JPH0920714A - Eudesmane derivative and inducer for production of nerve growth factor containing the same as active ingredient - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new eudesmane derivative having inducing effects on the production of a nerve growth factor(NCF) by carrying out prescribed extracting and fractionating treatments of a fruit body or a mycelium of Dictyophora indusiata Fisch. SOLUTION: This eudesmane derivative of formula I or II. The derivative of formula I has the following physicochemical properties: molecular weight: 216 (C15 H20 O); solubility: soluble in benzene, chloroform, acetone and ethyl acetate and insoluble in water; color and property: colorless, amorphous, neutral, etc. Furthermore, the derivative of formula II has the following physicochemical properties: molecular weight: 250 (C15 H22 O3 ); solubility; soluble in benzene, chloroform, acetone and ethyl acetate and insoluble in water; color and property: colorless, amorphous, neutral, etc. The derivative of formula I or II is obtained by carrying out a suitable extracting treatment of a fruit body or a mycelium of Dictyophora indusiata Fisch. which is a mushroom belonging to the genus Dictyophora of the family Phallaceae.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an eudesman (eudesman) isolated from the fruiting bodies and mycelia of Dictyophora indusiata, a mushroom of the genus Phallaceae and genus Dictyophora.
e) A derivative and a nerve growth factor (NGF) production inducer containing the eudesmane derivative as an active ingredient.

[0002]

2. Description of the Related Art Conventionally, there have been several reports on compounds isolated from mushroom fruiting bodies and their drug effects. For example, kawaratake (Po
The ergosterol derivative isolated from the fruiting body of lyporus versicolor) has a cell-killing effect on liver cancer cells tetrahedron 39, 277.
9-2785 (1983). In addition, the mushroom mushroom (Agaricus blazei), which is a mushroom in the agaric family.
The ergosterol derivative isolated from the fruiting body of) has a cytocidal effect on cervical cancer cells. Phytochemistry 27, 2777-278
9 (1988). The same is reported in JP-B-48-6766, JP-B-55-71702, JP-B-58-62118, and the like.

[0003] As for the mushrooms belonging to the mushroom family Ganoderma lucidum, the octadecenoic acid derivatives, isoindolinone derivatives and phthalide derivatives isolated from the fruiting bodies of the mushrooms have a cytocidal effect on cervical cancer cells. -157347, JP-A-3-157367,
It is reported in JP-A-3-157379.

[0004]

However, except for Japanese Patent Application No. 6-234270, there is no report on a compound isolated from fruiting bodies or mycelium of Phellinus linteus and its drug effect.

[0005]

DISCLOSURE OF THE INVENTION
In view of the above circumstances, as a result of diligent research on the compound isolated from the fruiting body and mycelium of Phellinus linteus and its drug effect, when a predetermined extraction treatment and fractionation treatment are applied to the fruiting body and mycelium of Phellinus linteus, New Oidesman (eu
A desmane derivative has been isolated and the eudesm
It was found that the ane) derivative has a nerve growth factor (NGF) production-inducing effect.

That is, the present invention relates to an eudesmane derivative represented by the following formula 1 or formula 2 and a nerve growth factor (NGF) production inducer containing the eudesmane derivative as an active ingredient.

[0007]

(Equation 1)

[0008]

(Equation 2)

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The eudesmane derivative represented by the formula (1) or (2) can be obtained by treating fruit bodies and / or mycelia of Phellinus linteus as follows. First, raw or dried fruiting bodies of Phellinus linteus and /
Alternatively, the mycelium is subjected to extraction treatment with a homogeneous mixed solvent of water and a water-soluble organic solvent, solid-liquid separation is performed by filtration or centrifugation to obtain an extract, and the water-soluble organic solvent is evaporated from the extract to form an aqueous layer. To get As the homogeneous mixed solvent, 80-85% methanol aqueous solution, ethanol aqueous solution, 85% acetone aqueous solution or the like can be used. The extraction is usually performed at room temperature, but it may be heated under reflux, and the extraction time is usually 1 to 72 hours. For example, dried fruit bodies of Phellinus linteus are added to an 85% ethanol aqueous solution, homogenized, left to stand at room temperature for 24 hours, filtered to obtain an extract, and the extract is heated under reduced pressure at 40 to 45 ° C. Then, the water layer is obtained by evaporating the ethanol.

Next, the water layer is subjected to a liquid-liquid partition extraction process using a mixed solvent of water and a water-insoluble organic solvent, and the water-insoluble organic solvent layer is separated from the water-insoluble organic solvent layer. A dry solid is obtained by evaporating the water-insoluble organic solvent. In this case, chloroform, ethyl acetate, diethyl ether or the like can be used as the water-insoluble organic solvent. For example, ethyl acetate was added to the above aqueous layer, the mixture was shaken and allowed to stand, then the separated ethyl acetate layer was separated, and the ethyl acetate layer was concentrated under reduced pressure to 4
A dry solid is obtained by heating at 0 to 45 ° C and evaporating ethyl acetate.

The dry matter itself has a nerve growth factor (N
GF) is effective as a production inducer, but in order to remove impurities from the dried solid matter and enhance the effect of inducing nerve growth factor (NGF) production, the dried solid matter is subjected to chromatographic fractionation treatment,
The chromatographically fractionated product is further re-fractionated to isolate the desired eudesmane derivative.
In this case, benzene, chloroform, hexane, acetone, ethyl acetate,
Chloroform / acetone, chloroform / methanol,
Chromatographic fractionation can be performed by silica gel column chromatography using hexane / ethyl acetate as a mobile phase, thin layer chromatography, etc., and re-fractionation can be performed by high performance liquid chromatography using an ODS column. .

As will be described in detail in Examples, two kinds of compounds are isolated by the re-fractionation treatment as described above. Of the two compounds isolated, the physicochemical properties and the structural analysis results of the first compound are as follows. (1) Molecular weight: 216 (C ₁₅ H ₂₀ O) (2) Infrared absorption spectrum (cm ⁻¹ ): 1261, 16
64, 2925 (3) Nuclear magnetic resonance spectrum ( ¹ H-NMR, δ):
1.14 (3H, s), 1.76 (3H, s), 2.0
4 (3H, d, J = 0.99), 2.30 (1H, d,
J = 15.84), 2.37 (1H, d, J = 15.8)
4), 2.98 (1H, m), 4.79 (1H, s),
4.81 (1H, s), 5.85 (1H, s), 6.0
2 (1H, d, J = 2.64) (4) Nuclear magnetic resonance spectrum ( ¹³ C-NMR, δ): 2
0.3, 20.5, 23.7, 25.7, 36.0, 3
7.7, 45.3, 52.4, 110.8, 125.
7, 133.3, 141.1, 148.4, 153.
3, 199.2 (5) Solubility in solvent: benzene, chloroform,
Soluble in acetone and ethyl acetate, insoluble in water. (6) Basic, acidic, neutral: neutral (7) Color and properties: colorless, amorphous

From the above physicochemical properties and the results of structural analysis, it was determined that the first compound isolated was the eudesmane derivative represented by the formula 1.

The physicochemical properties and structural analysis results of the second compound of the two isolated compounds are as follows. (1) Molecular _{weight: 250 (C 15 H 22 0} 3) (2) Infrared absorption spectrum (cm ^-1): 887,103
1,1066, 1442, 1650, 2938, 340
5 (3) Nuclear magnetic resonance spectrum ( ¹ H-NMR, δ):
1.08 (3H, s), 1.30 (1H, m), 1.5
5 (2H, m), 1.70 (1H, m), 1.77 (3
H, m), 1.83 (1H, m), 2.05 (1H,
m), 2.10 (1H, d, J = 17.16), 2.6
0 (1H, m), 2.80 (1H, d, J = 17.1)
6), 4.30 (1H, dd, J = 13.51, 1.3
2), 4.52 (1H, dd, J = 13.51, 1.3
2), 4.79 (2H, s), 6.05 (1H, s) (4) Nuclear magnetic resonance spectrum ( ¹³ C-NMR, δ): 2
0.9, 22.1, 25.2, 32.9, 33.1, 3
9.4, 39.7, 48.3, 62.3, 73.1, 1
09.3, 125.3, 149.3, 161.0, 19
9.6 (5) Solubility in solvent: benzene, chloroform,
Soluble in acetone and ethyl acetate, insoluble in water. (6) Basic, acidic, neutral: neutral (7) Color and properties: colorless, amorphous

From the above physicochemical properties and structural analysis results, it was determined that the second compound isolated was the eudesmane derivative represented by the formula 2.

As will be described later in detail in the Examples, the eudesmane derivative of the present invention has an effect of inducing nerve growth factor (NGF) production, and the nerve growth factor (NG)
F) A compound having a production inducing effect has been attracting attention as a therapeutic agent for senile dementia.

[0017]

【Example】

Test Category 1 Extraction and isolation of eudesmane derivatives:
To 1200 g of dried fruiting bodies of Pleurotus cornucopiae, 5 liters of an 85% ethanol aqueous solution was added, homogenized, and allowed to stand overnight at room temperature, followed by filtration to obtain an extract. To the residue was added 5 liters of 85% ethanol aqueous solution, and the extraction treatment was performed in the same manner. The same extraction treatment was repeated a total of 5 times, and the extracts were combined. Then, the combined extracts were heated under reduced pressure at 40 to 45 ° C. to evaporate ethanol to obtain an aqueous layer.

2 l of ethyl acetate was added to the above aqueous layer,
After shaking and allowing to stand, the separated ethyl acetate layer was separated. Ethyl acetate (2 liters) was added to the residue, and liquid-liquid partition extraction treatment was performed in the same manner to separate the ethyl acetate layer. The same liquid-liquid partition extraction process was repeated three times in total, and the respective ethyl acetate layers were combined. The combined ethyl acetate layers were heated under reduced pressure at 40 to 45 ° C. to evaporate ethyl acetate, and further dried with a desiccator to obtain 20.2 g of a dry solid.

The dried product was dissolved in chloroform and subjected to silica gel column chromatography (carrier is Wakogel C-200, trade name, manufactured by Wako Pure Chemical Industries, Ltd.). On this occasion,
As a mobile phase, chloroform, chloroform / acetone = 9/1, 8/2, 6 / so that the polarities gradually increase.
4 (volume ratio), chloroform / methanol = 9/1, 8
/ 2 (volume ratio), 6 sections in total, 200 ml for each, 10 for each
A total of 12 fractions were obtained from 0 ml fractions.

The third fraction of the above total 12 fractions was further subjected to silica gel column chromatography (carrier is FL-60D, trade name, manufactured by Fuji Devison Chemical Co., Ltd.). At this time, 200 ml of benzene was used as the mobile phase and 50
A total of 8 fractions were obtained in ml. The second fraction was used as a mobile phase and was subjected to high performance liquid chromatography using methanol / water = 85/15 (volume ratio) (carrier was YM.
CR & D D-ODS-5-A, trade name, manufactured by YMC Co., Ltd., eudesmane represented by Formula 1
0.8 mg of the derivative was isolated.

The tenth fraction of the above total 12 fractions was further subjected to silica gel column chromatography (carrier is FL-60D, trade name, manufactured by Fuji Devison Chemical Co., Ltd.).
At this time, as the mobile phase, hexane / ethyl acetate = 9 /
A total of 2 fractions of 1,85 / 15 (volume ratio) were used in 200 ml portions, and 25 ml fractions were obtained in total of 8 fractions. 4th of these
The fraction was subjected to high performance liquid chromatography using methanol / water = 60/40 (volume ratio) as a mobile phase (carrier is YMC R & D D-ODS-5-A, trade name, manufactured by YMC), and the formula 2 was obtained. Eudesman shown (eudesm
1 mg of the ane) derivative was isolated.

Test Category 2 Eudesmane derivative nerve growth factor (N
GF) production inducing effect: Furukawa's method {biochemical
Biochemical and Biophysical Research Commu
nications), 136, 57-63 (1986)}, primary astroglial cells prepared from the cerebral cortex of late embryonic (19-day-old) rats were treated with Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum. It was cultivated aseptically. The culture was carried out for 1 to 2 weeks by changing the medium every 3 days. Upon reaching confluence, the medium was changed to DMEM containing 0.5% bovine serum albumin, and the cells were further cultured for several days to obtain a culture base. Separately, the eudesmane derivative isolated in Test Category 1 was dissolved in dimethyl sulfoxide, and the solution was changed to DMEM containing 0.5% bovine serum albumin.
Then, these were administered to the above culture base to prepare a total of two groups of administration groups. Each treatment group was treated with eudesm
The concentration of the ane) derivative was adjusted to 1 mM. For comparison, eudesmane derivative was not dissolved, but dimethyl sulfoxide alone was added to DMEM containing 0.5% bovine serum albumin, which was administered to the above-mentioned culture base to prepare a control group. Was prepared. After culturing a total of two groups of administration groups and one group of control groups for 24 hours, the culture solution was collected and the method of Furukawa et al. {Journal of Neurochemistr
y), 40, 734-744 (1983)}, and the nerve growth factor (NGF) concentration was measured by the enzyme assay method. A t-test was performed between a control group cultured without administration of the eudesmane derivative and each culture group cultured with 1 mM of the eudesmane derivative. As a result, each administration group was significantly tested as effective at a risk rate of 1%.

[0023]

As is apparent, the eudesmane derivative of the present invention described above is effective as a nerve growth factor (NGF) production inducer.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yukio Ishiguro 5-96 16 Higashimishima, Nishinasuno-cho, Nasu-gun, Tochigi Prefecture

Claims (3)

[Claims] 1. An eudesman represented by the following formula 1 (eu
desmane) derivative. (Equation 1) 2. An eudesman represented by the following formula 2 (eu
desmane) derivative. (Equation 2) 3. The eudesman (eu according to claim 1 or 2)
nerve growth factor (NG) containing a derivative of desmane as an active ingredient
F) Production inducer.