JP2003155236A - Use of pterocarpan - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition containing a compound derived from natural materials and free from safety problems as an active component, having osteogenesis promoting action and antiarteriosclerotic action and useful as pharmaceuticals and health foods. SOLUTION: The invention provides a composition containing pterocarpan and having osteogenesis promoting action and antiarteriosclerotic action, a food containing pterocarpan and an antioxidation agent containing pterocarpan.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to pterocarpan (p
terocarpan), more specifically, to a composition containing pterocarpan and useful as a medicine, health food, functional food, antioxidant and the like.

[0002]

2. Description of the Related Art Pterocarpan is a compound contained in Astragalus (Astragalus) belonging to the legume family, such as Ougi. Ougi has long been used as a herbal medicine.
In particular, it is used by being mixed with a prescription suitable for elderly people, and its effects include diuresis and tonic action. It is also known to be effective against cardiovascular diseases including hypertension and hyperlipidemia. Regarding the pharmacological action of the compound isolated from Astragalus sinensis, aminobutyric acid has an antihypertensive action, L-canavanine has a silkworm metamorphosis inhibiting action, a saponin component has an anti-inflammatory action, and plasma cyclic adenylate (cyclic
AMP) concentration increasing action is known. However, nothing is known about the bone formation promoting action and the anti-atherogenic action.

[0003]

Since Japan is aging society in recent years, it is expected that the number of osteoporosis, arteriosclerosis, etc. associated with aging will increase in the future. In order to prevent or ameliorate these diseases, it is effective to ingest foods having a bone formation promoting action or an anti-atherogenic effect. An object of the present invention is to provide a composition having an osteogenesis promoting effect and an anti-atherogenic effect as a medicine, a health food or the like.

[0004]

[Means for Solving the Problems] Isoflavones such as daidzein, glycitin, and genistein, which have female hormone-like actions, have already been found in soybeans, and data have been shown that these prevent myocardial infarction. Therefore, the inventors of the present invention investigated the isoflavone-related compounds, which are more active than soybean isoflavones, from natural products by using the osteogenesis promoting action and anti-atherosclerotic action as indexes. Regarding the osteogenesis promoting action, using MC3T3-E1 which is an osteoblast culture line, and regarding the anti-atherosclerotic action,
The activity was evaluated using a cell culture system of vascular endothelial cells, vascular smooth muscle cells and macrophages. That is, pterocarpan or another isoflavone compound isolated and purified from C. ougi was added to the cell culture system, and the bone was examined using the alkaline phosphatase activity which is a calcification promoting enzyme as an index. Regarding anti-atherogenic effects, vascular endothelial cells were tested for their inhibitory effect on the decrease in cell viability due to LPS stimulation and the function of the fibrinolytic system, and vascular smooth muscle cells were evaluated for their activity using the proliferative capacity as an index. did.

In the present invention, pterocarpan used as a raw material is isolated and purified from C. japonica. As a result, it was found that pterocarpan has an excellent bone formation promoting action, anti-atherogenic action and antioxidant action,
The present invention has been completed. The present invention provides the inventions described below.

(1) A composition having an osteogenesis promoting action and an anti-atherogenic effect, which contains pterocarpan. Claim 2: A food product characterized by containing pterocarpan. Claim 3: The food according to claim 2, which is a health food or a functional food having a bone formation promoting action. Claim 4: The food according to claim 2, which is a health food or a functional food having an anti-atherogenic effect. Claim 5: An antioxidant characterized by containing pterocarpan.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION As described above, the present invention relates to pharmaceuticals, foods and antioxidants containing pterocarpan. The pterocarpan according to the present invention comprises (6a
R, 11aR) -3-hydroxy-9,10-dimethoxy pterocarpane, which is represented by the following structural formula (1).

[0008]

[Chemical 1]

The present invention is basically based on the use of pterocarpan alone for the above-mentioned uses, but it also includes the use of other isoflavone compounds obtained from, for example, corium as well as pterocarpan, if necessary. Even in this case, an excellent effect can be obtained. Here, as another isoflavone compound, formononetine (formononetine, 7-hydroxy) represented by the following structural formula (2) is used.
-4'-methoxy isoflavone) and 7,3'-dihydroxy-4'-methoxy isoflavone represented by the following structural formula (3).

[0010]

[Chemical 2]

[0011]

[Chemical 3]

The above-mentioned pterocarpan and other isoflavone compounds used in the present invention can be isolated by extracting them from pearl millet. Pterocarpan and the like are contained not only in the roots of Astragalus membranaceus but also in the leaves above the ground.
One example of a method for isolating these compounds from sugi (Cryptomeria japonica) is as follows. Shredded sugi (Cryptomeria japonica) was extracted with methanol, the obtained extract was dissolved in water, defatted with ether, and then extracted with butanol to give pterocarpan. And its related compounds can be obtained. The isolated pterocarpan and other isoflavone compounds can be used as a crudely purified product as they are, but in consideration of their uses and the like, they can be purified by a conventional method as necessary to obtain a purified product. For example, it can be purified by silica gel column chromatography using a mixed solvent of chloroform and methanol and by sequentially increasing the methanol concentration.

When pterocarpan or a drug containing pterocarpan and another isoflavone compound is a composition having an osteogenesis promoting action, the content of pterocarpan in the composition is 0.1 to 100 mg, preferably 0.1. ~ 50 mg, more preferably 0.3-10 mg
Is. When the content of the active ingredient, pterocarpan, or other isoflavone compound is within the above range, no cytotoxicity is observed and there is no possibility of harming the living body. But,
If it is less than the lower limit, the bone formation promoting action is not sufficiently exhibited. On the other hand, even if it is added over the upper limit, the effect corresponding to the added amount is not exhibited.

Next, when the drug is a composition having an anti-atherogenic effect, the content of pterocarpan in the composition is 0.1 to 100 mg, preferably 0.1 to 100 mg.
50 mg, more preferably 0.3 to 10 mg. When the content of the active ingredient, pterocarpan, or other isoflavone compound is within the above range, no cytotoxicity is observed and there is no possibility of harming the living body. However, if it is less than the lower limit value, the anti-atherosclerotic effect is not sufficiently exerted. On the other hand, if the amount exceeds the upper limit, the effect corresponding to the amount added cannot be obtained.

In the case of foods containing pterocarpan or pterocarpan and other isoflavone compounds, such as health foods and functional foods, the content of pterocarpan in the foods is 0.1 to 5 mg, preferably 0.1. -3 mg, more preferably 0.3-1 mg. When the content of the active ingredient, pterocarpan, or the isoflavone compound is within the above range, no cytotoxicity is observed and there is no possibility of harming the living body. However, if it is less than the lower limit, the bone formation promoting action and the anti-atherogenic action are not sufficiently exhibited. On the other hand, even if the amount exceeds the upper limit, sufficient effects commensurate with the amount added cannot be obtained.

In the antioxidant containing pterocarpan or pterocarpan and another isoflavone compound, the content of pterocarpan is 0.1 to 5 mg, preferably 0.1 to 3 mg, more preferably 0.3 to 1 mg. . When the content of the active ingredient, pterocarpan, or the isoflavone compound is within the above range, no cytotoxicity is observed and there is no possibility of harming the living body. However, if it is less than the lower limit, the desired antioxidant effect is not sufficiently exhibited. On the other hand, if the amount exceeds the upper limit, the effect corresponding to the amount added cannot be obtained.

The pterocarpan and isoflavone compounds used in the present invention are derived from natural products and have been used in various prescriptions as Kampo medicines since ancient times, and there is no problem in terms of safety. There is no worry even if you take foods that you use for a long period of time. The permissible daily amount of pearl millet is 3 to 5 g, and pterocarpan is contained in the amount of about 0.3 to 0.5 mg per volume. Also,
When using the above-ground part not designated as a crude drug to make tea, pterocarpan is 0.1 compared to the daily dose.
It is considered that about 1 mg is ingested. From the above, it is considered that the above-mentioned effects are sufficiently exhibited even when ingested as a crude drug or tea.

[0018]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited thereto. Example 1 The osteoblast cell culture strain MC3T3-E1 was added to α-MEM.
5 × 10 ⁵ cells / 10 ml / 10 in% FCS medium
The cells were inoculated with a 0 mm dish and cultured at 37 ° C. in 5% CO ₂ for 3 days, and the cells were confirmed to be in a saturating state (confluent) to complete the culture. Then, the cells were placed in a 12-well plate at 1 × 10 ⁵ cells /
2 ml / well inoculation or 3 in 60 mm dish
× 10 ⁵ cells / 4 ml / 60 mm dish was inoculated, and 3 times in the same manner as above with α-MEM-added 10% FCS medium.
After culturing for one day, it was washed with phosphate buffer (PBS).
Then, the medium was replaced with 1% FCS medium containing α-MEM, and at the same time, 1 × 10 ⁻⁵ M of pterocarpan was added to start the culture. As a control, the same test was performed in the case where pterocarpan was not added.

After culturing for 3 days, the culture supernatant and the cell layer were separated by centrifugation and collected. Then, alkaline phosphatase activity (ALP activity) in the supernatant and in the cell layer was measured. The cell layer was sonicated in 0.25 M sucrose to destroy the cells, and then used as a sample for intracellular ALP measurement. ALP measurement in the culture supernatant is 1 per sample
M MgCl ₂ 0.5 μl, 0.25 M sucrose 50 μl, 0.5 M carbonate buffer (pH 10.4
1) 50 μl, culture supernatant 250 μl and water 49.5 μl
0.1 ml of 8 mM p-nitrophenol disodium phosphate was added to the mixed solution containing and the reaction was started at 37 ° C.

Intracellular ALP measurement was carried out with 0.5 μl of 1 M MgCl ₂ , 50 μl of 0.25 M sucrose and 0.5 M Tris-HCl (pH) per sample.
9.2) 50 μl, sample 125 μl and water 174.
To the mixed solution containing 5 μl, 0.1 ml of 8 mM disodium p-nitrophenol phosphate was added and the reaction was started at 37 ° C. The reaction was stopped by adding 1.5 ml of 0.6 N NaOH for both reactions, and the released p-nitrophenol was measured by a spectrophotometer (420 nm).
The enzyme activity was determined by measuring the amount of p-nitrophenol (nmol) produced by the reaction for 1 minute of incubation to the cellular protein (m
It is shown around g). As a control, levamisole was added to each sample. Statistical processing method A significant difference test of each measured value was performed using Student's test. Those with a risk rate of 5% or less were considered to have a significant difference. The measurement results of ALP activity in the culture supernatant are shown in FIG.
The measurement result of P activity is shown in FIG. As is clear from these results, addition of pterocarpan increases ALP activity, which is an index of bone formation.

Example 2 Bovine brain capillary endothelial cells were added to a 96-well plate in 10% FB.
The cells were cultured in S-containing Dulbecco's MEM medium (hereinafter sometimes abbreviated as DMEM medium) until the cells were saturated. After culturing, the medium was replaced with a serum-free DMEM medium and treated with or without lipopolysaccharide (LPS) and pterocarpan for 24 hours. After treatment, commercially available 3- (4,5-dimethylthiazol-2-yl) -2,
5-diphenyl tetrasodium bromide (hereinafter M
It may be abbreviated as TT. ) The cell viability was quantified using an assay kit (Chemicon). The operation was performed according to the attached instruction manual, and the outline is as follows. 10 μl of the MTT solution was added to each well and reacted for 4 hours. After completion of the reaction, 100 μl of 0.04 N HCl and 100 μl each of isopropanol were added, and the mixture was further reacted for 10 minutes. Using an ELISA plate reader (Corona), the absorbance at 570 nm was 630 n.
m was used as a reference wavelength. The result (bovine brain capillary endothelial cell survival rate) was expressed as a relative value (%) with respect to the control group, with the cell survival rate of the control group being 100. The results are shown in Fig. 3. Note that * in the figure represents a significant difference (p <0.05) from the group treated with LPS alone.

As is clear from the figure, the presence of pterocarpan is effective in reducing cell viability. That is, it is known that LPS, which is a constituent component of the outer membrane of Gram-negative bacteria, has the property of acting on vascular endothelial cells to cause a decrease in cell viability, and accelerating the lumen of blood vessels to promote coagulation. However, the presence of a predetermined amount of pterocarpan (5 to 50 µM) can prevent the decrease in vascular endothelial cell viability due to LPS. Vascular endothelial cells are cell types that are in constant contact with blood, and not only prevent contact between blood and subendothelial tissues, but also prostacyclin (prostaglandin I ₂ ) and fibrinolytic system (proteolytic enzymes such as plasmin). It is speculated that pterocarpan enhances the antithrombotic defense response of endothelial cells against LPS procoagulant activity and contributes to the maintenance of blood vessel homeostasis, since it releases many anticoagulant factors such as proteins It

Furthermore, tissue changes of vascular endothelial cells in the presence of LPS alone or in the presence of LPS and pterocarpan were confirmed by Giemsa staining method. That is, bovine brain capillary endothelial cells were added to DME containing 10% FBS in a 60 mm dish.
The cells were cultured in M medium until the cells became saturated. After culturing, the medium was replaced with a serum-free DMEM medium, and treated with or without LPS and pterocarpan for 24 hours. After the treatment, the medium was removed, the cell layer was washed twice with PBS, and the cells were fixed with methanol. After removing the methanol and drying the cells, add Giemsa staining solution to 20-
Stained for 30 minutes. After staining, wash the cells with running water,
After drying, it was observed under a microscope.

As a result, as shown in FIGS.
In the presence of pterocarpan, voids and dead cells were not observed in the presence of pterocarpan, whereas the pebble-like morphology observed in vascular endothelial cells was damaged by PS alone treatment, and the increase in voids and dead cells was observed. It was confirmed that the morphology was almost the same. In the figure, a is a control, b is LPS (1.0 μg / m
1) treated product, c is LPS (1.0 μg / ml) and pterocarpan (10 μM) treated product, d is LPS (1.0 μm)
g / ml) and pterocarpan (25 μM) treated product, e
Is LPS (1.0 μg / ml) and pterocarpan (5
0 μM) treated products are shown. The result is the MT
Correlation with the results of the T method indicates that pterocarpan has a strong inhibitory effect on LPS-induced vascular endothelial cell injury.

Example 3 In this example, the inhibitory effect of pterocarpan on the proliferation ability of vascular smooth muscle cells was examined. In this case,
The amount of 5-bromo-2'-deoxyuridine (BrdU) incorporated into DNA was examined as an index. First,
Rat fetal thoracic aortic smooth muscle cells (A10) were cultured in a 96-well plate in 100 μl of DMEM medium containing 10% FBS until subconfluent. After culturing, serum-free DME was added to transfer the cells from the growth phase to the stationary phase.
After changing to M medium and incubating for 48 hours, 5%
The medium was exchanged with FBS-containing DMEM medium and treated in the presence or absence of pterocarpan for 46 hours. After the treatment, cell proliferation was quantified using a commercially available Cell Proliferation ELISA Kit (manufactured by Roche Diagnostics). The operation was performed according to the attached instruction manual, and the outline is as follows.

10 μl of BrdU standard solution was added to each well, and after further culturing for 2 hours, the medium was removed and the cells were fixed with methanol. Removes methanol and anti-BrdU
An antibody was added and reacted for 90 minutes. After washing each well, a substrate solution was added and reacted for 10 minutes. Then 1MH
After stopping the reaction with ₂ SO ₄ , the absorbance was measured at an absorbance of 450 nm using an ELISA plate reader (Corona) as a reference wavelength. For comparison,
Formonenonetin and 7,3'-dihydroxy-4'-
Methoxy isoflavone was tested in the same manner. Results are shown in FIG. * In the figure represents a significant difference (p <0.05) from the LPS alone treatment group. As is clear from the figure, pterocarpan suppressed the growth promotion of vascular smooth muscle cells in a concentration-dependent manner. On the other hand, formononetin and 7,3′-dihydroxy-4′-methoxyisoflavone were not effective enough, but it was revealed that the presence of high concentration of formononetin was effective.

Next, the same test as described above was conducted using daidzein and genistene as controls. Results are shown in FIG. The result (proliferation of A10) was expressed as a relative value (%) with respect to the control group, with the cell survival rate of the control group being 100.
Note that * in the figure indicates a significant difference (p <
0.05). As a result, it was revealed that the inhibitory effect of pterocarpan on the proliferation ability of vascular smooth muscle cells was stronger than daidzein and genisten, and the effect of suppressing the proliferation of the cells was large.

Furthermore, the inhibitory effect of pterocarpan (25 μM) on the proliferation of vascular smooth muscle cells was examined daily. First, 4 × 10 ³ A10 cells were seeded in a 30 mm dish in 1 ml of DMEM medium containing 10% FBS, and cultured for 48 hours. After completion of the culture, in order to shift the cells from the growth phase to the stationary phase, the cells were exchanged with a serum-free DMEM medium, incubated for 48 hours, and then D containing 5% FBS was added.
The medium was replaced with MEM medium and treated in the presence or absence of pterocarpan. After the treatment, trypsin-EDTA solution was added to detach the cells, and the cells were collected in a centrifuge tube. After collecting the cells, centrifuge at 1000 rpm and trypsin-E
After removing the DTA solution, it was resuspended in 200 μl of DMEM medium containing 5% FBS. 0.5% trypan blue staining solution (manufactured by Nacalai Tesque, Inc.) was added to the resuspended solution at a ratio of 1: 1, and unstained cells were used with a Thoma hemocytometer (manufactured by Elma Sales Co., Ltd.). Was measured. The results are shown in Fig. 7. As is clear from the figure, pterocarpan stably inhibited cell growth during the test period, but in the case of the control, the cell growth inhibitory effect was not observed halfway.

Since the promotion of vascular smooth muscle cell proliferation is an important phenomenon that promotes arteriosclerosis, it was found that pterocarpan exhibiting an inhibitory effect on this is useful as an anti-atherosclerotic agent. Up to now, many products have been used as drugs having an anti-atherosclerotic effect, but it is desired to develop a drug that effectively enhances the function of endothelial cells and, on the contrary, weakens the responsiveness of vascular smooth muscle cells. Was there.
The above results prove that pterocarpan is sufficient to meet this need.

[0030]

INDUSTRIAL APPLICABILITY According to the present invention, it is useful as a medicine, a health food, a functional food, an antioxidant, etc., which contains as an active ingredient pterocarpan, which has been used as a crude drug and has no problem in safety. Different compositions are provided. In particular, it is a new finding that pterocarpan has excellent bone formation-promoting action and anti-atherogenic action, and is expected to be widely used in the fields of pharmaceuticals, foods, and the like.

[Brief description of drawings]

FIG. 1 shows the effect of pterocarpan on ALP in the culture supernatant.

FIG. 2 shows the effect of pterocarpan on ALP in the cell layer.

FIG. 3 shows the effect of pterocarpan that blocks LPS-induced reduction in vascular endothelial cell viability.

FIG. 4 shows the effect of pterocarpan on the tissue changes of vascular endothelial cells.

FIG. 5 shows the inhibitory effects of various compounds on the proliferation ability of vascular smooth muscle cells.

FIG. 6 shows the inhibitory effect of various compounds on the proliferation ability of vascular smooth muscle cells.

FIG. 7 shows the growth inhibitory effect of pterocarpan on vascular smooth muscle cells.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C07D 493/04 106 C07D 493/04 106A C09K 15/06 C09K 15/06 // A61K 35/78 A61K 35 / 78 J (72) Takashi Yamagishi, 165 Koencho, Kitami-shi, Hokkaido Kitami Institute of Technology On-campus (72) Inventor Yasuyuki Nomura Kita 12-jo Nishi 6-chome, Kita-ku, Sapporo, Hokkaido (72) Inventor, Graduate School of Pharmaceutical Sciences (72) Okuma Osamu Kita-ku, Sapporo, Hokkaido Kita-ku, Nishi 6-chome, Hokkaido University Graduate School of Pharmaceutical Sciences, Hokkaido University (72) Inventor Takashi Uehara Kita-ku, Kita-ku, Kita-ku, Nishi 6-chome, Hokkaido University F Term (Reference) ) 4B018 MD08 MD61 ME04 ME05 ME06 4C071 AA01 AA07 BB01 BB06 CC12 EE05 FF17 LL01 4C086 AA01 AA02 CA01 MA01 MA04 MA52 NA14 ZA45 ZA96 ZB01 4C088 AB59 AC 01 BA08 NA10 NA14 ZA45 ZA96 ZB01 4H025 AA51 AC04 AC07 BA01

Claims (5)

[Claims] 1. A composition having an osteogenesis promoting action and an anti-atherosclerotic action, which comprises pterocarpan. 2. A food containing pterocarpan. 3. The food according to claim 2, which is a health food or a functional food having a bone formation promoting action. 4. The food according to claim 2, wherein the food is a health food or a functional food having an anti-atherogenic effect. 5. An antioxidant containing pterocarpan.