JP2000256205A - Antimutagenic agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an antimutagenic agent which exerts an excellent activity inhibitory effect against mutagenic substances in foods or exhaust and is effective in preventing carcinogenesis by including an extract from Cirsium maritimum Makino as an active ingredient. SOLUTION: This antimutagenic agent is obtained by including an extract obtained by extracting Cirsium maritimum Makino belonging to the family Compositae the genus Cirsium by the use of e.g. a mixed solvent or the like of a polar solvent and a nonpolar solvent as an extracting solvent. Further, as the compound of formula I and/or II contained in the above extract is an active ingredient in the objective antimutagenic agent, the synthesized above compound may be used as the active ingredient. The daily dose in the case that the above antimutagenic agent is used as a medicine, is preferably determined so as to be 0.005-20 g/kg (body weight) as an extract included when the extract is used, 0.0015-1.5 g/kg (body weight) in the content when cirsimarin is used and 0.0005-0.5 g/kg (body weight) when cirsimaritin is used.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an anti-mutagenic agent,
An object of the present invention is to provide an anti-mutagenic agent which exhibits an extremely good activity-suppressing action on mutagenic substances present in foods and exhaust gas and can be effectively used for preventing carcinogenesis.

[0002]

2. Description of the Related Art In most cases, cancer is caused by mutagens in the environment. This mutagen is a chemical substance that induces mutation at a higher frequency than mutations that occur naturally in mammalian cells and the like, and its occurrence is deeply involved in daily life such as eating and smoking. For example, when cooking meat and fish,
Amino-1,4-dimethyl-5H-pyrido [4,3-
b] indole (Trp-P-1) or 3-amino-1-
Methyl-5H-pyrido [4,3-b] indole (Tr
p-P-2), 2-amino-6-methyldipyrido [1,
2-a: 3 ', 2'-d] imidazole (Glu-P-1)
And 2-amino-6-dipyrido [1,2-a: 3 ', 2'-
d] Imidazole (Glu-P-2), 2-amino-3
-Methylimidazo [4,5-f] quinoline (IQ) and 2-amino-3,4-dimethylimidazo [4,5-f]
Quinoline (MeIQ), 2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline (MeIQx)
And 2-amino-3-methyl-α-carboline (MeAα
Heterocyclic amines such as C) have strong mutagenic properties.

In addition, benzo [a] pyrene (benzpyrene, B [a]) present in cigarette smoke and automobile exhaust gas
P), 1-nitropyrene and 1,6 present in diesel exhaust gas and in used engine oil of gasoline passenger cars.
-Nitropyrene, furthermore sodium azide (SA), aflatoxin B ₁ (AFB ₁ ), 2-aminoanthracene (2-AA), 2-aminofluorene (2-AF),
2-nitrofluorene (2-NF), 4-NQO, AF
-2, MNNG, etc. are also strong mutagens.

[0004] As described above, the main cause of the occurrence of cancer is mutagen, but on the other hand, since the generation of mutagen is deeply involved in daily life, it is difficult for mutagen to enter the body. It is extremely difficult to prevent. Therefore, studies on anti-mutagenic substances that suppress the mutagenicity of mutagens have been actively conducted, and various anti-mutagenic substances have already been discovered. For example, it has been reported that extracts of European herbs and Chinese vegetables, fruits, teas, flavors and the like have strong antimutagenic properties. The present inventors have also disclosed in Japanese Patent Application Laid-Open No.
inus officinalis L.,) has strong antimutagenic properties.
No. 5066, Baccharis trimera
Less.) And its extracts have strong anti-mutagenic properties.

[0005]

DISCLOSURE OF THE INVENTION The present inventors have further conducted intensive studies on anti-mutagenic substances. As a result, it has been found that extracts of Cirrusium maritimum Makino have strong anti-mutagenic properties. And also thistle (Cirsiu
m maritimum Makino) extract was found to contain a large amount of silcimarin and silcymaritin as active ingredients, leading to the completion of the present invention.

[0006]

Means for Solving the Problems The invention according to claim 1 relates to an anti-mutagenic agent characterized by comprising an extract of milk thistle (Cirsium maritimum Makino) as an active ingredient. The present invention relates to an anti-mutagenic agent characterized by comprising silcimarin and / or silsimaritin.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The anti-mutagenic agent according to the present invention has a milk thistle (Cirsium maritimum Makino) as an active ingredient.
Extract is blended. Thistle (Cirsium maritimu)
m Makino) is a perennial plant of the family Asteraceae, which grows on coastal gravel and windy grasslands, such as the Pacific coast of Japan. The plant height is as low as 15 to 60 cm, the side branches are spread laterally and are branched, and the leaves are fleshy, shiny, feather-like, deeply split and have hard thorns. The roots are straight roots and penetrate deep into the ground, and are called burdock because they are burdock-like, and are also used as substitutes for burdock. It can also eat soft new shoots and leaves in early spring, and is widely planted for food. Whole grass is also used as a folk medicine for the treatment of gonorrhea, beriberi and burns.

[0008] Also, thistle (Cirsium maritimum Maki)
Thistle plants, including no), are called "Ji" in Chinese medicine and are used for stomachic, tonic, detoxifying, diuretic and hemostatic. Furthermore, it is also known to have a blood pressure lowering action and an antibacterial action.

In the present invention, thistle (Cirsium
maritimum Makino) can be used in all parts of the above ground and underground parts, even if all parts are used or leaves, stems, roots,
You may use each part, such as a flower part, individually or in mixture.
Further, both those in a dry state and those in a non-dry state can be suitably used.

The above milk thistle (Cirsium maritimum)
Makino) extract is used as an active ingredient, but the form of use is not particularly limited, and the extract may be used as it is or may be used as an extract by concentration or drying.

The extraction solvent used for obtaining the extract may be water or an alcohol such as methanol or ethanol, or a polar solvent such as ketones such as acetone, ethyl acetate or diethyl ether, esters, ethers, acids or alkalis. A mixed solvent of one or two or more non-polar solvents such as n-hexane, petroleum ether, cyclohexane, chloroform, dichloromethane, benzene, etc., and a mixture of a polar solvent and a non-polar solvent As a preferred example, a mixed solvent and the like can be mentioned, but there is no particular limitation. The extraction temperature at the time of obtaining the extract is not particularly limited, either heating extraction or non-heating extraction is possible.

Thistle (Cirsium maritimum Makino)
As is clear from the test examples described below,
It contains silcymarin represented by the following formula 3 (chemical formula 3) and silcimarinin represented by the following formula 4 (chemical formula 4).

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[0013] As is clear from the test examples described later, cilsimaritin has extremely effective antimutagenicity against mutagenic substances. On the other hand, silymarin is in vit
Although no activity is observed in ro, this silcimarin is a glycoside of silsimaritin and can be a precursor of silsimaritin, so it is hydrolyzed by stomach acid and some intestinal bacteria after ingestion into the body, It is thought that it may be converted to and express anti-mutagenic activity. It is also possible to chemically or enzymatically hydrolyze the silymarin in the extract to produce an extract with an increased content of silymarin. Thus, although indirect,
Silsimarin can also be expected to have the same antimutagenicity as silcimaritin.

By the way, anti-mutagens are largely desmutagens and biological anti-mutagens depending on the mode of action.
(bio-antimutagen). Mutagens are antimutagens that act before the mutagen is incorporated into the DNA. In addition, the mutagen forms a direct adduct with the mutagen to inactivate the mutagen and to inhibit metabolic activating enzymes. Those that hinder the conversion of the mutagen precursor to the ultimate mutagen and those that inactivate the activated form after metabolic activation are subdivided. Biological antimutagens, on the other hand, are antimutagens that act on the repair system after DNA has been damaged by the mutagen.

Thistle (Cirsium maritimum Makino)
As is clear from the test examples described later, cilsimaritin contained in the extract is a mutagen, and more specifically,
Inactivates the mutagen by directly acting on the mutagen to form an adduct, or inhibiting the metabolic activating enzyme to prevent the conversion of the mutagen precursor to the ultimate mutagen It shows mutagenicity.

[0016] Accordingly, cilsimaritin is converted to Trp-P-
1, Trp-P-2, IQ, MeIQ, MeIQx, M
eAαC, B [a] P, AFB ₁ , 2-AA and 2-A
F shows excellent antimutagenicity against indirect mutagens such as F, but 2-NF, 4-NQO and AF-2, MNN
It shows almost no antimutagenicity against direct mutagens such as G and SA.

In the present invention, the above-mentioned silcymarin and / or silcymaritin can be blended as an active ingredient. It should be noted that both silcimarin and silsimaritin can be suitably used, both of natural origin and of synthetic origin.

The dosage form of the anti-mutagenic agent according to the present invention includes:
Examples thereof include a solution, a soft extract, and a powder, but are not particularly limited. The administration form is also not particularly limited, and may be added as appropriate pharmaceuticals such as powders, granules, tablets, capsules, syrups, etc., according to a usual method of preparation, by adding optional excipients and auxiliaries, or Drinks, as specified health foods expected to have mutagenic effect
Drinks such as juice and foods such as confectionery may be used.

When the anti-mutagenic agent of the present invention is used as a pharmaceutical, the daily application rate is as follows.
It is 0.005 to 20 g / kg (body weight). When silcimarin is used, the content is adjusted to be 0.0015 to 1.5 g / kg (body weight).
When cilsimaritin is used, the application rate may be determined so that the content is 0.0005 to 0.5 g / kg (body weight).

In addition, thistle (Cirsium maritimum Maki)
no) and its extracts, and furthermore, silymarin and silcymaritin are very safe because they are derived from natural products,
When the acute toxicity (LD ₅₀ ) was examined using mice, the plant itself was 10 g / kg (body weight), the plant extract was 5 g / kg (body weight), and the silymarin was 1 g / kg.
(Body weight), 700 mg / kg (body weight) of silcimaritin
Even when the excessive amount was administered, no death cases of the mice were observed, and it was impossible to measure the LD ₅₀ value. Therefore,
It can be said that the anti-mutagenic agent according to the present invention is also excellent in safety.

[0021]

Test Examples Hereinafter, the present invention will be described in detail based on test examples. The milk thistle (Cirsium maritimum Makino) used in the test examples shown below was collected at a farm in Muroto in June 1997 and collected at 4 ° C or lower to prevent the decomposition of components. Transported and used for testing immediately upon arrival.

[Preparation of extract] Thistle (Cirsium ma
ritimum Makino) 24.83 g above the ground
After removing contaminants, the mixture was extracted with 99.32 liters of ethanol at room temperature. The obtained extract was filtered to obtain 71.587.
One liter of the filtrate was obtained, and 99.32 liters of ethanol was added again to the extraction residue to carry out warm extraction. The obtained extract was filtered in the same manner to obtain a filtrate of 75.665 liters. The two filtrates were combined and concentrated under reduced pressure until the alcohol was completely distilled off to obtain 855.6 g of an extract. 55.6 g of this extract was used for anti-mutagenic test, and 800 g for anti-mutagenic isolation.

[Anti-mutagenicity test] The anti-mutagenic activity of the above extract was measured by the modified Ames method using Trp-P-2 (manufactured by Wako Pure Chemical Industries, Ltd.) as a mutagenic substance. The modified Ames method is to test whether Salmonella, which has become a histidine auxotrophy (His ⁻ ) due to a mutation, will revert to His ⁺ ,
This is a method for examining antimutagenic activity.

The above milk thistle (Cirsium maritimum Maki)
The extract of no) was diluted with dimethyl sulfoxide (DMSO) to 2.5 mg / ml, and then diluted with a membrane filter (PTFE, 0.2 μm, Sartorius, Minisart SRP15, Gottingen, G
ermany) to obtain a sample solution of Example. DMSO was similarly sterilized by filtration as a control solution. Test strains include Salmonella typhimurium T
A98 (a strain causing a frameshift mutation) was used. The preparation of this suspension of Salmonella TA98 was carried out according to the method described in JP-A-7-53397.

Trp-P-2 is 1.5 μg / m in distilled water.
to a mutagen solution, and then a membrane filter (cellulose acetate, 0.45 μm, Advantec
The solution was sterilized by filtration using TOYO (Dismic 25CS). Also, Tr
p-P-2 undergoes metabolic activation to give N-OH-Trp-
Since it is known to be an indirect mutagen that becomes P-2 and shows mutagenicity, a metabolic activating enzyme S-9mix is added to the anti-mutagen test. It was prepared by the method described in Japanese Unexamined Patent Publication No. Hei 9-17609.

The sample solution prepared by the above-described method was used to prepare 0.1.
1 ml, TA98 bacterial suspension 0.1 ml, Trp-P-2
(1.5 μg / ml) 0.1 ml, S-9 mix 0.5
Then, the mixture was added to 3.0 ml of soft agar kept at 50 ° C., rapidly stirred, and sown on MBB agar medium. As a control, DMSO was used instead of the sample solution, and the resultant was similarly spread on an MBB agar medium. In addition, the soft agar described above was 3.0 g of NaCl and 3.5 g of Agar.
After dissolving in 00 ml of distilled water and sterilizing in an autoclave,
It was kept at 50 ° C. The MBB agar medium was prepared by the method described in JP-A-7-53397. After culturing at 37 ° C. for 3 days, the number of colonies of a histidine non-auxotrophic strain (His ⁺ ) that had been remutated was counted. In addition, three plates were prepared for each treatment section, and the average value of the number of colonies was calculated.

Using the average number of colonies calculated by the above method, the anti-mutagenic activity (mutation suppression rate) was calculated based on the following equation (1).

(Equation 1) C: H of DMSO solution in the presence of Trp-P-2
is ⁺ number of colonies N; number of His ⁺ colonies due to spontaneous mutation S; His in the presence of Trp-P-2 and sample solution
⁺ Number of colonies A; number of His ⁺ colonies in the absence of Trp-P-2

Further, the sample solutions of the examples and the T
In order to examine the presence or absence of toxicity to A98 bacteria, the number of viable bacteria was measured by the method described below. 0.1 ml of bacterial suspension serially diluted to 10 ⁵ with phosphate buffer pH 7.0
And 0.1 ml of a sample solution (or DMSO) was added to 3.0 ml of a soft agar kept at 50 ° C. as described above,
Plated on MBB agar. After culturing at 37 ° C. for 3 days, the number of viable cells was counted, and the effects of the sample solution and DMSO on the growth of Salmonella TA98 were examined. In addition, about the number of viable bacteria, three plates were prepared for each treatment section, and the average value was calculated.

Table 1 summarizes the results of the antimutagenic activity and viable cell count of the examples.

[Table 1]

From the results in Table 1, it was found that thistle (Cirsium ma
ritimum Makino) extract has strong anti-mutagenicity against Trp-P-2.

[Isolation of anti-mutagen] Next, the milk thistle (Ci
The above-ground extract of R. maritimum Makino) was purified according to FIG. 1 using the anti-mutagenic activity as an index. First, thistle (Cirsium mari) obtained by the above method
6.4 liters of hot water was added to 800 g of the extract of timum Makino), followed by warm extraction. This was repeated four times, and then separated into a hot water extract and a residue by filtration. The residue remaining on the filter paper was dissolved in a mixture of methanol and ethyl acetate (2: 1), and concentrated under reduced pressure until the solvent was completely distilled off to obtain fraction 1 (90 g). On the other hand, the hot water extract (filtrate) was concentrated under reduced pressure to obtain fraction 2. Fraction 2 subsequently crystallized, which was recrystallized from methanol to isolate 1.174 g of compound 1.

Fraction 1 showed strong antimutagenicity as a result of the same antimutagenic test as described above, and was further purified by chromatography on a silica gel column (4.5 cm × 60 cm). That is, using a chloroform-methanol solvent as a developing solvent, fraction A (14.96 g), fraction B (43.28 g), and fraction C (2
8.01 g) and fraction D (4.02 g). The silica gel used was manufactured by Merck, Germany.

Furthermore, fractions A to D were also subjected to an anti-mutagenic test. As a result, remarkable anti-mutagenic activity was observed in fractions B and C.
Sephadex LH-20 column (4.5 cm × 60 cm) subjected to chromatography, fraction B-1 (1.37 g), fraction B-2 (37.9 g), fraction B-3
(2.35 g), fraction C-1 (3.52 g),
Fraction C-2 (14.41 g) and fraction C-3 (3.53 g) were fractionated. In addition, Sephadex LH-20
Used was one manufactured by Pharmacia Biotech, Sweden.

Fractions B-1 to B-3 and C-1
C-3 was also subjected to an anti-mutagenic test, and as a result, strong anti-mutagenic activities were observed in fractions B-2 and B-3.
It was subjected to Sephadex LH-20 column chromatography, and fraction B-2-a (3.8 g), fraction B-2
-B (6.02 g), fraction B-2-c (8.9)
7g), fraction B-2-d (3.96 g), fraction B-2-e (16.32 g), fraction B
-3-a (270 mg), fraction B-3-b (8
6 mg) and fraction B-3-c (1.9 g). Fraction C-3 was recrystallized from methanol to give compound 1 (1.41 g).

The fraction B-2-b is composed of Polyamide C-20.
0 column (4.5 cm × 60 cm) As a result of chromatography, fraction α-1 (3.36 g), fraction α-2 (1.56 g), fraction α-3 (750 m
g). Incidentally, Polyamide C-200 manufactured by Wako Pure Chemical Industries, Ltd. was used. Furthermore, fraction α-3 is Sephad
Compound 2 (9) was obtained by ex LH-20 column chromatography.
5 mg).

The fractions B-2-c and B-2-d
And B-2-e were combined, subjected to Polyamide C-200 column chromatography, and fraction β-1 (7.
63g), fraction β-2 (4.93 g) and fraction β-3 (9.16 g). Fraction β-3 was prepared using Sephadex L using water-methanol solvent.
As a result of subjecting to H-20 column chromatography, compound 2 was obtained.
(130 mg).

[Identification of anti-mutagens] Next, Compounds 1 and 2 isolated as anti-mutagens were identified. Compound 1 was white crystals with a melting point of 243.5-244.5 ° C, and Compound 2 was pale yellow crystals with a melting point of 255-256 ° C.

Compounds 1 and 2 were analyzed by ultraviolet absorption spectrum (UV) analysis, infrared absorption spectrum (IR) analysis,
It was identified by mass spectrometry (FAB-MS or EI-MS), ¹ H nuclear magnetic resonance analysis ( ¹ H-NMR), and ¹³ C nuclear magnetic resonance analysis ( ¹³ C-NMR). The results of the analysis are shown in Tables 2 and 3 and FIGS. 2 is an ultraviolet absorption spectrum of Compound 1, FIG. 3 is an infrared absorption spectrum of Compound 1, FIG. 4 is a mass spectrum of Compound 1, FIG. 5 is a ¹³ C- ¹ H-NMR spectrum of Compound 1, and FIG. FIG. 7 is an infrared absorption spectrum of Compound 2, FIG. 8 is a mass spectrum of Compound 2, FIG.
Is a ¹³ C- ¹ H-NMR spectrum of Compound 2.

[Table 2]

[Table 3]

The melting point of Compound 1 was in agreement with the literature value, and the signal in the NMR spectrum was very similar to that of silcymarin except for the sugar moiety. Identified as silymarin.

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Compound 2 was identified as silsimaritin represented by the following formula (6) because the melting point and the analytical value of 〜 coincided with the literature values.

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[Anti-mutagenic test] The isolated Compound 1 (silcimarin) and Compound 2 (silcimaritin) were
An anti-mutagen test was performed using Trp-P-2 as a mutagen. Table 4 shows the results. In the table, inactive is D
The number of His ⁺ colonies was larger than that of MSO (control), indicating that no anti-mutagenicity was observed. (The same applies to Tables 5 to 8 below)

[Table 4]

From the results in Table 4, it can be seen that cilsimaritin is not Trp
-It has strong anti-mutagenicity against P-2, but its glycoside, silcimarin, has weak anti-mutagenicity.

Next, heterocyclic amines other than Trp-P-2, specifically, Trp-P-1, IQ,
Using MeIQ, MeIQx and MeAαC (all manufactured by Wako Pure Chemical Industries, Ltd.) as mutagenic substances, antimutagenicity tests were performed on compound 1 (silcimarin) and compound 2 (silcimaritin). The test method was the same as that using Trp-P-2 described above, except for the dilution concentration of the mutagen. That is, Trp-P-1 is 2.5 μm.
g / ml, IQ to 0.5 μg / ml, MeIQ to 0.1 μg / ml, MeIQx to 0.5 μg / ml
Then, MeAαC was diluted to 200 μg / ml with distilled water to obtain a mutagenic solution. The results are shown in FIG. 10 together with the results when Trp-P-2 was used.

From the results shown in FIG.
It can be seen that the mutagenicity of heterocyclic amines other than p-P-2 is also strongly suppressed.

Further, similarly using other mutagenic substances, an anti-mutagenic test was carried out on compound 1 (silcimarin) and compound 2 (silcimaritin). First, Salmonella TA98 was used as a test strain, and B
[A] P, AFB ₁ , 2-AA, 2-AF, 2-NF,
Anti-mutagenicity tests were performed using 4-NQO and AF-2. In addition, B [a] P, 2-AA, 4-NQO and AF-
2 was from Wako Pure Chemical Industries, Ltd., AFB ₁ was from SIGMA, USA, and 2-AF and 2-NF were from Aldrich chemical, USA. The test method was as follows: B [a], which is an indirect mutagen
P, AFB ₁ , 2-AA and 2-AF are performed in the same manner as in the case of using Trp-P-2 described above, except for the method of diluting the mutagenic substance, and the direct mutagen, 2-N
Regarding F, 4-NQO and AF-2, Trp-P-2 was used except for the point that S-9mix required for metabolic activation of the mutagen was not added and the method of diluting the mutagen. An anti-mutagenic test was performed in the same manner. The mutagen dilution method is as follows. That is, B [a] P is 2
50 μg / ml, AFB ₁ 40 μg / ml, 2-AA
Is 100 μg / ml, 2-AF is 100 μg / ml, 2
-NF is 100 µg / ml, 4-NQO is 2 µg / m
1 and AF-2 were diluted to 1 μg / ml using DMSO. The results are shown in FIG.

Next, Salmonella TA100 was used as the test strain.
(Strain causing a base pair substitution mutation), and B [a] P, AFB ₁ , 2-AA, 2-A
F, 2-NF, MNNG, 4-NQO, AF-2 and S
A was used to conduct an anti-mutagenic test. In addition, B [a] P, A
_{FB 1, 2-AA, 2} -AF, 4-NQO and AF-2
Was the same as above, MNNG was from Tokyo Chemical Industry Co., Ltd., and SA was from Wako Pure Chemical Industries, Ltd. The test method is the same as that for Trp-P-2 except that B [a] P, AFB ₁ , 2-AA and 2-AF, which are indirect mutagens, are the same as those described above except for the method of dilution of the mutagen.
Performing in the same manner as in the case of using -P-2, the direct mutagens, 2-NF, MNNG, 4-NQO, AF-2 and SA, are required for the metabolic activation of the mutagens.
The anti-mutagenicity test was performed in the same manner as in the case of using Trp-P-2, except that -9mix was not added and the method of diluting the mutagen. The mutagen dilution method is as follows. That is, B [a] P is 250 μg / ml, AFB
₁ is 40 μg / ml, 2-AA is 100 μg / ml, 2
-AF is 100 μg / ml, 2-NF is 100 μg / m
1, 50 μg / ml for MNNG, 2 μg / ml for 4-NQO
ml and AF-2 were each diluted with DMSO to 1 μg / ml, and SA was diluted with distilled water to 10 μg / ml. The result is shown in FIG.

From the results shown in FIGS. 11 and 12, cilsimaritin was an indirect mutagen other than B [a] P (AFB ₁ , 2-A
A, 2-AF), but also directly mutagenic (2-NF, MNNG, 4-NQO, AF-2, S
Not show antimutagenic at all for the A), whereas a glycoside Shirushimarichin Shirushimarin it can be seen that no activity against mutagen than AFB _1.

[Examination of anti-mutagenic action mechanism] Subsequently, the anti-mutagenic action mechanism of silcimaritin and silsimarin was
Investigation was performed using a modified pre-incubation method of the mes method. still,
Mutagen solution of Trp-P-2, S-9mix, bacterial suspension, soft agar and MBB used in the following tests
The agar medium is the same as that used in the anti-mutagenic test described above. DMSO was used as a control test.

(Test of Mutagenicity) Direct adduct formation with mutagen: 0.1 ml of a sample solution of silsimaritin or silcimarin diluted to 2.5 mg / ml with DMSO, and 1.5 μg / ml of distilled water 0.1 ml of a Trp-P-2 mutagen solution diluted to a concentration was preincubated at 37 ° C. for 30 minutes. Subsequently, 0.5 ml of S-9mix and 0.1 ml of the bacterial suspension were added, and the mixture was further incubated at 37 ° C for 20 minutes. 2. Soft agar kept at 50 ° C.
In 0 ml, the mixture was rapidly stirred and overlaid on MBB agar medium.

Inhibition of metabolic activation 0.1 ml of the same sample solution used above and S-9
After pre-incubating the mix at 37 ° C for 30 minutes, 0.1 ml of a Trp-P-2 mutagen solution and 0.1 ml of a bacterial suspension were added, and the mixture was further incubated at 37 ° C for 20 minutes. The soft agar kept at 50 ° C was 3.0m
and agitated quickly and overlaid on MBB agar medium.

Inactivation of Ultimate Mutagen First, N-OH-Trp-P-2, which is the ultimate mutagen of Trp-P-2, was prepared by the following method.
First, 0.1 ml of Trp-P-2 (30 μg / m
l), 0.5 ml of S-9mix was added thereto, and the mixture was added at 37 ° C for 20 minutes.
Activated with shaking for minutes. Next, the reaction was stopped by adding 0.6 ml of acetone, and then left on ice for 15 minutes. After centrifugation at 3500 rpm for 15 minutes, the supernatant was taken and acetone was distilled off under reduced pressure. After the obtained concentrate was dissolved in 0.1 ml of distilled water, the solution was sterilized by filtration through a membrane filter (cellulose acetate, 0.45 μm), and this was used as an N-OH-Trp-P-2 solution.

The N-OH-Trp obtained by the above method
0.1 ml of the P-2 solution, 0.1 ml of the bacterial suspension, 0.1 ml of the same sample solution used above, and 0.5 ml of the phosphate buffer were mixed and incubated at 37 ° C. for 20 minutes. This mixed solution was added to 3.0 ml of soft agar kept at 50 ° C., rapidly stirred, and overlaid on an MBB agar medium.

The plates were cultured at 37 ° C. for 3 days, and the demutagenic activity (mutation suppression rate) was calculated in the same manner as in the antimutagenic test described above. Table 5 shows the test results for, Table 6 shows the test results for, and Table 7 shows the test results for.

[Table 5]

[Table 6]

[Table 7]

(Biological antimutagenicity test) Bacterial suspension 0.1
ml, 0.1 ml of a mutagenic solution of Trp-P-2, and S
0.5 ml of −9 mix was incubated at 37 ° C. for 20 minutes to mutate TA98 cells. 3900
After centrifugation at 15 rpm for 15 minutes, the cells were washed three times with 13.8 ml of a phosphate buffer, and Trp-P-2 and S-9mi were washed.
x was removed. The washed cells were resuspended in 0.7 ml of a phosphate buffer, and 0.7 ml of the suspension and 0.1 ml of the same sample solution used above were incubated at 37 ° C. for 40 minutes. This was added to 3.0 ml of soft agar kept at 50 ° C., rapidly stirred, and overlaid on the MBB agar medium. After culturing at 37 ° C. for 3 days, the biological antimutagenic activity (mutation suppression rate) was calculated in the same manner as in the above-described antimutagenic test. Table 8 shows the results.

[Table 8]

From the results in Tables 5 to 8, it can be seen that sillsimaritin does not show any activity in the biological antimutagenicity test and shows activity in the negative mutagenicity test. bio-antimutagen)
It turns out to be a strong demutagen. In addition, cilsimaritin is the ultimate mutagen, N-OH-Tr
Rather than inactivating p-P-2 (see Table 7),
Inactivation by adduct formation on rp-P-2 (Table 5)
By suppressing the conversion of the mutagen precursor to the ultimate mutagen by inhibition of the metabolic activating enzyme S-9mix (see Table 6), resulting in the production of N-OH-Trp-P-2. The amount can be reduced, thereby indicating strong demutagenicity. That is, in the above test example, the result that although cilsimaritin exhibited excellent antimutagenicity against indirect mutagens, but hardly exhibited antimutagenicity against direct mutagens, the result that Supported by mutagen.

[0056]

As described in detail above, the present invention according to claim 1 relates to an anti-mutagenic agent characterized by comprising an extract of milk thistle (Cirsium maritimum Makino) as an active ingredient. The present invention relates to an anti-mutagenic agent characterized by comprising cilsimarin and / or cilsimaritin, and has the following effects.

That is, thistle (Cirsium maritimum Ma
kino) extract contains silcimarin and silsimaritin, and silsimaritin is a mutagen that exhibits excellent antimutagenicity against indirect mutagens.
Trp-P-1, Trp-P-2, IQ, MeIQ, M
eIQx, MeAαC, AFB ₁ , 2-AA and 2-A
It exhibits excellent antimutagenicity against mutagen such as F. In addition, since silymarin is a glycoside of silymaritin and can be a precursor of silymaritin, it is hydrolyzed by stomach acid and some intestinal bacteria after ingestion into the living body, converted to sylymaritin and converted to an antimutagenic substance. It is thought that the activity may be exhibited. Therefore, thistle (Cirsium maritimum Makino) and / or its extract,
Alternatively, an anti-mutagenic agent containing silcimarin and / or silsimaritin can be effectively used as a drug or food for the purpose of preventing carcinogenesis and the like, and is highly safe because it is derived from a natural product. It has an excellent effect.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram showing a step of isolating an anti-mutagen from an extract of an above-ground part of thistle (Cirsium maritimum Makino).

FIG. 2 is an ultraviolet absorption spectrum of Compound 1 contained in an extract of the above-ground part of thistle (Cirsium maritimum Makino).

FIG. 3 is an infrared absorption spectrum of Compound 1 contained in an above-ground extract of thistle (Cirsium maritimum Makino).

FIG. 4 is a mass spectrum of Compound 1 contained in an extract of the above-ground part of thistle (Cirsium maritimum Makino).

FIG. 5: ¹³ C- ¹ H-NM of compound 1 contained in an extract of the above-ground part of thistle (Cirsium maritimum Makino)
It is an R spectrum.

FIG. 6 is an ultraviolet absorption spectrum of Compound 2 contained in an extract of the above-ground part of thistle (Cirsium maritimum Makino).

FIG. 7 is an infrared absorption spectrum of Compound 2 contained in an extract of the above-ground part of thistle (Cirsium maritimum Makino).

FIG. 8 is a mass spectrum of compound 2 contained in an extract of the above-ground part of thistle (Cirsium maritimum Makino).

FIG. 9: ¹³ C- ¹ H-NM of compound 2 contained in an above-ground extract of thistle (Cirsium maritimum Makino)
It is an R spectrum.

FIG. 10 is a graph showing the results of an anti-mutagenicity test on silcymaritin and silcimarin against heterocyclic amines.

FIG. 11 is a graph showing the results of an anti-mutagenicity test on sirsimaritin and silcimarin using Salmonella TA98 as a test strain.

FIG. 12 is a graph showing the results of an anti-mutagenicity test for silcimaritin and silsimarin using Salmonella TA100 as a test strain.

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Claims (2)

[Claims] 1. Thistle (Cirsium maritimum Makin)
An anti-mutagenic agent characterized by comprising the extract of o) as an active ingredient. 2. An anti-mutagenic agent characterized by comprising a cilsimarin represented by the following formula 1 (chemical formula 1) and / or a cilsimaritin represented by the following formula 2 (chemical formula 2). Embedded image Embedded image