JPH10265376A - Inhibitor for activation of carcinogenic virus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject medicine capable of inhibiting activation of a carcinogenic virus as an anticarcinogenic promotor by including capsanthin and/or its fatty acid ester. SOLUTION: This medicine contains capsanthin extracted and isolated from the fruit of plants belonging to Capsium genus and/or its fatty acid ester such as a fatty acid monoester or a fatty acid diester, as an active principle. The capsanthin fatty acid ester is at least one kind selected from a group of palmitic acid ester, lauric acid ester and myristic acid ester, and has a inhibiting effect on the activation of the Epstein-Barr virus genome. Those compounds have an anticarcinogenic and preventive effect and an antivirotic effect, and are utilized as pharmaceuticals, cosmetics, health foods, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an oncogenic virus activation inhibitor.

[0002]

BACKGROUND OF THE INVENTION Cancer is the number one cause of death in Japanese people.
Prevention of carcinogenesis has become a very important issue for the health of the population. It is generally believed that cancer arises through two distinct phases, initiation and promotion. That is, initiators such as ultraviolet rays, radiation, and mutagens cause damage that cannot repair the DNA of normal cell genes (initiation), and the resulting potential cancer cells are repeatedly stimulated by chemicals called promoters. Being (promotion) cancerous. Therefore, cancer can be prevented by blocking either the initiation or promotion process.

However, it is impossible to completely remove the initiator because ultraviolet rays, radiation, and various mutagens which act as the initiator are present in the environment. In addition, since it is considered that an adult already has an initiated cell, inhibition of the initiation process is not effective from the viewpoint of preventing carcinogenesis. On the other hand, since the promotion process is long-term, inhibiting this process is considered to be an effective method for preventing carcinogenesis.

[0004]

From the above viewpoints, attempts have been made to search for an antitumor promoter, and it has been reported that many plant extracts and their components act as antitumor promoters. It is desired to provide a safe plant extract derived from an edible plant having a virus activation inhibitory action. In addition, examples have been reported in which crude extracts of many plants such as Huangbai can suppress experimental carcinogenesis, and their active ingredients are being elucidated as exemplified by β-carotene.

[0005] An object of the present invention is to provide a novel oncogenic virus activation inhibitor having an oncogenic virus activation inhibitory action as an anti-carcinogenic promoter.

[0006]

As a primary screening test for an anti-tumor promoter, there is an Epstein-Barr virus genome activation suppression test. In this test method, tetradecanoyl phorbol acetate (hereinafter abbreviated as TPA) is used as a promoter in a culture system of Raji cells, which are cultured cells derived from Perkit's lymphoma containing the Epstein-Barr virus genome. It is based on the suppression of viral genome expression. This method is rapid and quantitative, and allows for the detection of active substances in trace amounts.

[0007] Epstein-Barr virus is a virus belonging to the family Herpesviridae and is considered to cause Parkin's lymphoma and nasopharyngeal carcinoma. The virus is not only detected in these cancer patients, but is extremely ubiquitous and universal in humans worldwide, and it is said that almost all adults are infected with the Epstein-Barr virus. ing. Epstein-Barr virus has been shown to blast into normal human B lymphocytes as an infection target, conferring infinite proliferative potential on it, and has been shown to provide humans with a built-in oncogenic potential for B lymphocytes. Defined as a resident virus factor.

[0008] The inventors of the present invention have conducted systematic studies and experiments on a large number of microorganisms and plants using the Epstein-Barr virus genome activation inhibitory effect as an index. As a result, capsanthin, a kind of carotenoid, particularly its fatty acid ester Have obtained a compelling finding that they have a strong inhibitory effect on Epstein-Barr virus genome activation, and thus completed the present invention. That is, the present invention of claim 1 is an oncogenic virus activation inhibitor containing capsanthin as an active ingredient. The invention of claim 2 is an oncovirus activation inhibitor comprising a fatty acid ester of capsanthin as an active ingredient.

Further, the present inventors have studied the structure of capsanthin fatty acid ester and found that, in addition to the capsanthin fatty acid monoester, especially the capsanthin fatty acid diester has a stronger inhibitory effect on Epstein-Barr virus genome activation. I have gained knowledge that I need to fight. The invention of claim 3 is an oncovirus activation inhibitor comprising a fatty acid monoester of capsanthin as an active ingredient. The invention according to claim 4 is an oncogenic virus activation inhibitor comprising a fatty acid diester of capsanthin as an active ingredient.

Further, as a result of a detailed study of the action of the fatty acid ester of capsanthin on the activation of the Epstein-Barr virus genome, it was found that esters of palmitate, laurate and myristic acid exerted the action of inhibiting the activation of anticancer virus. I found something to do. The invention of claim 5 is the oncogenic virus activation inhibitor according to claims 2, 3 or 4, wherein the fatty acid ester of capsanthin is at least one of palmitic acid ester, lauric acid ester and myristic acid ester.

[0011]

DETAILED DESCRIPTION OF THE INVENTION Capsanthin and its fatty acid esters can be isolated from the fruits of plants of the genus Capsicum. Among them, paradicsom pap
rika) contains a large amount of capsanthin and the like, and those separated therefrom can be suitably used. In the present invention, capsanthin and its fatty acid ester contained in an extract obtained by extracting paradicom paprika with acetone or the like are preferable. However, the present invention is not limited to these, and there is no problem whether it is extracted from other plants or a synthetic product. The extraction method is not particularly limited, and another extraction solvent may be used.

The above paradicom paprika (paradics)
om paprika) is a vegetable native to Hungary.
Its original seed is clover-shaped, deep red, has a glossy and waxy surface, and has the property of not losing color when heated. In addition, the pulp is thick, has a high sugar content, and has no green odor. In addition, as an example of the component analysis, vitamin A potency (IU) is 780, vitamin B ₂ is 0.23 mg, vitamin C is 189 mg, and iron is 0.62 per 100 g of edible portion.
mg. Paradigm paprika (pa)
radicsom paprika) refers to the original species of paradigm paprika, and in the present invention, a variety that has a particular fruit among the original species is repeatedly selected and fixed to improve the reliability of fertility. (Hereinafter referred to as pure strains). In addition, pure type,
It is classified into scientific name Capsicum annuumL.var.grossum by taxonomy. In addition, a species obtained by crossing any of the large bell type, the Pimento type, the Hungarian paprika type, or the large neapolitan type peppers with such a pure paradigm paprika (hereinafter referred to as F1 type).
And F1 species (hereinafter referred to as quaternary hybrids) and reverted species of these hybrids (hereinafter F2)
Seeds. ) Is defined. In other words, the term "paradigm paprika" as used in the present invention means, in addition to the original species, pure strains, paradigm paprika of F1, quaternary hybrids, F2 hybrids, and hybrids thereafter.

The present inventor separated the extract by liquid chromatography and analyzed the components in order to examine the structures of capsanthin and its fatty acid ester contained in the above plant extract, and found that the hexane extract contained carotenoids. It was found that a large amount of capsanthin, a fatty acid monoester of capsanthin, and a fatty acid diester of capsanthin were contained.

[0014] Capsanthin and fatty acid esters of capsanthin isolated from these plant extracts exhibited a strong inhibitory effect on oncogenic virus activation. Therefore, since these carotenoids inhibit the expression of the Epstein-Barr virus genome, they exert carcinogenesis preventive effects and antiviral effects. Therefore, they can be widely used in the fields of pharmaceuticals, cosmetics, health foods and the like from the viewpoint of cancer prevention.

The Epstein-Barr virus
The effect of inhibiting the expression of the genome was observed by the following method. That is, the above-mentioned raji cell culture system is cultured by adding TPA, which is a carcinogenesis promoter, and n-butyric acid, which has a synergistic action for expressing the activity, and a non-detectable substance, and activated by TPA to be expressed on the cell surface. Epstein-Barr virus early antigens detected were detected by indirect immunofluorescence using antibodies from nasopharyngeal carcinoma patients.

[0016]

EXAMPLES Capsanthin or a fatty acid ester thereof is extracted by the following procedure as an example. In the extraction of the present embodiment, the aforementioned paradicom paprika (paradics
ompaprika) (scientific name Capsicum annuum L.var.g
rossum). That is, 800 g of the edible portion of paradicom paprika was cut, immersed in acetone, allowed to stand in a dark place at room temperature, and permeated occasionally to obtain a red acetone extract. Acetone was added again to the extraction residue, and the same operation was repeated three more times to collect an acetone extract. The extract was concentrated under reduced pressure to dryness to obtain an acetone extract. In addition, a methanol extract extracted using methanol instead of acetone was also produced. Hexane was added to these to dissolve and hexane-soluble fractions were collected to obtain a hexane extract. This was concentrated under reduced pressure to obtain a hexane extract.

Further, as a result of quantifying these extracts, the edible portion of paradicom paprika was changed from 800 g to 120 mg.
It has been found that carotenoids can be extracted as an extract. In order to analyze these components, hexane extract was subjected to column chromatography using silica gel as an adsorbent, and 40 mg of a fatty acid diester of capsanthin and 40 mg of a hexane-ether (5: 2) were eluted with a fraction eluted with hexane-ether (8: 2). 24 mg of the fatty acid monoester of capsanthin was separated from the fraction eluted in 5), and 12 mg of capsanthin was separated from the fraction eluted with ether-acetone (8: 2).

Using this capsanthin, a fatty acid monoester of capsanthin and a fatty acid diester of capsanthin, the expression inhibitory activity of the Epstein-Barr virus genome was measured under the following conditions. A culture solution of Epstein-Barr virus latently infected human lymphoblastoid cell line and Raji cells was prepared by adding fetal serum and antibiotics to PRMI1640. Under these culture conditions, the spontaneous incidence of Epstein-Barr virus early antigen is 0.
1% or less. Raji cells adjusted to a concentration of 1 × 10 ⁶ cells / ml were treated with 4 mM n-butyric acid, 20 ng / ml T-cell.
PA and 1000mol ratio / TPA (20ng = 32pmol / ml)
The cells expressing the Epstein-Barr virus early antigen were detected by indirect immunofluorescence using serum of a nasopharyngeal carcinoma cultured at 37 ° C. for 48 hours in the above culture medium to which the test substance was added. Was calculated with respect to a control to which no test substance was added, and was defined as a virus / genome reproduction inhibitory activity. Further, the concentration of the test substance is set to 500 mol ra
tio / TPA (20 ng = 32 pmol / ml), 100 mol ratio / TPA (20 n
g = 32 pmol / ml) and 10 mol ratio / TPA (20 ng = 32 pmol / ml). Table 1 shows the results.

[0019]

[Table 1]

The capsanthin, the fatty acid monoester component of the capsanthin and the fatty acid diester component of the capsanthin extracted from paradicom paprika exhibited a strong oncogenic virus activation inhibitory activity almost similar to that of β-carotene, which is known as an antitumor promoter. . Among them, the fatty acid diester component of capsanthin was found to have a stronger inhibitory effect on carcinogenic virus activity than β-carotene. In addition, the raji cell survival rate was maintained at 70%, and it was found that there was almost no toxicity to cells.

The capsanthin used in the present invention is a compound having a structure in which R ₁ and R ₂ are H atoms in the following structural formula. The extract obtained from the extract of Paradichom paprika of the present invention contains derivatives of fatty acid monoesters and diesters of capsanthin.
Fatty acids or H atoms are bonded to ₁ and R ₂ respectively. Capsanthin, a fatty acid monoester of capsanthin and a fatty acid diester of capsanthin were prepared by UV-VI
S, mass spectrum (hereinafter abbreviated as MS), ¹ H
The structures were identified by NMR and ¹³ C NMR.

[0022]

Embedded image

FIG. 1 shows MS data of capsanthin, FIG. 2 shows ¹ H NMR data of capsanthin, and FIG. 3 shows ¹³ C NMR data of capsanthin.

The fatty acid monoester of capsanthin is MS
From the results of NMR and NMR, it was found that the compound was capsanthin-3′-ester and R ₁ was H. FIG. 4 shows MS data of the fatty acid monoester of capsanthin, FIG. 5 shows ¹ HNMR data of the fatty acid monoester of capsanthin, FIG.
Shows ¹³ C NMR data of a fatty acid monoester of capsanthin. Constituent fatty acid and the composition ratio of R ₂ to find from these data are shown in Table 2.

[0025]

[Table 2]

The fatty acid diester of capsanthin was identified as a constituent fatty acid from the results of MS and NMR. However, these data alone cannot identify the constituent fatty acids of R ₁ and R _2, but merely indicate a combination. FIG.
Is the MS data of the fatty acid diester of capsanthin, FIG.
Shows the ¹ H NMR data of the fatty acid diester of capsanthin. R ₁ and R ₂ found from these data
Combinations of the constituent fatty acids and their composition ratios are as shown in Table 3.

[0027]

[Table 3]

As shown in Table 1, the fatty acid esters of capsanthin have a high anticancer activity. Among them, as shown in Tables 2 and 3, these are largely due to esters of myristine, lauric acid, and palmitic acid. Furthermore, it is clear that the composition ratio of those containing myristic acid as a constituent fatty acid is large. That is, among the capsanthin fatty acid esters exhibiting an anti-cancer activity, the above three fatty acids are preferable, and myristic acid having a large composition ratio is most suitable, and is considered to be highly active.

Further, in order to clarify the effect of suppressing carcinogenesis,
A skin cancer suppression test using mice was performed. That is,
The mouse skin two-stage carcinogenesis inhibition test was performed under the following conditions.

A group of 15 ICR female mice (6 weeks old)
24 hours after shaving the back body hair of 7,12-dimethylbenz [α] anthracene (hereinafter abbreviated as DMBA) dissolved in acetone (0.1 ml) on the back skin.
(100 μg, 390 nmol) was applied for initiation, and one week later, each experimental group was treated as follows. Group 1: TPA (1 μm) dissolved in acetone (0.1 ml)
g, 1.7 nmol) twice a week for 20 weeks. At this time, acetone (0.1 ml) is applied to the same site one hour before the application of TPA (positive control group). Groups 2 to 4: Test samples (capsanthins) dissolved in acetone (0.1 ml) twice a week for 20 weeks, 1 hour before application of TPA (1 μg, 1.7 nmol), as in group 1 85 nmol (separated by skipping the solvent of methanol extract) is applied. The test samples were group 2: capsanthin, group 3: capsanthin monoester, group 4
Group: Capsanthin diester.

Start of promotion by TPA application 20
Until the week after, papillomas generated on the backs of the mice were observed every week, and the ratio of mice having papillomas generated and the average number of papillomas generated per mouse were evaluated in comparison with the positive control group. The results are shown in FIGS.

From the test results shown in FIG. 9, it was found that in the positive control group, the first tumor was formed on the 7th week of the promotion and the tumor was formed on all mice on the 11th week, whereas the second group (capsanthin-treated) was formed. In group 7, the formation of tumor was observed only in week 9, and in group 3 (capsanthin monoester-treated group) and group 4 (capsanthin diester-treated group), tumor formation was delayed only at week 9. It became clear. In addition, even after 20 weeks at the end of the test, no tumor formation was observed in 13.3% of the mice in the fourth group.

From the results shown in FIG. 10, the average number of tumors per mouse was 20 weeks later, and that in the positive control group was 9.
While the number was one, the number was 7.2 in the second group, 6.5 in the third group, and 5.0 in the fourth group.
3%, 31% and 45% of the carcinogenesis inhibitory effects were observed.

[0034]

According to the present invention, it is possible to provide an excellent anti-Epstein-Barr virus genome inactivating agent of natural origin. Therefore, the novel anti-Epstein-Barr virus genome inactivator according to the present invention can be expected to have anti-carcinogenic and anti-viral effects, and can be used in a wide variety of applications depending on the fields of pharmaceuticals, cosmetics, and health foods. Becomes

[Brief description of the drawings]

Fig. 1 MS data of capsantin

FIG. 2 ¹ H NMR data of capsanthin

FIG. 3 ¹³ C NMR data of capsanthin

FIG. 4 MS data of fatty acid monoester of capsanthin

FIG. 5. ¹ HNM of fatty acid monoester of capsanthin
R data

FIG. 6 ¹³ CNM of fatty acid monoester of capsanthin
R data

FIG. 7: MS data of fatty acid diester of capsanthin

FIG. 8 ¹ H NMR of fatty acid diester of capsanthin
data

FIG. 9 is a graph showing the relationship between the elapsed weeks after the start of the experiment and the proportion of papilloma-bearing mice.

FIG. 10 is a graph showing the relationship between the elapsed weeks after the start of the experiment and the number of papillomas generated in mice.

Continuation of front page (72) Inventor Harukun Tokuda 3rd, Shimogamokitaencho, Sakyo-ku, Kyoto-shi, Kyoto (72) Inventor Yoshihiro Ito 37-3 Takaya Takeyacho, Sakyo-ku, Kyoto-shi, Kyoto

Claims (5)

[Claims] 1. An oncovirus activation inhibitor comprising capsanthin as an active ingredient. 2. An oncovirus activation inhibitor comprising a fatty acid ester of capsanthin as an active ingredient. 3. An oncovirus activation inhibitor comprising a fatty acid monoester of capsanthin as an active ingredient. 4. An oncogenic virus activation inhibitor comprising a fatty acid diester of capsanthin as an active ingredient. 5. The oncogenic virus activation inhibitor according to claim 2, wherein the fatty acid ester of capsanthin is at least one of palmitic acid ester, lauric acid ester and myristic acid ester.