JPH06305970A - Inhibitor for tumor promotion - Google Patents

Abstract

PURPOSE:To provide the inhibitor exhibiting remarkable inhibition of an expression of EBV genome, capable of being used as an inhibitor for viral genome, applicable to a treating and prophylactic preparation for viral and tumor diseases as an inhibitor for cancergenesis promotion and useful as a health food or functional food. CONSTITUTION:The inhibitor for cancergenesis promotion contains, as an active component, a sterol compound whose structural formula has cholestane and ergostane structure and/or stigmastane structure without having an unsaturated substituent at 24-position as an active component. The sterol compound having the cholestane structure in its structural formula, that is cholestanol, is an active component which is extracted from a polifera.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a carcinogenic promotion inhibitor containing a sterol compound having a specific skeleton in the structural formula as an active ingredient. More specifically, the present invention relates to a new carcinogenic promotion inhibitor which is excellent in safety and also has an inhibitory activity on a carcinogenic promoter such as skin cancer.

[0002]

2. Description of the Related Art Conventionally, various anticancer agents have been developed, and those chemically synthesized from natural products,
Furthermore, a wide variety of products, including those produced by genetic engineering techniques, have been proposed as having anticancer activity. In parallel with the development of such an anti-cancer agent, the elucidation of the carcinogenic mechanism has been promoted, and the center of gravity has been shifted to the development of an anti-cancer agent in accordance with the mechanism of this carcinogenic mechanism.

[0003] The knowledge about the process leading to the development of cancer up to now includes the steps of initiation in which normal cells undergo damage to the chromosome by the carcinogenic initiator and become potential tumor cells, and then the tumor cells are converted into tumor cells by the action of the carcinogenic promoter. The two-stage theory of carcinogenesis is generally accepted. Although various oncogenic promoters have been found in the studies to date, they are widely present in the environment surrounding humans, and it is difficult to completely prevent their ingestion. It has been shown that the inhibition of G.p.c. may be important for suppressing the onset of cancer. In such a situation, the development of an anticancer agent different from the conventional mechanism of action is desired, and in particular, it has an action of suppressing the onset of cancer, and the development of a highly safe anticarcinogenic promotion inhibitor is demanded. It was

Under these circumstances, in addition to the development of anti-cancer agents used as therapeutic agents for cancers, the practical development of anti-carcinogenic substances having a carcinogenic inhibitory activity has become an important issue. The need is also sought from a clinical perspective. For example, skin cancer, which causes carcinogenicity over a long period of time due to environmental factors, has been rare in Japanese people in the past, but has recently been on the increase, and various therapeutic methods using anticancer agents have been investigated. . However, there are not many cases that lead to complete cure, and there are safety problems such as side effects,
Recently, it has been considered effective to focus on the suppression of carcinogenesis and to examine means for preventing the development of cancer. Under such circumstances, studies have been conducted on substances that act on anti-carcinogenic promoters in marine organisms, but there are still few studies,
Sarcophytol A slightly derived from C. serrata mushrooms (Toshio Kuroki, Cancer Bioscience 3. Carcinogenesis and Cancer Cells P. 20, 1991), Fucosterol, 24-Methylenecholesterol, and Salingosterol from Wakame Rice are known. It's just being done. (Chemistry and Biology Vo
l. 29, No. 9 P. 598-603 (199
1))

[0005]

Therefore, the present invention is
Paying attention to the mechanism of chemical carcinogenesis, we developed an anti-carcinogenic promoter that is an important factor of its carcinogenesis-suppressing action in skin cancer and the like. It is also intended to be provided as something that can also be used. This invention provides a novel carcinogenic promotion inhibitor containing a sterol compound having a specific skeleton in the structural formula as an active ingredient.

[0006]

That is, the present invention is
The present invention was completed through the following studies, based on the discovery of the present inventors regarding the inhibition of carcinogenic promotion of sponge extracts. First, the inventor found that in the Raji strain, which is a cultured cell derived from Burkitt's lymphoma containing the EBV (Epstein-Barr virus) genome, most of the compounds that inhibit the expression of the EBV genome are mouse. We focused on the fact that it acts as an anti-carcinogenic promoter in a two-stage carcinogenesis experiment for skin cancer. Then, we searched for a virus / genome inactivating substance that inhibits the expression of EBV / genome from deep-sea marine organisms.

This method, which focuses on the inhibitory effect on the expression of the EBV genome, is a method in which a carcinogenic promoter TPA (tetradecanoylphorbol acetate) and n-butyric acid acting as a synergistic effect in the Raji strain culture system, and This is a method in which a test substance is added and cultivated, and detection is performed by an indirect fluorescent antibody method using an antibody derived from cells activated by TPA. This method is quick and quantitative, and in addition,
It is an excellent method in that it can detect trace amounts of active ingredients.

[0008] The inventors of the present invention, as a result of further study by this method, found an EBV / genome expression inhibitory action in a sponge extract. The sponge extract in the present invention is extracted with a hydrophilic solvent such as water or alcohol, and is further fractionated with various combinations of water, a hydrophilic solvent, and a hydrophobic solvent. The above-mentioned EBV / genome expression inhibitory action is remarkable for each of the extracted fractions. It was confirmed that the substance was cholestanol by searching for an effective substance in the extracted fraction.

As the extraction solvent, alcohols such as methanol, ethanol, propanol and isopropanol,
Appropriate substances such as ether, ester, chloroform and hexane can be used. For example, in this invention,
The methanol extract component of sponge can be exemplified as one in which hexane / water, chloroform / water and ethyl acetate / water systems were sequentially distributed to obtain an extract fraction of hexane, chloroform, ethyl acetate and water.

The origin of sponge is not particularly limited. Also, as the means for extraction, any appropriate means can be adopted including conventionally known means. In the carcinogenic promotion inhibitor of the present invention containing a sponge extract as an active ingredient, the active ingredient is the organic solvent fraction and the water-soluble fraction extracted by the organic solvent and water as described above, and a combination thereof. Is included.
The active ingredient in any of the active fractions was single and was cholestanol.

In this way, as an anti-carcinogenic promoter substance (EBV-EA expression suppressing substance) from glass sponge,
We identified cholestanol, a type of sterol. Cholestanol is about 50% EB at 1000 equivalents of TPA.
The inhibitory effect on V-EA expression was shown.

The inhibitor of the present invention is useful as an antiviral agent, a carcinogenesis inhibitor, etc., and can be used not only as a pharmaceutical formulation or a preventive formulation, but also as a health food or a functional food. Is also possible. Hereinafter, the present invention will be described in more detail with reference to examples.

[0013]

EXAMPLES The present invention will be described with reference to examples. Activity Test Method According to the procedure shown in FIG. 1, Raji cells were added with TPA and n-butyric acid, and then with a test substance obtained in the below-mentioned Example, and cultured at 37 ° C. for 48 hours. . In this case, the test substance was dissolved in water, ethanol or dimethylsulfoxide and added to the Raji culture medium.

After the completion of the culture, the EBV. Genome expression inhibitory activity was measured. Example 1 As a sponge raw material, New Zealand eastern sea area, depth 570
The glass sponge sampled in m was finely ground in a frozen state, and methanol extraction was performed according to the procedure shown in FIG. The obtained extract was filtered off and the solid was extracted again with methanol. The methanol extracts were combined and the solvent was distilled off under reduced pressure to obtain a methanol extract.

The extract was distributed between water and n-hexane, and the solvent in the hexane layer was distilled off to obtain a hexane fraction.
This hexane fraction was tested for EBV
-The genome expression rate was measured. The results are shown in Table 1.
As is clear from Table 1, it was confirmed that EBV / genome expression was significantly inhibited.

Example 2 The water distribution layer of Example 1 was partitioned with water and chloroform as shown in FIG. 2, and the solvent of the chloroform layer was distilled off to obtain a chloroform fraction. Using this, the EBV / genome expression rate was measured in the same manner. The results are shown in Table 1. As in Example 1, it was confirmed that EBV / genome expression was significantly inhibited.

Example 3 The water distribution layer from Example 2 was partitioned with water and ethyl acetate as shown in FIG. 2, and the solvent of the ethyl acetate layer was distilled off to obtain an ethyl acetate fraction. The EBV / genome expression rate was measured, and the results shown in Table 1 were obtained. It was confirmed that EBV / genome expression was inhibited.

Example 4 The water distribution layer of Example 3 was desalted and freeze-dried to obtain a freeze-dried powder. The EBV / genome expression rate was also measured for this. As a result, as shown in Table 1, inhibition of EBV / genome expression was also confirmed in this water-soluble fraction.

[0019]

[Table 1]

The n-hexane fraction of Example 1 was subjected to chromatography using silica gel (MS GEL SIL) as a carrier, and hexane: diethyl ether (85: 1).
5), hexane: diethyl ether (65:35), ethyl acetate, and methanol were successively eluted, and the most active fraction Fr. Got 1. This fraction was a fraction containing sterol as a main component. Fr. 1 was again subjected to the same silica gel chromatography, and hexane: diethyl ether (7
0:30), hexane: diethyl ether (60: 4)
0), hexane: diethyl ether (50:50), ethyl acetate and methanol were eluted in this order, and fractions showing activity Fr. Got 2. This Fr. 2 was further fractionated by high performance liquid chromatography using a silica gel column (Develosil 100-5) and hexane: ethanol (90:10) as a mobile phase. Fractions showing activity were collected and collected on Fr. It was set to 3. The action of each fraction is shown in Table 2.
This Fr. 3 was recrystallized using hot methanol to obtain colorless needle crystals. When the 1 H-NMR spectrum of this product was measured, it was almost the same as the spectrum of β-cholestanol. The main component of the three recrystallized products was identified as β-cholestanol. Furthermore, this product was acetylated with acetic anhydride, and as a result of electron impact ionization mass spectrometry, this product contained β-cholestanol as a main component,
It was suggested that the fraction contained ergostanol as an impurity.

Fr. In order to confirm that the main component of the 3 recrystallized product is β-cholestanol from its activity, a commercially available reagent β-cholestanol (GR-grade manufactured by Nacalai Tesque, Inc.) was purchased and their activities were compared. . As shown in Table 3, Fr. The 3 recrystallized product showed almost the same activity as commercial β-cholestanol.

As described above, from the glass sponge extract, E
Β-cholestanol was identified as a BV-EA expression inhibitor. Therefore, in order to examine the activity of other related sterols, 7 types of commercially available sterols (manufactured by Nacalai Tesque, Inc., SIGME CHEMICAL) were purchased and the activity was measured. The results are shown in Table 4. The sterols used in the test all showed an action at 1000 equivalents of TPA, and especially β-sitosterol and ergosterol were β-.
A stronger effect was observed than cholestanol. Table 5 shows these structures.

[0023]

[Table 2]

[0024]

[Table 3]

[0025]

[Table 4]

[0026]

[Table 5]

[0027]

As described in detail above, according to the present invention, EBV / genome expression is significantly inhibited, and
It can be used as a genome inactivating agent, can be applied as a carcinogenic promotion inhibitor, as a prescription or preventive prescription for antivirus or anticancer, or as a health food or a functional food.

[Brief description of drawings]

FIG. 1 is a method diagram showing a test method for EBV / genome expression inhibitory activity in vitro.

FIG. 2 is a method diagram showing an extraction / separation process as an example.

[Procedure amendment]

[Submission date] July 19, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Correction content]

[Claims]

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0010

[Correction method] Change

[Correction content]

The origin of sponge is not particularly limited. Also, as the means for extraction, any appropriate means can be adopted including conventionally known means. In the carcinogenic promotion inhibitor of the present invention containing a sponge extract as an active ingredient, the active ingredient is the organic solvent fraction and the water-soluble fraction extracted by the organic solvent and water as described above, and a combination thereof. Is included.

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] 0022

[Correction method] Change

[Correction content]

As described above, β-cholestanol was identified as an EBV-EA expression inhibitor in the glass sponge extract. Therefore, in order to examine the activity of other related sterols, 7 kinds of commercially available sterols (manufactured by Nacalai Tesque, SIGMA CHEMICAL) are available.
Was purchased and the activity was measured. The results are shown in Table 4. The sterols used in the test all showed an action at 1000 equivalents of TPA, and in particular, β-sitosterol and ergosterol were found to have a stronger action than β-cholestanol. Table 5 shows these structures and the structure of cholestanol.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0026

[Correction method] Change

[Correction content]

[0026]

[Table 5]

Claims (4)

[Claims] 1. A carcinogenic promotion inhibitor containing a sterol compound having a cholestane skeleton, an ergostane skeleton and / or a stigmastane skeleton having no unsaturated substituent at the 24-position in the structural formula as an active ingredient. 2. The carcinogenic promotion inhibitor according to claim 1, wherein the sterol compound having a cholestane skeleton in its structural formula is cholestanol. 3. The carcinogenic promotion inhibitor according to claim 2, wherein cholestanol is an organic solvent fraction of sponge extract. 4. The carcinogenic promotion inhibitor according to claim 2, wherein cholestanol is a water-soluble fraction of sponge extract.