JP2657883B2 - Sheatan derivative, nerve growth factor production inducer containing the same as active ingredient, and antibacterial agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention
ae), a cyathane derivative contained in a cultured mycelium of Hericium erinaceum, a mushroom of the genus Coricum (Hericium), a nerve growth factor (NGF) production inducer containing the derivative as an active ingredient, and an antibacterial agent .

[0002]

2. Description of the Related Art Conventionally, there have been several reports on compounds contained in fruit bodies of mushrooms and their drug effects. For example, the mushroom of the scrophulariaceae, Agaricus (Pol)
Yporusversicolor) contains an ergosterol derivative, which has a cytocidal effect on liver cancer cells. Tetrahedron 39, 2779-2785 (1)
983). The fruit body of Agaricus blazei, a mushroom belonging to the agaricaceae family, also contains an ergosterol derivative, and it has been shown that the ergosterol derivative has a cell killing effect on cervical cancer cells. 2
7, 2777-2789 (1988). The same is reported in JP-B-48-6766, JP-B-55-71702, JP-B-58-62118, and the like.

[0003] With respect to the mushrooms of the Agaricaceae family, the fruit bodies of Yamabushitake also include octadecenoic acid derivatives, isoindolinone derivatives, and phthalide derivatives, which have a cell killing effect on cervical cancer cells. There are some cases in Japanese Unexamined Patent Application Publication No.
157367 and JP-A-3-157379.

[0004]

However, there is no report on the compounds contained in the cultured mycelium of Yamabushitake and their drug effects.

[0005]

Means for Solving the Problems Thus, the present inventors have
In view of the above circumstances, as a result of intensive studies on the compounds contained in the cultured mycelium of Yamabushitake and their drug effects, a novel cyathane derivative having a specific chemical structure was found in the cultured mycelium of Yamabushitake. It has been found that the cyathane derivative 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 a cyathane derivative:
ane) Nerve growth factor (NGF) containing a derivative as an active ingredient
It relates to a production inducer and an antibacterial agent.

[0007]

(Equation 1)

The cyathane derivative represented by the formula 1 is obtained by treating the mycelium of Yamabushitake as follows. First, the mycelium of Yamabushitake was
4 weeks at 30 ° C. in C medium (glucose, starch, corn steep liquor, KH ₂ PO ₄ , CuSO ₄ , ZnCl ₂ , thiamine hydrochloride and aqueous pH = 5.3-5.5 liquid medium) Incubate. The obtained cultured mycelium is separated into a supernatant and a cultured mycelium as 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 an organic solvent,
80-85% methanol, ethanol, 85% acetone and the like. The extraction is usually carried out at room temperature, but it may be heated to reflux, and the extraction time is usually 1 to 72 hours. For example, 8
Add the cultured mycelium of Yamabushitake in 5% ethanol,
After homogenizing and leaving it at room temperature for 24 hours,
Filter to obtain an extract, and extract the extract under reduced pressure at 40-45 ° C.
To evaporate the organic solvent to obtain an aqueous phase.

Next, the aqueous phase is subjected to liquid-liquid distribution extraction 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 dried product. . in this case,
Examples of the organic solvent include chloroform, ethyl acetate, diethyl ether and the like. For example, adding ethyl acetate to the aqueous phase, shaking, and leaving to separate the separated ethyl acetate layer, heating the ethyl acetate layer under reduced pressure at 40 to 45 ° C. to evaporate the ethyl acetate. To obtain a dried product.

[0010] The above dried product is itself a nerve growth factor (N
GF) is effective as a production inducer and an antibacterial agent, but in order to remove impurities from the dried product to enhance its nerve growth factor (NGF) production inducement effect and antibacterial effect,
The dried product is preferably subjected to a chromatographic fractionation treatment, and more preferably, the chromatographically fractionated product is further fractionated to isolate a target cyathane derivative. In this case, as will be described later in detail in Examples, chromatographic fractionation can be performed using silica gel column chromatography or thin layer chromatography using chloroform / acetone, hexane / ether or the like as a developing solvent. Refractionation can be performed by high performance liquid chromatography using a column.

The physicochemical properties and structural analysis results of the compound thus isolated by the refractionation treatment are as follows. (1) Molecular _{weight: 432 (C 25 H 36 O} 6) (2) Infrared absorption spectrum: 3421,1691,1
616 cm ^-1 (3) Nuclear magnetic resonance spectrum ( ¹ H-NMR, δ): 0.1.
97 (3H, d, J = 6.59), 0.98 (3H,
d, J = 6.23), 0.99 (3H, s), 1.02
(3H, s), 1.50 (2H, m), 1.53 (2
H, m), 1.58 (2H, m), 2.28 (3H,
m), 2.75 (2H, m), 3.20 (1H, dd,
J = 8.42, 8.79), 3.35 (1H, dd, J
= 11.72, 10.62), 3.60 (1H, dd,
J = 8.79, 8.79), 3.69 (1H, m),
3.82 (1H, d, J = 9.71), 4.03 (1
H, dd, J = 11.72, 5.86), 4.58 (1
H, d, J = 8.42), 5.14 (1H, d, J =
9.71), 6.92 (1H, br.s), 9.56
(1H, s) (4) Nuclear magnetic resonance spectrum (< ¹³ > C-NMR, [delta]): 1
6.4, 21.4, 21.9, 24.4, 27.1, 2
7.7, 28.4, 29.7, 36.4, 38.1, 4
1.6, 41.9, 49.4, 66.9, 70.7, 7
0.9, 71.1, 74.9, 79.1, 98.5, 1
35.9, 140.2, 142.1, 155.4, 19
3.4 (5) Solubility in solvent: chloroform, acetone,
Soluble in ethyl acetate and methanol, slightly soluble in ethanol, insoluble in water (6) Basic, neutral and acidic: neutral substance (7) Color and properties: colorless oil

From the above-mentioned physicochemical properties and structural analysis results, the isolated compound is a siatan (cya) represented by the formula 1.
thane) derivative.

As will be described in detail later in the examples, the cyathane derivative of the present invention is a nerve growth factor (NG).
F) Compounds having a production inducing effect and an antibacterial effect, and having a nerve growth factor (NGF) production inducing effect, are attracting attention as therapeutic agents for senile dementia, and compounds having an antibacterial effect are considered as natural antibacterial agents. The use for food is drawing attention.

[0014]

【Example】

Extraction and Isolation of Cyathane Derivatives Mycelia of Yamabushitake were cultured in SGC medium (described above) for 4 weeks to obtain cultured mycelia. 85% ethanol 1
110 g (wet weight) of the cultured mycelium was added to 1 liter, homogenized, left at room temperature for 24 hours, and filtered to obtain an extract. One liter of 85% ethanol was added to the residue, and an extraction treatment was performed in the same manner to obtain an extract, which was combined with the first extract. Then, the combined extract was heated at 40 to 45 ° C. under reduced pressure to evaporate ethanol to obtain an aqueous phase.

[0015] 1 liter of ethyl acetate is added to the aqueous phase,
After shaking, the mixture was allowed to stand, and the separated ethyl acetate layer was separated. Ethyl acetate (1 L) was added to the residue, and liquid-liquid partitioning and extraction were performed in the same manner to separate the ethyl acetate layer, which was combined with the first ethyl acetate layer. The combined ethyl acetate layer was heated at 40 to 45 ° C. under reduced pressure to evaporate the ethyl acetate, and further dried in a desiccator to obtain 2.1 g of a dried product.

The above 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, chloroform / acetone was used as a developing solvent so that the polarity would gradually increase.
9: 1, 8: 2, 7: 3, 1: 1 with 60 ml each, 10
A total of 24 ml fractions were obtained. The fraction eluted with 9: 1 chloroform / acetone was further subjected to silica gel column chromatography, using 80 ml of chloroform / acetone = 7: 3 as a developing solvent to obtain a total of 8 fractions of 10 ml. From the second fraction, 31.9 mg of a cyathane derivative represented by the formula 1 was isolated.

Induction effect of cyathane derivative on nerve growth factor (NGF) production Method of Furukawa et al. {Biochemical and Biophysical Research Communications (Biochemical)
and Biophysical Research Communications), 13
6, 57-63 (1986)}, the primary embryonic astroglial cells of rat embryos (19 days old) were cultured in an incubator in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (1-2). The medium was replaced every three days for a week), and when the cells reached confluence, the cells were cultured for several days by changing to DMEM containing 0.5% bovine serum albumin.
A cyanate (cyathane) derivative dissolved in dimethyl sulfoxide (DMSO) was prepared to a predetermined concentration in DMEM containing 0.5% bovine serum albumin,
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) Nerve growth factor (NGF) concentration was measured by an enzyme assay according to｝. A control group cultured without administration of a cyathane derivative and a cyathane derivative
ane) A t-test was performed with a group cultured by administering 1 mM of the derivative. As a result, the administered group was significantly tested as effective at a 1% risk rate.

Antibacterial effect of cyathane derivative PH 7.2 containing meat extract 1.0%, polypeptone 0.5%, sodium chloride 10.25%, agar 1.5%
Prepare 7.4 normal agar medium and add 15 ml and 6
Dispense each ml and autoclave (121 ° C for 20 minutes)
Was subjected to steam sterilization under pressure. 15 ml of a medium sterilized under pressure and steam was aseptically introduced into a sterilized petri dish and fixed on a flat surface to form a base layer. Bacterium (Bacillus subtilis), yeast (Saccharomyces cerevisiae), and fungi cultured by shaking and separately cultivating 6 ml of a medium autoclaved and steam sterilized at 30 ° C. for 16 hours in 5 ml of a liquid medium and cooling to 55 to 60 ° C. (Aspergillus niger) was inoculated in an amount of 50 μl, mixed well, and then infused as a bacterial layer onto the above-mentioned base layer to obtain an assay medium. A paper disc in which a 10 mg / ml solution of a cyathane derivative was impregnated and the solvent was dried was gently placed on the assay medium with tweezers, and cultured for 24 hours in a constant temperature room at 30 ° C., and the inhibition circle formed. Was measured, and 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 1 against bacteria (Bacillus subtilis).
0 mm, 2 mm for yeast (Saccharomyces cerevisiae) and 4 mm for mold (Aspergillus niger), and the cyathane derivative is derived from these bacteria,
It was tested for its antibacterial effect against yeast and mold.

[0019]

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

 ──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Yukio Ishiguro 5-96-19 Higashimishima, Nishinasuno-machi, Nasu-gun, Tochigi Prefecture (56) References JP-A-1-67182 (JP, A) JP-A-7-70133 ( JP, A) Japanese Patent Publication No. 6-96520 (JP, B2) Japanese Patent Publication No. 8-26010 (JP, B2) Abstracts of Natural Organic Compounds Symposium (35th), 1993, P.A. 393-400, TETRAHEDRON LETTE RS, VOL. 35, NO. 10, (1994) p. 1569-1572

Claims (3)

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