JP2562092B2 - Mutagen inhibitor - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to 3,4 ', on mutagenicity inhibitor containing 5 trihydroxystilbene.

[0002]

2. Description of the Related Art Conventionally, many studies have been carried out on a carcinogenic inhibitory substance consisting of an extract extracted from a plant,
As an extract from a monocotyledonous plant, a plant of Gramineae plant, Liliaceae plant, or Iridaceae plant origin is known. For example, in the origin of the Liliaceae plant, Cape Aloe leaves from South Africa (Japanese Patent Publication No. 46-28092), garlic or onions (Japanese Patent Laid-Open Nos. 48-98010 and 48).
-98011), which originates from the plant of the genus Tritonia, in the case of the origin of the iris family (JP-B-54
No. 28806) is known. In addition to this,
Ingredients extracted from medicinal carrot (JP-A-55-334)
56), an extract of Hiyodoribana belonging to the Asteraceae plant (JP-A-55-45652), a component extracted from Achacha-zuru (JP-A-58-59921), and a component contained in Psycho (Shosho Sho). 62-187408) and other extracts containing saponins are known as substances having an anti-cancer effect.

As a stilbene derivative having an antitumor effect, 4,4'-bis (13.5-triazyl-6) is used.
-Amino) stilbene-2,2'-disulfonic acid derivative is known (Japanese Patent Publication No. 46-15503).

[0004]

However, the above-mentioned stilbene derivative has a very complicated structure, and its production is also difficult, and the appearance of a substance having a mutagen inhibitory effect that can be produced or extracted more easily has not been revealed. preferable. The present invention has been made in view of the above viewpoints, and an object thereof is to provide a mutagen inhibitor having a simple structure and an excellent substance .

[0005]

The present inventors have completed the present invention as a result of various studies on substances having a mutagen suppressing action. That is, mutagenicity inhibitor of the present invention, 1 to show 3-4'-5-trihydroxystilbene (hereinafter, simply referred to as a trihydroxystilbene.) Of features that you contain. Here, "consisting of trihydroxystilbene" is used to mean not only trihydroxystilbene simple substance but also the case of containing trihydroxystilbene (eg, solution, mixed powder containing other powders, etc.). It Usually, a solution as it is is used, or a solution prepared by dissolving powder or the like in a predetermined solvent immediately before use is used.

[0006]

[Chemical 1]

[0007]

The present invention will be described below in detail with reference to examples. In this example, each sample (S) having the sample concentrations shown in Table 1 and the following was prepared, and addition of this sample caused induction of the SOS reaction by a certain amount of mutagen (activated T _rp -P-1). I investigated how much it was suppressed. This inhibitory effect was examined by a mutagen detection method (umu test) using the induction of SOS reaction as an index. Here, the "umu test" is a new type of mutagen detection test that utilizes the SOS reaction that is observed during DNA damage in Escherichia coli. This test method is described below, and has many advantages such as results in a short time. That is, 3 in LB medium (tryptone 1%, yeast extract 0.5%, salt 0.5%)
Test bacterium solution cultured overnight at 7 ° C (test bacterium Salmone
lla typhimurium TA1535 / pS
K1002) in TGA medium (tryptone 1%, sodium chloride 0.
5%, glucose 0.2% and ampicillin 50 μg /
1/50 amount was inoculated into the mixture (added in the amount of ml) and cultured at 37 ° C. with shaking.

When the bacterial concentration reaches the logarithmic growth phase (A ₆₀₀ is 0.25 to 0.30), 2.0 ml of the bacterial solution is added.
Take each into a test tube and add 0.1 ml of mutagen (activated T _rp -P-1 prepared as shown below),
0.1 ml of each sample, 0.1 M phosphate buffer (pH 7.
0) 0.3 ml was added and cultured at 37 ° C. for 2 hours. After culturing, the bacterial solution was spun down to collect the cells, and then the bacterial cells were resuspended in physiological saline.
-Galactosidase activity was measured. Here, the logarithmic growth phase is the time when the number of bacteria and cells increases logarithmically, and is also called the exponential growth phase.

The β-galactosidase activity is measured by Mi
Leller's method (Miller, J .: Experim
ents in molecular genetic
s, Coldspring Harbor Labor
atory, New York, P352-355 (19)
72)). That is, 2.25 ml of Z buffer
After adding 0.25 ml of the above test bacterial solution to the
odium dodecyl sulphate aqueous solution 5
0 μl and 10 μl of chloroform were added and vigorously stirred.
Substrate (o-nitro-phenyl-β-D) was added to the solution.
-Galactopyranoside 4 mg / m
l) 0.25 ml was added and reacted at 28 ° C. Then, after 15 minutes, 1.25 ml of 1 M Na ₂ CO ₃ was added to stop the reaction, and A ₄₂₀ , A ₅₅₀ and A were measured by a spectrophotometer.
₆₀₀ (absorbance) was measured.

Here, the β-galactosidase activity value is
It was calculated by the following formula. β-galactosidase activity value (unit) = 1000 (A ₄₂₀ −1.75 × A ₅₅₀ ) /1.5×A ₆₀₀ The SOS reaction inhibition rate was calculated by the following formula. SOS reaction inhibition rate (%) = [1- (A−C) / (B−C)] × 100 (%) However, A in the above formula is β− when the sample (S) is added to the mutagen. The galactosidase activity value, B the β-galactosidase activity value induced only by the mutagen, and C the control β-galactosidase activity value, respectively.

The sample concentration is 10 mg / sample of the above sample (S).
Dissolved in pure water to a concentration of ml to make a stock solution, 1 mg (trihydroxystilbene) /2.5 m
1, 0.75mg / 2.5ml, 0.5mg / 2.5m
3 concentrations of 1 (ie 0.4 mg / ml, 0.3 m, respectively)
g / ml, 0.2 mg / ml), and this 3 full-scale sample was examined. The same amount of pure water was used as a control. Each test was conducted as one set of trials, and the average thereof was taken. The activated T _rp -P-1 was prepared by dissolving 5 mg of T _rp -P-1 in 20 ml of pure water, adding 20 ml of S9mix, and culturing at 37 ° C. After 20 minutes, 40 ml of acetone was added to stop the reaction, and the denatured protein was removed by centrifugation. This centrifugal supernatant was decompressed and concentrated (without acetone) by a rotary evaporator under ice cooling, and this concentrated solution was activated T _rp.
-P-1. And the activated T _rp -P-1 in the table
The solution had a T _rp -P-1 concentration of 0.4 mg / ml. The activated _T rp -P-1 / trihydroxystilbene mixture shows a _T rp -P-4 concentration trihydroxystilbene concentration and 0.4 mg / ml are shown in Table. The results of the above tests are shown in Table 1.

[0012]

As shown in Table 1 above, the concentration of trihydroxystilbene is directly proportional to the SOS reaction inhibition rate, and particularly when the concentration is 0.4 mg / ml,
The suppression rate improved to 98.0%. From this, it is clear that trihydroxystilbene has a strong inhibitory effect on the induction of the SOS reaction by activated _Trp- P-1. The present invention is not limited to the specific examples described above, and various modifications may be made within the scope of the present invention depending on the purpose and application. That is,
The concentration of trihydroxystilbene may exceed 0.4 mg / ml, and the solvent for forming the solution is not limited to water, and other solvents or mixed solvents may be used depending on the purpose. Further, various additives may be added to the use solution.

[0014]

INDUSTRIAL APPLICABILITY As described above, 3-4'-5-trihydroxystilbene has a simple structure and an excellent mutagen inhibitory action. Therefore, a mutagen inhibitor containing this can be expected sufficiently as a carcinostatic substance. When the trihydroxystilbene concentration is 0.3 mg / ml or more, the SOS reaction inhibition rate is 70% or more, which is an extremely excellent effect.

Claims (2)

(57) [Claims] 1. A 3,4 ', mutagenic inhibitor characterized by containing 5-trihydroxy stilbene. Wherein said 3,4 ', mutagenic inhibitor according to claim 1, wherein the concentration of 5-trihydroxy stilbene is 0.3 mg / ml or more.