction V (Sigma)
Protease inhibitor cocktail tablets (Roche)
Phospho-PKC Antibody Sampler Kit (Cell Signaling)
MAPK Family Antibody Sampler Kit (Cell Signaling)
Phospho-MAPK Family Antibody Sampler Kit (Cell Signaling)
Anti-mouse IgG HRP conjugated F (ab ') ₂ fragments (Anti mouse IgG HRP-linked F (ab') 2 fragment) (Amersham Biosciences)
Anti-rabbit IgG HRP conjugated F (ab ') ₂ fragments (Anti rabbit Ig, HRP-linked F (ab') 2 fragment) (Amersham Biosciences)
Kaleidoscope Prestained Standards (BIO-RAD)
Precision Plus Protein ^TM Standard (BIO-RAD)
ECL ^TM (Amersham Biosciences)
ECL Advance ^TM (Amersham Biosciences)
DBX Developer and Replenisher (KODAK)
DBX Fixer and Replenisher (KODAK)
Diagnostic Film (X-OMAT AR) (Kodak)
High sensitivity film (Hyperfilm ^TM ) (Amersham Biosciences)
Mini Trans-Blot (registered trademark) Cell (BIO-RAD)
Mini-PROTEAN (registered trademark) II Cell (BIO-RAD)
Micro BCA ^™ Protein Assay Reagent Kit (PIERCE)

・ Common reagents
Na ₂ HP0 ₄ , NaCl, KCl, HCl, NaOH, Glycerol, Methanol, Ethanol, Tris (Tris (hydroxymethyl) aminomethane), NaHC0 ₃ , Na ₂ C0 ₃ , BPB, NaF, Na ₃ V0 ₄ , Nonidet P- 40, Sodium deoxycholate and sodium molybdenum were special grades manufactured by Wako Pure Chemical Industries, Ltd. or equivalent.

・ Measuring instrument METTLER balance (AG245) (METTLER TOLEDO)
Auto high pressure sterilization personal clave (HA240MIV) (Hirayama Manufacturing Co., Ltd.)
Centrifuge (CT-110) (Sakuma Manufacturing Co., Ltd.)
pH meter (HM-20S) (Toa Denpa Kogyo Co., Ltd.)
Spectrophotometer (UV-190) (Shimadzu Corporation)
CO ₂ incubator (Juji Chemical Industry Co., Ltd.)
Clean bench (NS-18BW) (Juji Chemical Industry Co., Ltd.)
Phase contrast microscope (IMT-2) (OLYMPUS)
Fluorescence phase contrast microscope (IX 71) (OLYMPUS)
High-speed centrifuge himac (CR21) (Hitachi, Ltd.)
Microplate reader (MPR-A4i) (Tosoh Corporation)

・ Abbreviations
SDS: Sodium dodecyl sulfate
PBS: Phosphate buffered saline (pH 7.4)
TBS: Tris buffered saline (pH 8.0)
T-TBS: 0.05% Tween-20 in TBS
DMEM: Dulbecco's modified Eagle medium
FBS: Fetal Bovine Serum
HSF: Human skin fibroblast
PKC: Protein kinase C
PTK: Protein tyrosine kinase
PKA: Protein kinase A
PKG: Protein kinase G
MAPK: Mitogen-activated protein kinase
ERK: Extracellular signal-regulated kinase
JNK: c-Jun N-terminal kinase
SAPK: Stress-activated protein kinase
MEK: MAP kinase / ERK-activating kinase
Bidence Pilosa: Bidence Pilosa hot water extract

1. Extraction conditions of Centaurein and Jacein from Bidence Pyrrosa Extracts and cell proliferation from Bidence Pyrrosa powder (ground powdered leaves of Miyakojima were treated with powder, prepared by Musashino Immunology Institute) Sought the relationship.
Extraction of drug Bidence Pilosa is extracted with purified water at a rate of 1 g / 10 mL for 12 hours at 100 ° C., then centrifuged at 1500 rpm for 10 minutes, and the supernatant is used as a hot water extract. Moreover, it extracted at room temperature for 24 hours using ethanol, and processed similarly, and obtained the ethanol extract. All the extracts were used after sterilization with a filter (0.2 μm).

Cell count method
HSF was seeded at 3 × 10 ³ cells / well in each well of a 96-well microtiter plate, and cultured using 10% FBS DMEM under conditions of 37 ° C. and 5% CO ₂ . After 24 hours, the cells were washed twice with PBS and cultured in 0.5% dialyzed FBS DMEM again under the same conditions to introduce into the stationary phase. Further, 24 hours later, 0.5% dialyzed FBS DMEM to which Hidden Firth / Pirosa hot water extract or ethanol extract (0.01, 0.1, 1 mg / mL) was added was treated. To control the ethanol extract, ethanol was added to 1%. The same treatment was performed 24 hours later. 48 hours after the drug treatment, 10 μL of the cell measurement kit-8 was added to each well, and the number of cells was measured with a microplate reader using the absorbance (measurement wavelength: 450 nm, control wavelength: 650 nm) as an index. The cell number of the control (bidence pillowa = 0 mg / mL) was taken as 100%, and the number of cells resulting from the treatment with the Bidence pillowa hot water extract or ethanol extract was converted to% and displayed. These differences were tested by Student's t-test (FIG. 1: data, mean ± SE; n = 6; Student's t-test (** p <0.01, *** p <0.001).

result
By treating HSF with each concentration of B. pirosa hot water extract for 48 hours, an increase in the number of cells was observed compared to the control. The cell growth rate of HSF was larger as the treatment concentration was higher in the Bidence / Pirosa hot water extract, and the growth rate was 225 and 385% at 0.1 and 1 mg / mL, respectively, when the control was 100% (FIG. 1-black). bar). On the other hand, the Vidence / Pyrosa ethanol extract (FIG. 1-white bar) did not show the cell growth promoting action like the hot water extract.

2. As mentioned above, it was shown that the hot water extract of Bidence pirousa promoted the growth of human skin fibroblasts, so that it was further divided into each fraction and proliferated against HSF. The effect was examined.
The Bidence-Pirosa hot water extract was passed through a porous resin column (Diaion HP-20), the passing fraction was designated as fraction C, and the methanol elution fraction was designated as fraction D. Further, the fraction D was eluted by changing the concentration of methanol using an ODS column to obtain fractions D1 to D7 (FIG. 2).
Preparation of Drug Each fraction obtained from the Bidence Pilosa hot water extract was used as a 10 mg / mL solution using purified water as a solvent and sterilized with a filter (0.2 μm).

Cell proliferation assay
Each well of a 96-well microtiter plate was seeded with HSF at 3 × 10 ³ cells / well and cultured in 10% FBSDMEM at 37 ° C. and 5% CO ₂ . After 24 hours, the cells were washed twice with PBS and cultured in 0.5% dialyzed FBS DMEM again under the same conditions to introduce into the stationary phase. Further, 24 hours later, HSF was treated with 0,5% dialyzed FBS DMEM added with fraction C and fraction D (0.01, 0.1 mg / mL) or fractions D1 to D6 (0.1 mg / mL), respectively. The same treatment was performed 24 and 48 hours later. 72 hours after the drug treatment, 10 μL of the cell measurement kit-8 was added to each well, and the number of cells was measured with a microplate reader using the absorbance (measurement wavelength: 450 nm, control wavelength: 650 nm) as an index.

Results Comparison of the growth effect of fraction C and fraction D on HSF revealed that both fractions promoted the growth of HSF, but fraction D showed a stronger growth promotion effect than fraction C. When control (fraction C or D = 0 mg / mL) was taken as 100% and the growth rates of 0-1 mg / mL treatment were compared, fraction C was 198% and fraction D was 398%.
In addition, fractions D1 to D6 were treated with HSF at a concentration of 0.1 mg / mL for 72 hours. As a result, fractions D1 to D5 showed a slight tendency to promote growth, but Bidence Pilosa hot water extract or fraction A strong growth promoting action as obtained by the treatment of D was not observed. On the other hand, the treatment with fraction D6 greatly promoted the growth of HSF (Fig. 3; 1: 0.5% dialyzed FBS DMEM treatment, 2: fractions D1, D2, D3 0.1 mg / mL 0.5% dialyzed FBS DMEM treatment, 3: fractions. D4 0.1 mg / mL 0.5% dialyzed FBS DMEM treatment, 4: fraction D5 0.1 mg / mL 0.5% dialyzed FBS DMEM treatment, 5: fraction D6 0.1 mg / mL 0.5% dialyzed FBS DMEM treatment; data, mean ± SE N = 6; Student's t-test (* p <0.05, ** p <0.01, *** p <0.001)).
Fraction D6 contained flavonoids Jacein and Centaurein as main components. (From extraction, structure determination, and comparison data with standard. Data not shown.) (See Natural Medicines 57 (3), 100-104 (2003)).

3. Centaurein and Jacein promote cell growth
A comparative study was conducted on the cell growth promoting effects of Centaurein and Jacein.
Drug preparation
Jacein and Centaurein (provided by Prof. Toru Okuyama, Department of Natural Medicine, Meiji Pharmaceutical University) made purified water a 1 mg / mL solution in a solvent and used it after sterilizing with a filter (0.2 μm).

Cell proliferation assay
Each well of a 96-well microtiter plate was seeded with HSF at 3 × 10 ³ cells / well, and cultured using 10% FBS DMEM under conditions of 37 ° C. and 5% CO ₂ . After 24 hours, the cells were washed twice with PBS and cultured in 0.5% dialyzed FBS DMEM again under the same conditions to introduce into the stationary phase. After 24 hours, 0.5% dialyzed FBS DMEM supplemented with Jacein (0.1 mg / mL) or Centaurein (0.01 mg / mL) was treated with HSF. 24 hours after the drug treatment, 10 μL of the cell measurement kit-8 was added to each well, and the number of cells was measured with a microplate reader using the absorbance (measurement wavelength: 450 nm, control wavelength: 650 nm) as an index.

result
As a result of treatment with Jacein and Centaurein for 24 hours, both results showed that cell proliferation of HSF was significantly promoted. The drug treatment concentrations in this experiment were Jacein 0.1 mg / mL and Centaurein 0.01 mg / mL, and Centaurein exerted its action at a lower concentration (FIG. 4; 1: 0.5% dialyzed FBS DMEM treatment (control), 2 : Jacein 0.1 mg / mL, 0.5% dialyzed FBS DMEM treatment, 3: Centaurein 0.01 mg / mL, 0.5% dialyzed FBS DMEM treatment; data, mean ± SE; n = 6; Student t-test (* p <0.05, *** p <0.001))).

4). Examination of signal transduction pathways in human dermal fibroblasts (1) Examination of signal transduction pathways using protein kinase inhibitors Many cases where receptors bound to ligands transmit signals to intracellular by activation of specific enzymes Protein kinase phosphorylates a substrate protein and activates or inactivates it to transmit the signal to the next (Toru Akiyama, et al., “I understand the signal transduction”, Satoshi Teido, pp.18-62 (2001)).
In previous experiments, it was shown that Bidence Pilosa hot water extract and fraction D6 and Centaurein contained in it promote the proliferation of human skin fibroblasts.
In this experiment, various protein kinase inhibitors involved in intracellular signal transduction were used, and signal transduction pathways were examined using changes in HSF proliferation rate and protein expression by Bidence Pilosa and Centaurein as indicators.

Cell proliferation assay
Each well of a 96-well microtiter plate was seeded with HSF at 3 × 10 ³ cells / well and cultured using 10% FBS DMEM at 37 ° C. and 5% CO ₂ . After 24 hours, the cells were washed twice with PBS and cultured in 0.5% dialyzed FBS DMEM again under the same conditions to introduce into the stationary phase. After 24 hours, the drug treatment shown in (A) or (B) below was performed on HSF. The protein kinase inhibitor was pretreated at each concentration for 30 minutes, and DMSO, an inhibitor solvent, was added to the control. 48 hours after the drug treatment, 10 μL of the cell measurement kit-8 was added to each well, and the number of cells was measured with a microplate reader using the absorbance (measurement wavelength: 450 nm, control wavelength: 650 nm) as an index.
(FIG. 5A)
1.0.5% dialyzed FBS DMEM in 0.5% DMSO (control)
2. Bidenspirosa 1mg / mL in 0.5% dialyzed FBS DMEM
3.Genisine 2.6μM and bidenspirosa 1mg / mL in 0.5% dialyzed FBS DMEM
4. KT5720 56nM and bidenspirosa 1mg / mL in 0.5% dialyzed FBS DMEM
KT5823 234nM and Bidenspirosa 1mg / mL in 5.0.5% dialyzed FBS DMEM
6. Calphostin C 50nM and bidenspirosa 1mg / mL in 0.5% dialyzed FBS DMEM
(Fig. 5B)
1.0.5% dialyzed FBS DMEM in 0.5% DMSO (control)
2.0.5% dialyzed FBS DMEM center urein 0.01mg / mL
3. Calphostin C 50nM and centaurein 0.01mg / mL in 0.5% dialyzed FBS DMEM

Sample preparation
HSF was seeded in a 100 mm dish at 4 × 10 ⁵ cells / well, and cultured in 10% FBS DMEM under conditions of 37 ° C. and 5% CO ₂ for 24 hours. After washing twice with PBS, the cells were again cultured in serum-free DMEM under the same conditions to introduce into the stationary phase. After 24 hours, serum-free DMEM supplemented with Bidence Pilosa (1 mg / mL) or Centaurein (0.01 mg / mL) was treated with HSF. Each inhibitor was pretreated for 30 minutes, and DMSO, an inhibitor solvent, was added to the control. 15 minutes after drug treatment, HSF was washed twice with ice-cold PBS and 100 μL of ice-cold RIPA buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, O. 25% deoxy] Sodium cholate, protease inhibitor, 10 mM NaF, 2 mM Na ₃ VO ₄ , 40 mM, β-glycerophosphate, 10 mM sodium molybdate]. After standing on ice for 1 hour, the intracellular protein elution fraction was obtained by centrifuging at 15,000 rpm for 5 minutes. The obtained intracellular protein was quantified with a Micro BCA kit and then diluted with 2 × Laemmli sample buffer [4% SDS, 25% glycerol, 0.01% BPB, 0.12M Tris-HCl (pH 6.8)].

SDS-PAGE (SDS-polyacrylamide gel electrophoresis)
The sample was boiled at 100 ° C. for 5 minutes, and subjected to electrophoresis on 7.5% and 10% acrylamito gels according to the molecular weight of the sample to separate proteins.

Western blot
After SDS-PAGE, the gel protein was transferred to a membrane filter. After blocking the filter with T-TBS containing 5% skim milk [50 mM Tris-HCl (pH 8.0), 0.9% NaCl, 0.05% Tween-20], anti-phospho-PKC diluted 1,000-fold with T-TBS containing 5% skim milk (pan) Treated with antibody. After washing with T-TBS, it was treated with a secondary antibody diluted 2,000 times with T-TBS containing 5% skim milk. After washing with T-TBS, an ECL (registered trademark) coloring reagent was treated, and the film was exposed and developed.

result
HSF treated with tyrosine kinase (PTK), protein kinase A (PKA), protein kinase G (PKG), and protein kinase C (PKC) inhibitors at 50% inhibitory concentration (IC50), and growth rate by Bidence Pilosa The change of was examined. As a result, PTK inhibitor (Genistein), PKA inhibitor (KT5720), and PKG inhibitor (KT5823) did not suppress growth. On the other hand, the growth of PKC inhibitor (calphostin C) was suppressed to the same extent as the control. In addition, when Centaurein was similarly treated with calphostin C and examined, growth suppression of HSF was observed (FIGS. 5A and 5B; data, mean ± SE; n = 6; Student t-test (** * p <0.001 (compared to control), ^### p <0.001 (compared to Bidence Pilosa (A) or Centaurein (B)))).
Protein kinase C (PKC) phosphorylation was observed in the treatment with Bidence Pilosa or Centaurein from the results of protein expression studies by Western blotting. This PKC activation was suppressed by calphostin C (FIGS. 6A and 6B; HSF cells were treated with Vidence Pilosa (1 mg / mL) (A) or Centaurein (0.01 mg / mL) (B) at 37 ° C., 15 (The same treatment was performed using HSF cells pretreated with calphostin C (50 nM) for 30 minutes).

(2) Influence of MAPK signal transduction pathway Many proliferation stimuli from inside and outside the cell transmit a proliferation signal from the cytoplasm to the nucleus through the MAP kinase cascade while regulating protein phosphorylation.
In this experiment, the MAP kinase, ERK1 / 2 (p44 / 42), which is involved in cell growth / differentiation in response to extracellular stimuli such as growth factors, is specifically tested using an inhibitor of its activation enzyme MEK (PD98059). The effect of treatment with Pilosa or Centaurein on cell proliferation and protein expression was examined.

Cell proliferation assay
Each well of a 96-well microtiter plate was seeded with HSF at 3 × 10 ³ cells / well and cultured using 10% FBS DMEM at 37 ° C. and 5% CO ₂ . After 24 hours, the cells were washed twice with PBS and cultured in 0.5% dialyzed FBS DMEM again under the same conditions to introduce into the stationary phase. After an additional 24 hours, 0.5% dialyzed FBS DMEM supplemented with Bidence Pilosa (1 mg / mL) was treated with HSF. The MEK inhibitor PD98059 (30 μM) was pretreated for 30 minutes, and DMSO, an inhibitor solvent, was added to the control. 48 hours after the drug treatment, 10 μL of the cell measurement kit-8 was added to each well, and the number of cells was measured with a microplate reader using the absorbance (measurement wavelength: 450 nm, control wavelength: 650 nm) as an index. Centaurein (0.01 mg / mL) was treated in the same manner, and the number of cells 24 hours after the drug treatment was measured.

Sample preparation
HSF was seeded in a 100 mm dish at 4 × 10 ⁵ cells / well, and cultured in 10% FBS DMEM under conditions of 37 ° C. and 5% CO ₂ for 24 hours. After washing twice with PBS, again with serum-free DMEM. It was introduced in the stationary phase by culturing under the same conditions. After 24 hours, serum-free DMEM supplemented with Bidence Pilosa (1 mg / mL) or Centaurein (0.01 mg / mL) was treated with HSF. Calphostin C (50 μM) and PD98059 (30 μM) were pretreated for 30 minutes, and DMSO, an inhibitor solvent, was added to the contronil. 15 minutes after drug treatment, HSF was washed twice with ice-cold PBS and 100 μL of ice-cold RIPA buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.25% deoxycholic acid Sodium, protease inhibitor, 10 mM NaF, 2 mM Na ₃ VO ₄ , 40 mM β-glycerophosphate, 10 mM sodium molybdate]. After standing on ice for 1 hour, the intracellular protein elution fraction was obtained by centrifuging at 15,000 rpm for 5 minutes. The obtained intracellular protein was quantified with the Micro BCA Kit and then diluted with 2 × Laemmli sample buffer [4% SDS, 25% glycerol, 0.01% BPB, 0.12 M Tris-HCl (pH 6.8)].

SDS-PAGE (SDS-polyacrylamide gel electrophoresis)
Samples were boiled at 100 ° C. for 5 minutes, and proteins were separated by electrophoresis on 7.5% and 10% acrylamito gels according to the molecular weight of the samples, respectively.

Western blot
After SDS-PAGE, the gel protein was transferred to a membrane filter. The filter was blocked with T-TBS containing 5% skim milk [50 mM Tris-HCl (pH 8.0), 0.9% NaCl, 0.05% Tween-20], and diluted to 1,000-fold with T-TBS containing 5% BSA. Treated with 42 antibody, anti-phospho-p44 / 42 antibody. After washing with T-TBS, it was treated with a secondary antibody diluted 2,000 times with T-TBS containing 5% skim milk. After washing with T-TBS, an ECL (registered trademark) coloring reagent was treated, and the film was exposed and developed.

Results Proliferation of HSF promoted by treatment with Bidence Pilosa showed significant suppression as a result of inhibition of MEK with PD98059 (FIG. 7A; 1: 0.5% DMSO and 0.5% dialyzed FBS DMEM treatment (control), 2: Bidence・ Pirosa 1 mg / mL, 0.5% dialyzed FBS DMEM treatment, 3: PD98059 30 μM and Bidence Pilosa 1 mg / mL, 0.5% dialyzed FBS DMEM treatment; data, mean ± SE; n = 6; Student t-test (** * p <0.001 (compared to control), ^### p <0.001, ^## p <0.01 (compared to Bidence Pilosa (A)))). In addition, Centaurein, which showed a strong cell growth promoting action, was also inhibited by simultaneous treatment with PD98059 (FIG. 7B1: 0.5% DMSO and 0.5% dialyzed FBS DMEM treatment (control), 2: Centaurein 0.01 mg / mL, 0.5% dialyzed FBS DMEM treatment, 3: PD98059 30 μM and Centaurein 0.01 mg / mL, 0.5% dialyzed FBS DMEM treatment; data, mean ± SE; n = 6; Student t-test (*** p <0.001 (Comparison with control), ^### p <0.001, ^## p <0.01 (compared to Bidence Pilosa (A)))).
As a result of examining the activation of ERK from protein kinase C by Western blotting, phosphorylation of ERK1 / 2 by treatment with Bidence Pilosa or Centaurein was inhibited by calphostin C and PD98059, respectively (FIGS. 8A and 8B; HSF) Cells were treated with Bidence Pilosa (1 mg / mL) (A) or Centaurein (0.01 mg / mL) (B) for 15 minutes at 37 ° C. HSF cells were also treated with calphostin C (50 nM) and / or PD98059 (30 μM). ) And the same treatment was performed using the pre-treated for 30 minutes.

Conclusion
Centaurein significantly promoted human skin fibroblast proliferation.
On the other hand, this growth-promoting action was suppressed to the control and identification level by simultaneous treatment with calphostin C, which is a protein kinase C inhibitor, and further suppressed by PD98059 treatment, which is a MEK inhibitor. In addition, it was found that Centaurein enhances phosphorylation of protein kinase C and ERK1 / 2 by examining protein expression changes by Western blotting. Moreover, phosphorylation of ERK1 / 2 was suppressed by PD98059D and calphostin C. JNK / SAPK was not expressed in human dermal fibroblasts. Although p38 was expressed in human dermal fibroblasts, its phosphorylation was not observed (data not shown). Therefore, it is considered that Centaurein promotes the proliferation of human dermal fibroblasts via protein kinase C and ERK.

5. Centaurein and Jacein's effect of promoting wound healing Wound healing is a repair reaction in vivo when tissue is damaged for some reason. The wound healing process is divided into the bleeding / coagulation phase, the inflammation phase, the proliferative phase, and the reconstruction phase according to the time course39). In the proliferative phase, fibroblasts and vascular endothelial cells are actively proliferated by cell growth factors released in the inflammatory phase to form granulation tissue and new blood vessels.
As mentioned above, it has been shown that Centaurein, which is a constituent of Bidence Pirosa hot water extract and fraction D6, promotes the proliferation of human skin fibroblasts, and Bidence Pirosa and Centaurein are found in fibroblasts. On the other hand, it is speculated that wound healing is promoted in cell migration / extension and cell proliferation. In this experiment, we investigated the wound healing effect of Centaurein's cell proliferation.

Effect of Centaurein on in vitro wound healing model
The wound healing promoting effect of Centaurein due to cell proliferation was examined using an in vitro wound healing model. In addition, the signal transduction pathway at this time was examined under the same conditions as cell proliferation.

Experimental method Preparation of in vitro wound healing model
A 35 mm dish was seeded with HSF at 1 × 10 ⁴ cells / dish and cultured to confluence using 10% FBS DMEM under conditions of 37 ° C. and 5% CO ₂ . This medium was changed every two days. After confluence, the cells were washed twice with PBS and cultured in serum-free DMEM again under the same conditions to introduce into the stationary phase. After another 24 hours, the cells were replaced with PBS, and a certain range of cells were diagonally detached using a pipette tip (10 μL) so as to draw an “x”, which was used as an in vitro wound healing model (LiouG, I., Matragoon S ., Samuel S., Behzadian M A., Tsai NT, GuX., Roon P, Hunt DM, HuntR.C., Caldwell RB, Marcus DM, Mol.Vis., 8, 483-493 (2002); Javelaud D ., Laboureau J., Gabison E., Verrecchia F, Mauviel A., J.Bio1.Chem., 278, 24624-24628 (2003); Minori Sakurai, et al. Japanese Biopharmaceutical Society 47th Annual Meeting, Tokyo, Abstracts of Lectures , Pp.106 (2000)). After thoroughly washing with PBS, Bidence Pilosa (1 mg / mL), fraction D6 (0.1 mg / mL), and Centaurein (0.01 mg / mL) were each treated. Calphostin C (50 nM) and PD98059 (30 μM) were pretreated at each concentration for 30 minutes. Twenty-four hours after these treatments, the state of the grooves produced by cell detachment and the surrounding cells were observed with a microscope.

Results FIG. 9 shows the results of examination of migration / extension and proliferation effects of Centaurein on HSF in an in vitro wound healing model. Treatment with Centaurein (Fig. 9B; Calphostin C (50nM) and Centaurein (0.01mg / mL) treatment in DMEM) compared to control (Fig. 9A; Centaurein (0.01mg / mL) treatment in DMEM)) Changes were observed in which the width of the groove narrowed and the density of cells around the wound increased. That is, Centaurein was found to promote HSF migration, extension, and proliferation. On the other hand, when the PKC inhibitor calphostin C or the MEK inhibitor PD98059 was simultaneously treated, the effects described above were suppressed (FIG. 9C; PD98059 (30 nM) and Centaurein (0.01 mg / mL) treatment in DMEM). .

Discussion Skin tissue is a barrier from external stimuli such as wounds and infections, and prompt recovery is required upon injury. Wound healing begins with the blood clotting reaction associated with platelet activation, and the important process is covering the cell defect site by migration of surrounding cells and subsequent cell growth, wound contraction, angiogenesis, Synthesis of extracellular matrix. In particular, angiogenesis and fibroblast migration / proliferation are important processes that occur prior to the formation of the extracellular matrix, and involve various growth factors and cytokines.
Bidence Pilosa, which has been reported to have antioxidant and anti-inflammatory effects, is thought to eliminate free radicals and promote wound healing. In addition, the proliferation of vascular endothelial cells and fibroblasts during the growth phase is an important reaction for angiogenesis and granulation (Makoto Tamura, Protein Nucleic Acid Enzyme, 45, 1145-1151 (2000)). It is thought to affect proliferation and improve wound healing.
According to an in vitro wound healing model, Centaurein promoted proliferation of cells around the wound site, and cell migration from both sides of the groove was observed, and the width of the cell defect site was narrowed (FIG. 9C).
From the examination of the intracellular signal transduction pathway in wound healing of Bidence Pilosa performed under the same conditions as the measurement of cell proliferation, both Calphostin C and PD98059 are cell migration to the cell defect site by Bidence Pilosa or Centaurein and cells around the wound site Inhibited growth. That is, inhibition of protein kinase C and ERK resulted in inhibition of wound healing. It has been reported that vascular endothelial cells, vascular smooth muscle cells and other cells involved in angiogenesis are accompanied by activation of protein kinase C during the wound healing process (Tang S., Morgan KG ,, Parker C., Ware JA., J. Biol. Chem., 272, 28704-28711 (1997); Wyatt TA .. Ito H., Veys TJ ,, Spurzem JR., Am. J. Physiol., 273, L1007-1012 (1997) ). Similar to the results obtained in cell proliferation measurements and protein expression studies, it was confirmed that in the in vitro wound healing model, Bidence Pilosa has cell migration and proliferation action through activation of protein kinase C and ERK. Bidence Pilosa is thought to promote wound healing by promoting the migration and proliferation of various cells. In addition, depending on the type of cells and proliferation stimuli, ERK has also been suggested to interact with other MAP kinases in wound healing, such as cell migration by p38 and JNK and contraction of collagen gel matrix (Javelaud D., Laboureau., Gabison E , Verrecchia F, Mauviel A., J. Bio1. Chem., 278, 24624-24628 (2003); Lee DJ, Rosenfeldt H. Grinnell F., Exp. Cell Res. 257, 190-l97 (2000)). In human dermal fibroblasts, JNK expression was not observed, but p38 expression was confirmed (data not shown). It has been reported that p38 is activated in corneal epithelial cells and vascular smooth muscle cells during inflammation or by trauma such as burns (Guru-Dutt Sharma, Jiucheng He, Haydee EP Bazan., J. Biol. Chem., 278, 21989-21997 (2003); Ju H., Nerurkar S., Sauermelch CF., Olzinski AR., Mirabile R., Zimmerman D., Lee JC., Adams J., Sisko J., Berova M ., Willette RN., J. Pharmacol. Exp. Ther., 301, 15-20 (2002); Ballard-Croft C., White DJ., Maass DL., Hybki DP., Horton JW., Am. J. Physiol. Heart Circ. Physiol., 280, H1970-1981 (2001)). In this study, activation of p38 by stimulation of Bidence pirousa proliferation was not confirmed in Western blotting (data not shown), but activation by inflammatory cytokines cannot be denied. Therefore, it is inferred that p38 is activated during the wound healing process in human skin fibroblasts, and interaction with ERK activated by Bidence Pilosa occurs.
The above results explain the wound healing in the wound model of Centaurein and its structural analog Jacein, which show growth promoting action on human dermal fibroblasts.

It is a figure which shows the human fibroblast proliferation effect | action of a biden spirosa extract. It is a figure which shows each extraction fraction of Bidence pillows. It is a figure which shows the human fibroblast proliferation effect | action of the fraction D among biden spirosa extracts. It is a figure which shows the human fibroblast proliferation effect of Centaurein and Jacein. It is a figure which shows the proliferation effect | action by simultaneous treatment of Bidence Pilosa or Centaurein and a protein kinase C inhibitor in a human fibroblast. It is a figure which shows the phosphorylation of protein kinase C by Vidence pillowa or Centaurein and the phosphorylation inhibitory effect by calphostin C in a human fibroblast. It is a figure which shows the proliferation effect | action by simultaneous treatment with Vidence pirousa or Centaurein and a MEK inhibitor in a human fibroblast. It is a figure which shows the phosphorylation of ERK1 / 2 by Bidence pillowa or Centaurein and the phosphorylation inhibitory effect by PD98059 in a human fibroblast. It is a figure which shows the cell growth promotion effect of Centaurein.

Claims (3)

A fibroblast proliferation promoter containing Centaurein (R ₁ = H, R ₂ = CH ₃ ) or Jacein (R ₁ = CH ₃ , R ₂ = H) represented by the following formula as an active ingredient. The fibroblast proliferation promoter according to claim 1, wherein the fibroblasts are human skin fibroblasts. A wound treatment composition comprising the fibroblast growth promoter according to claim 1 or 2.

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