JP2729638B2 - Virus / genome inactivator - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a virus / genome inactivating agent containing apigenin or chrysin as an active ingredient.

(Prior art and its problems) The two-stage theory of carcinogenesis, which is generally accepted as a carcinogenesis mechanism, consists of an initiator that damages the chromosome of normal cells and a cell that has been transformed into a potential tumor cell by the damage. It refers to an experimental system that produces a tumor by the action of a promoter that gives a synthetic effect and changes it into a tumor cell.

As an initiator, DMBA (dimethylbenzanthracene) or the like is known, and as a promoter, TPA (tetradecanoylphorbol acetate) or the like is known as a typical example. Research is underway to clarify the mechanism of carcinogenesis through carcinogenesis experiments using such promoters and initiators.

In particular, the promotion process promoted by a promoter is regarded as important in connection with the universality of oncogenes, and many attempts have been made to clarify this point to derive a new concept of anticancer properties. One of them is the search for an anti-tumor promoter that inhibits the action of a tumor promoter. So far, lipoxygenase inhibitors such as NDGA (nordihydroguaiaretic acid) and W
Calmodulin inhibitors such as -7, glucocorticoids, retinoids, and the like are known, but the search for such inhibitors is still in a late stage.

Attempts to search for anti-cancer promoters from nature have also been actively conducted, and it is known that crude extracts of plants suppress experimental carcinogenesis. Few.

On the other hand, in animal cell lines harboring the virus genome, the virus
A method for finding a substance that inhibits the expression of the genome and searching for an anti-tumor promoter using this as an index has also been developed.The method is excellent in that it is rapid and quantitative, and can detect trace amounts of active ingredients. is there.

(Object of the Invention) An object of the present invention is to search for a virus / genome inactivating substance that suppresses the expression of the virus / genome by TPA in a cultured cell line containing the EB virus / genome, and to obtain active ingredients so far. Based on the findings, the object is to provide these active ingredients as oncogene promoter inhibitors.

(Constitution of the Invention) The present inventors used Raji (Raji) strain, a cultured cell derived from Burkitt's lymphoma, which contains the genome of EBV (Epstein-Barr virus), and contained EBV contained in plants.
As a result of searching for a virus / genome inactivating substance that inhibits the expression of V. genome, it was found that a group of flavonoids inhibited the expression of virus and genome at a low concentration.

That is, the present invention is a virus / genome inactivating agent containing apigenin or chrysin as an active ingredient.

Plants containing apigenin include Satureia obovata, Acanthus ilicifolius, Globularia elongata, Artemisia assoana, and Polygonum hydropiper. (Pinus morrisonicola), Periploca nigrescens, Cotula cinerea (Cotu)
la cinerea), Hypericum perforatum (Hyperi)
cum Perforatum), Cartamas Glaucus (Carthamu)
s glaucus), Launaea tenuiroba (Launaea ten)
uiloba), Gutierrezia sa
rothrae), Betonica officinalis (Betonica offi)
cinalis, Passiflora incarnata (Passiflora in
carnata), Matricaria cham
omilla), Avena sativa, Haplormosia monophylla, Acacia farnesiana, Agrimonia eupatoria, Apium graveolens, Dahlia pinata (Dahlia pinata) bipinnatus), Fujimodoki (Daphne ge)
nkwa), Chrysanthem Morifolium (Chrysanthe)
mum morifolium) and Agrimonia pilos
a), Namban karamushi (Boehmeria nivea), Acacia ixiophylla, Inu saffron (Colchicum autumnale), Poplar (Populus nigr)
a), Onopordum acanthi
um), Achillea millefoliu
m), soybean (Glycine hispida), Valeria cristata, Ibuki (Juniperus chinens)
is), cornflower (Centaurea Cyanus), cornflower thistle (Centaurea iacea), lemon (Citrus limon),
Snapdragon (Antirrhinum majus) and the like are plants containing chrysin, such as Pinus morisonikola (Pinus mo
rrisonicola), Kenoposium Graveolence (Cheno)
podium graveolens), poplar (Populus nigra), great lizard (Cormus tschonoski), Orogishiram indicum (Oroxylum indicum), Pinus sibirica (Pinu)
s sibirica), Alnus sieboldian
a) and the like are known, and the extraction and production of apigenin or chrysin from the above plants can be achieved by a combination of known solvent extraction methods and various chromatographic methods.

For example, apigenin is obtained by extracting parsley leaves with methanol, partitioning the resulting extract with water and hexane, then with water and butyl acetate, subjecting the butyl acetate extract to silica gel column chromatography, and using a mixed solvent of hexane / ethyl acetate. And can be obtained from the fraction eluted.
Chrysin can also be extracted and isolated from plants containing them by the same operation.

When apigenin or chrysin is present as a glycoside in a plant, aglycone can be extracted with an acid or an enzyme at any stage of the isolation process.

Also, a plant extract containing apigenin or chrysin as an active ingredient can be used as a virus / genome inactivating agent.

For example, a plant extract containing apigenin can be used to remove a plant containing the compound from water, a hydrophilic organic solvent such as methanol, ethanol, propanol, butanol, isopropanol, acetone, methyl ethyl ketone, or a mixture thereof, or an acetate ester. It is obtained by using and extracting.

Furthermore, if necessary, a fraction in which the active ingredient apigenin is more concentrated can be obtained.

Therefore, the apigenin or chrysin of the present invention and a plant extract containing the same as an active ingredient can be used as a drug for inactivating a virus genome in the form of a composition containing an effective and non-toxic amount thereof. For example, as an oral preparation, it can be used as a solid preparation such as tablets, capsules, troches, granules, powders and the like, or a liquid preparation such as solutions and syrups. These various preparations can be produced by a known method using a conventional inorganic or organic, or solid or liquid carrier for a pharmaceutical preparation.

In addition, the apigenin or chrysin of the present invention and a plant extract containing the same as an active ingredient inhibit the expression of the EB virus genome, and thus may be used as an antiviral agent and an anticancer agent.

Furthermore, it has been shown that the action of inhibiting the action of a tumor promoter such as TPA can also be expected to prevent cancer, and apigenin or chrysin and a plant extract containing the same as an active ingredient are used as cancer preventive drugs and health foods. Application as a functional food is also possible.

On the other hand, the effect of inhibiting the expression of the EB virus genome is observed by the following method.

That is, in the Raji strain culture system described above, TPA, which is a tumor promoter, and n-butyric acid, which acts as a synergistic effect for the expression of activity, and a test substance are added and cultured, and activated by TPA and expressed on the cell surface. This is a method for detecting EBV-EA (EB / virus-early antigen) by an indirect fluorescent antibody method using an antibody derived from the serum of a nasopharyngeal carcinoma patient.

(Examples) Next, the expression inhibitory activity of apigenin or chrysin on the virus genome will be described by examples and comparative examples.

[Example 1] As a culture solution of human lymphoblastoid cell line Raji cells latently infected with EBV, a culture solution obtained by adding fetal serum and antibiotics to RPMI1640 was used. Under these culture conditions, the natural expression rate of EBV-EA is
0.1% or less. Raj adjusted to a concentration of 1 × 10 ⁶ cells / ml
The i-cells were incubated at 37 ° C. in the above culture medium containing 4 mM n-butyric acid, 20 ng / ml TPA, and 100 times the molar amount of apigenin relative to TPA.
Cultured for 48 hours. EBV-EA was stained by the indirect fluorescent antibody method using the serum of a nasopharyngeal carcinoma patient, and the percentage of positive cells was calculated with respect to a control without apigenin, to determine the virus / genome expression inhibitory activity. The activity of apigenin to inhibit the expression of the virus genome was 62.2%.

[Example 2] 4 mM n was added to the Raji cell culture solution prepared in the same manner as in Example 1.
-Butyric acid, 20 ng / ml of TPA and butyl acetate extract of parsley were added and incubated at 37 ° C for 48 hours. The expression inhibition activity of the virus genome was calculated in the same manner as in Example 1. As a result, at a concentration of 10 μg / ml, the butyl acetate extract of parsley showed an inhibition activity of 50% or more.

Example 3 The concentration of the test compound was changed in the same manner as in Example 1,
Table 1 shows the results of investigating the activity of various flavonoids to inhibit the expression of the virus genome.

Comparative Example Table 1 shows the results when the hydroxyl group was replaced with an ether bond in the general formula. The activity is significantly reduced.

(Effect of the Invention) As described above, apigenin or chrysin of the present invention is TP
Since A significantly inhibits the expression of EBV-EA by A, it can be used as a drug aimed at inactivating the viral genome.

That is, it can be used as an antiviral agent and an anticancer agent, and can also be applied as a cancer preventive drug, a health food, and a functional food.

Claims (1)

(57) [Claims] 1. A virus / genome inactivating agent comprising apigenin and chrysin as active ingredients.