JPH0770133A - Cyathane derivative and nerve growth factor production inducing agent and antigbacterial agent containing the derivative as active component - Google Patents

Abstract

PURPOSE:To obtain a new cyathane derivative existing in the cultured mycelium of Hericium erinaceum. CONSTITUTION:The cyathane derivative expressed by the formula and existing in the cultured mycelium of Hericium erinaceum which is a kind of mushroom belonging to the genus Hericium, family Hydnaceae. The cyathane derivative can be produced by culturing mycelium of Hericium erinaceum in medium, separating the obtained cultured mycelium into supernatant and precipitated mycelium by centrifugal separation, extracting the cultured mycelium with homogeneous system consisting of water and an organic solvent, heating the extracted liquid to evaporate the organic solvent to obtain an aqueous phase, subjecting the aqueous phase to liquid-liquid partition extraction with a mixture of water and an organic solvent, collecting the organic solvent layer, evaporating the solvent and fractionating the obtained dry solid by chromatography.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to Hydaceae (Hydnaceae).
ae), a cyathane derivative contained in the cultured mycelium of Hericium erinaceum, a mushroom of the genus Hericium, and a nerve growth factor (NGF) production inducer and antibacterial agent containing the same .

[0002]

2. Description of the Related Art Heretofore, there have been a number of reports on compounds contained in mushroom fruiting bodies and their drug effects. For example, the mushrooms of the Sarcobidae family
The fruiting body of yporus versicolor) contains an ergosterol derivative, and the fact that the ergosterol derivative has a cell-killing effect on liver cancer cells is tetrahedron (Tetrahedron) 39, 2779-2785 (1).
983). The fruiting body of Agaricus blazei, which is a mushroom of the agaricid family, also contains an ergosterol derivative, and the ergosterol derivative has a cell-killing effect on cervical cancer cells. Phytochemistry Two
7, 2777-2789 (1988). The same thing is reported in Japanese Patent Publication No. 48-6766, Japanese Patent Publication No. 55-71702, Japanese Patent Publication No. 58-62118.

[0003] As for the mushrooms belonging to the mushrooms, Ganoderma lucidum, octadecenoic acid derivatives, isoindolinone derivatives and phthalide derivatives are contained in the fruiting bodies of the mushrooms, which have a cytocidal effect on cervical cancer cells. There are some cases in JP-A-3-157347 and JP-A-3-157347.
157367 and JP-A-3-157379.

[0004]

However, there are no reports on the compounds contained in the cultured mycelium of Pleurotus cornucopiae and their drug effects.

[0005]

However, the present inventors have
In light of the above-mentioned circumstances, as a result of diligent research on compounds contained in cultured mycelium of Yamabushitake and its drug effect, a new cyathane derivative having a specific chemical structure was found in the cultured mycelium of Yamabushitake. It was found that the cyathane derivative contained therein has a nerve growth factor (NGF) production inducing effect and an antibacterial effect.

That is, the present invention provides a cyathane derivative represented by the following formula 1 and the cyathane derivative.
nerve growth factor (NGF) containing ane) derivative as an active ingredient
It relates to production inducers and antibacterial agents.

[0007]

[Formula 1]

The cyathane derivative represented by the formula 1 can be obtained by treating the mycelium of Pleurotus cornucopiae as follows. First, the mycelium of Yamabushitake is SG
C medium (a liquid medium prepared by adjusting glucose, starch, corn steep liquor, KH ₂ PO ₄ , CuSO ₄ , ZnCl ₂ , thiamine hydrochloride and aqueous PH = 5.3 to 5.5) at 30 ° C. for 4 weeks Incubate. The obtained cultured mycelium is separated into a supernatant and a cultured mycelium that is a precipitate by centrifugation, and the cultured mycelium is extracted with a homogeneous system of water and an organic solvent. In this case, as a homogeneous system of water and organic solvent,
80-85% methanol, ethanol, 85% acetone and the like. The extraction is usually performed at room temperature, but may be heated under reflux, and the extraction time is usually 1 to 72 hours. For example, 8
Add the mycelium of Yamabushitake culture to 5% ethanol,
After homogenizing and letting this stand at room temperature for a whole day and night,
The extract is obtained by filtration, and the extract is under reduced pressure at 40 to 45 ° C.
The aqueous phase is obtained by heating at to evaporate the organic solvent.

Next, the aqueous phase is subjected to a liquid-liquid partition extraction process with a mixed system of water and an organic solvent to separate an organic solvent layer, and the organic solvent is evaporated from the organic solvent layer to obtain a dry matter. . in this case,
Examples of the organic solvent include chloroform, ethyl acetate, diethyl ether and the like. For example, ethyl acetate is added to the above aqueous phase, after shaking, the ethyl acetate layer separated by standing is separated, and the ethyl acetate layer is heated under reduced pressure at 40 to 45 ° C. to evaporate the ethyl acetate. To obtain a dry solid.

The dry matter itself is a nerve growth factor (N
It is effective as a GF) production inducer and an antibacterial agent, but in order to remove impurities from the dried solid matter to enhance its nerve growth factor (NGF) production inducing effect and antibacterial effect,
It is preferable to subject the dried solid matter to a chromatographic fractionation, and it is more preferable to further subject the chromatographically fractionated product to a re-fractionation treatment to isolate the desired cyathane derivative. In this case, as will be described later in detail in the Examples, it can be subjected to chromatographic fractionation using silica gel column chromatography or thin layer chromatography using chloroform / acetone, hexane / ether, etc. as a developing solvent, and ODS. Re-fractionation can be performed by high performance liquid chromatography using a column.

The physicochemical properties and the results of structural analysis of the compound thus isolated by the re-fractionation treatment are as follows. (1) Molecular weight: 434 (C ₂₅ H ₃₈ O ₆ ) (2) Infrared absorption spectrum: 3380, 2960, 2
870, 1376, 1170, 1062, 1041, 1
010 cm ^-1 (3) Nuclear magnetic resonance spectrum ( ¹ H-NMR, δ): 0.
96 (3H, d, J = 6.60), 0.97 (3H,
d, J = 6.60), 1.00 (3H, s), 1.02
(3H, s), 1.50 (2H, m), 1.52 (1
H, m), 1.63 (1H, m), 1.66 (2H,
m), 2.27 (2H, m), 2.32 (1H, m),
2.41 (1H, m), 2.48 (1H, m), 2.7
5 (1H, heptet, J = 6.60), 3.31 (1H,
dd, J = 10.56, 10.88), 3.41 (1
H, dd, J = 8.58, 8.91), 3.56 (1
H, dd, J = 8.91, 8.58), 3.63 (1
H, m), 3.89 (1H, d, J = 9.74), 3.
99 (1H, dd, J = 10.56, 5.40), 4.
00 (1H, d, J = 11.88), 4.31 (1H,
d, J = 11.88), 4.58 (1H, d, J = 8.
58), 4.81 (1H, br.d, J = 9.74),
6.01 (1H, br.d, J = 7.26) (4) Nuclear magnetic resonance spectrum ( ¹³ C-NMR, δ): 1
6.5, 21.4, 21.9, 24.5, 26.9, 2
7.8, 28.3, 28.4, 36.5, 38.1, 4
1.5, 42.6, 49.4, 66.0, 66.9, 6
9.9, 71.3, 73.3, 74.7, 79.8, 9
8.7, 135.7, 136.8, 139.5, 13
9.8 (5) Solubility in solvent: chloroform, acetone,
Ethyl acetate, soluble in methanol, slightly soluble in ethanol, insoluble in water (6) Distinction between basic, neutral, and acidic: neutral substance (7) Color and properties: colorless oil

From the above physicochemical properties and the results of structural analysis, the isolated compound is represented by the formula 1
thane) was determined to be a derivative.

As will be described later in detail in Examples, the cyathane derivative of the present invention is a nerve growth factor (NG).
F) A compound having a production-inducing effect and an antibacterial effect and having a nerve growth factor (NGF) production-inducing effect has been attracting attention as a therapeutic agent for senile dementia, and a compound having an antibacterial effect is used as a natural antibacterial agent. Attention has been paid to its use for food.

[0014]

【Example】

Extraction and isolation of cyathane derivative The mycelium of Pleurotus cornucopiae was cultured in SGC medium (previously described) for 4 weeks to obtain a cultured mycelium. 85% ethanol 5
700 g (wet weight) of the cultured mycelium was added to 1 liter, homogenized, allowed to stand at room temperature for 24 hours, and then filtered to obtain an extract. To the residue was added 5 liters of 85% ethanol and the same extraction treatment was performed to obtain an extract, which was combined with the first extract. Then, the combined extracts were heated at 40 to 45 ° C. under reduced pressure to evaporate ethanol to obtain an aqueous phase.

1 liter of ethyl acetate was added to the above aqueous phase,
After shaking, the mixture was allowed to stand and the separated ethyl acetate layer was separated. Ethyl acetate (1 liter) 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 operation was repeated 3 times, and the separated ethyl acetate layer was combined with the first ethyl acetate layer. The combined ethyl acetate layers were heated under reduced pressure at 40 to 45 ° C. to evaporate ethyl acetate, and then dried with a desiccator to obtain 7.0 g of a dry solid.

The dried product was dissolved in hexane and subjected to column chromatography using Wakogel C-200 (manufactured by Wako Pure Chemical Industries, Ltd.). At this time, as a developing solvent, chloroform / acetone =
Use 9: 1, 8: 2, 7: 3, 1: 1 for each 200 ml, 5
A total of 16 fractions of 0 ml were obtained. The chloroform / acetone = 1: 1 elution fraction was further subjected to silica gel column chromatography, and 80 ml of chloroform / methanol = 9: 1 was used as a developing solvent to obtain a total of 8 fractions of 10 ml. From the second fraction, 44.6 mg of the cyathane derivative represented by the formula 1 was isolated.

Nerve growth factor (NGF) production-inducing effect of cyathane derivatives Furukawa et al.'S method {Biochemical and Biophysical Research Communications
and Biophysical Research Communications), 13
6, 57-63 (1986)}, and cultured in early incubator (19-day-old) rat cortical astroglial cells in a Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (1-2 The medium was replaced every 3 days for a week), and when confluent was reached, the medium was changed to DMEM containing 0.5% bovine serum albumin and cultured for several days.
Here, a cyathane derivative dissolved in dimethyl sulfoxide (DMSO) was prepared with DMEM containing 0.5% bovine serum albumin to a predetermined concentration,
Was administered. After culturing for 24 hours, the culture solution was collected and the method of Furukawa et al. {Journal of Neurochemistry (Jou
rnal of Neurochemistry), 40, 734-744 (1
983)}, the nerve growth factor (NGF) concentration was measured by the enzyme assay method. Control group and cyathane (cyathane) cultured without administration of cyathane derivative
An ane) derivative was administered at 1 mM, and a t-test was carried out with a group cultured. As a result, the administered group was significantly tested as effective at a risk rate of 1%.

Antibacterial effect of cyathane derivative PH7.2 containing meat extract 1.0%, polypeptone 0.5%, sodium chloride 0.25%, agar 1.5%.
4 ordinary agar medium was prepared, 15 ml and 6 ml of each were dispensed into a test tube, and autoclaved (20 minutes at 121 ° C.) to perform autoclave sterilization. 15 ml of a medium subjected to pressure steam sterilization was aseptically flown into a sterilized petri dish and fixed on a flat surface to form a base layer. Separately, 6 ml of medium sterilized by autoclaving was dissolved, cooled to 55-60 ° C, and inoculated with 50 μl of Bacillus subtilis cultivated in 5 ml of liquid medium at 30 ° C for 16 hours with shaking, and mixed well. After that, this was used as a fungal layer and flowed onto the above-mentioned base layer to obtain an assay medium. On the assay medium, a 10 mg / ml solution of a cyathane derivative was soaked in a solution and a paper disk dried with a solvent was gently placed with tweezers, and incubated for 24 hours in a thermostatic chamber at 30 ° C. The diameter of
The antibacterial activity was determined by the following formula 2.

[Formula 2] Antibacterial activity (mm) = measured diameter of blocking circle (mm)
-Diameter of paper disc (8 mm) antibacterial activity is 3 mm, cyathane derivative was assayed to have antibacterial effect against Bacillus subtilis.

[0019]

As is apparent, the cyathane derivative of the present invention described above is effective as a nerve growth factor (NGF) production inducer and an antibacterial agent.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Hideki Sakamoto 47 Izaguchi, Nishinasuno-cho, Nasu-gun, Tochigi 12 12 (72) Yukio Ishiguro 5-96 Higashimishima, Nishinasuno-cho, Nasu-gun, Tochigi 19

Claims (3)

[Claims] 1. A cyanate represented by the following formula 1 (cyath
ane) derivatives. [Formula 1] 2. The cyathane according to claim 1.
A nerve growth factor (NGF) production inducer containing a derivative as an active ingredient. 3. The cyathane according to claim 1.
An antibacterial agent containing a derivative as an active ingredient.