JPH11180867A - Antimutagenic agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an antimutagenic agent exhibiting extremely good deactivating actions against mutagenic substances contained in foods, exhaust gases, etc., and capable of being effectively utilized for preventing carcinogenesis. SOLUTION: This antimutagenic agent contains Baccharis trimera Less. and/or its extract as an active ingredient. The antimutagenic agent also contains at least one of genkwanin, cirsimaritin, hispidulin and apigenin.

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] Heterocyclic amines such as imidazole (Glu-P-2) have strong mutagenic properties. Benzo (α) pyrene (benzpyrene) present in cigarette smoke and automobile exhaust gas, and 1-nitropyrene and 1,6-nitropyrene present in diesel exhaust gas and used engine oil of gasoline passenger cars are also strong. It is a mutagen.

[0003] 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 medicinal plants, vegetables, fruits, tea, flavors, and the like have strong antimutagenic properties. In addition, the present inventors have disclosed in Japanese Patent Application Laid-Open No. 7-53397
e L.) The fact that squeezed liquids and extracts obtained from plants have strong anti-mutagenic properties, and Japanese Patent Application Laid-Open No. 7-101859 discloses that parsley (Petroselinum crispum (Mill.)) and dill (Anethum graveolens) L.) and the like, it was further disclosed in Japanese Patent Application Laid-Open No. 9-176609 that rosemary (Rosem
arinus officinalis L.,) extract has strong antimutagenic properties.

[0004]

SUMMARY OF THE INVENTION The present inventors have further conducted intensive studies on anti-mutagenic substances. As a result, it has been found that bacillus (Baccharis trimera Less.) And / or an extract thereof have strong anti-mutagenic properties. In addition, the extract of bacillus (Baccharis trimera Less.) Contains flavones such as gencanin, silsimaritin, hispidalin and apigenin, which are strong against mutagens. The present inventors have found that the present invention has excellent antimutagenic properties, and have completed the present invention.

[0005]

That is, the invention according to claim 1 comprises a cascade (Baccharis trimera Less.) And / or
Or an anti-mutagenic agent characterized in that an extract thereof is blended as an active ingredient, the invention according to claim 2,
The present invention relates to an anti-mutagenic agent characterized by comprising at least one of gencanin, silcymaritin, hispidalin and apigenin.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The anti-mutagenic agent according to the present invention comprises a
(Baccharis trimera Less.) And / or an extract thereof. Calcaja (Baccharis trimera Less.)
Is a sub-shrub with a height of 1 to 2 m, which is widely distributed in the central and southern parts of South America, especially in southern Brazil, and is a plant used for the treatment of liver disease and rheumatism.

[0007] In the present invention, the calcifier (Bac
All parts of the above-ground part and underground part of (charis trimera Less.) can be used, and all parts may be used, or a part may be used, such as using only the above-ground part or only the stem part. Further, both those in a dry state and those in a non-dry state can be suitably used.

[0008] Calculator (Baccharis trimera Less.)
The use form of is not particularly limited, and a dry powder or the like may be used as it is, or an extract may be used. When using an extract, the extract may be used as it is, or
It can be used as an extract by concentration or drying.

The extraction solvent used for obtaining the extract includes water or alcohols such as methanol and ethanol, and polar solvents such as ketones such as acetone, ethyl acetate and diethyl ether, esters, ethers, acids and alkalis. A mixed solvent of one or two or more non-polar solvents such as n-hexane, petroleum ether, ligroin, cyclohexane, carbon tetrachloride, chloroform, dichloromethane, toluene, and benzene;
Further, a preferred example is a mixed solvent of a polar solvent and a non-polar solvent, but the present invention is not particularly limited. The extraction temperature at the time of obtaining the extract is not particularly limited, either heating extraction or non-heating extraction is possible.

[0010] Calculator (Baccharis trimera Less.)
In the extract of, as is clear from the examples described below,
Genkwan (genkwan) represented by the following formula 5
in), cilsimaritin (c) represented by the following formula 6
irsimaritin), hispidulin represented by the following formula 7 (formula 7), and apigenin (apigenin) represented by the following formula 8 (formula 8), and these flavones are used for mutagenic substances. And extremely effective antimutagenicity.

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In the present invention, the above 4
A variety of flavones can be blended, and one kind may be used alone or two or more kinds may be used in combination. In addition, as these flavones, both those derived from natural products and those synthesized are suitable.

The dosage form of the antimutagenic agent according to the present invention includes:
Examples thereof include a solution, a soft extract, and a powder, but are not particularly limited. The dosage form is also not particularly limited, and may be added as appropriate excipients and auxiliaries, and may be made into powders, granules, tablets, capsules, syrups, and other drugs in accordance with ordinary methods for manufacturing pharmaceutical preparations, or The food for specified health use expected to have a mutagenicity-suppressing effect may be a drink such as a drink or a juice, or a food such as a confection.

[0013]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail based on embodiments. However, the present invention is not limited to these examples. In the following examples, the calcifier (B) obtained from the Centroflora Institute in Brazil was used.
accharis trimera Less.) was used.

[Preparation of extract]
2. The above-ground part of trimera Less.) is dried and shredded.
5 Kg was warm-extracted with 19 liters of methanol, and the extract was filtered to obtain 13.75 liters of filtrate. The extraction residue was heated and extracted again with 12.7 liters of methanol,
Filtration was performed in the same manner to obtain a filtrate of 12.85 liters. The two filtrates were combined, and methanol was completely distilled off by concentration under reduced pressure to obtain 346.82 g of an extract. 4 of this extract
6.82 g were subjected to the anti-mutagenic test and 300 g were subjected to the 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. And the modified Ames method, histidine auxotrophy (His ^-)
Is a method for examining the antimutagenic activity by testing whether or not Salmonella spp. Causes reversion to His ⁺ .

The test strains include Salmonella t
yphimurium) TA98 was used. This Salmonella TA
98 was prepared by the method described in JP-A-7-53397. Trp-P-2 was diluted with distilled water to a concentration of 1.5 μg / ml, and then filtered and sterilized with a membrane filter (cellulose acetate, 0.45 μm, manufactured by Advantec TOYO, Dismic 25CS). Also, Trp-P
-2 is N-OH-Trp-P-2 upon metabolic activation
It is known to exhibit mutagenicity. Therefore, a metabolic activating enzyme S-9mix is added to the anti-mutagenic test, and this S-9mix is described in JP-A-9-1760009. Prepared by the method described in The above extract was treated with dimethyl sulfoxide (DMSO) at 5 mg / ml, 2 mg / ml.
After diluting the membrane filter (PTFE, 0.2μ
m, Sartorius, Minisart SRP15, Gottingen, Germany) to obtain sample solutions of Examples 1 and 2. Also,
DMSO was similarly sterilized by filtration as a control solution.

The sample solution prepared by the above-described method is used to adjust the sample solution.
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. Similarly, 0.1 ml of DMSO, 0.1 ml of a suspension of TA98, and 0.5 ml of S-9mix were spread on MBB agar medium, and the number of His ⁺ colonies due to spontaneous mutation was counted. Further, 0.1 ml of the sample solution, 0.1 ml of the TA98 suspension, and 0.5 ml of S-9mix were spread on MBB agar medium, and the number of His ⁺ colonies in the sample solution in the absence of Trp-P-2 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 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

The sample solutions of Examples 1 and 2 and DMS
In order to examine whether or not O was toxic to TA98 bacteria, the viable cell count was measured by the method described below. pH
0.1 ml of the bacterial suspension and 0.1 ml of the sample solution (or DMSO) serially diluted to 10 ⁵ with the phosphate buffer of 7.0
3.0 ml of soft agar kept at 50 ° C. in the same manner as above
And seeded on MBB agar. After culturing at 37 ° C. for 3 days, the number of viable bacteria was counted, and each sample solution and DM
The toxicity of SO to Salmonella TA98 was tested. In addition, regarding the number of viable bacteria, plate
Each sheet was manufactured, and the average value was calculated.

Table 1 summarizes the results of the number of revertant colonies, the number of viable bacteria and the mutation suppression rate.

[Table 1]

From the results in Table 1, it can be seen that the calcifier (Baccharis
(Trimera Less.) extract has very strong anti-mutagenicity.

[Isolation of anti-mutagen]
(accharis trimera Less.) was purified according to FIG. 1 using the anti-mutagenic activity as an index. First, 1.2 liters of water was added to 300 g of the extract, and 5%
After adjusting the pH to 3 with hydrochloric acid, the mixture was separated three times with ethyl acetate. After neutralization, the aqueous phase was concentrated to 113.83 g to obtain fraction 1. The organic phase was concentrated to 24.69 g in 1/8 volume to give fraction 2. The remaining 7/8 volume is adjusted to pH 9 and then concentrated to 3.16 liters,
Extracted three times with one liter of 5% NaHCO ₃ . NaHC
The O ₃ phase was neutralized and desalted, and then concentrated to 109.4 g to obtain a fraction 3. After extraction with NaHCO ₃ , the organic phase was concentrated to 89.18 g to obtain fraction 4. Fractions 2 and 4 were found to have remarkable anti-mutagenic activity as a result of an anti-mutagenic test performed on fractions 1 to 4.

Next, the fraction 4 was further purified. 80 g of the fraction 4 was subjected to silica gel chromatography using a chloroform-methanol solvent, and fractionated into four types of fractions A to D shown in Table 2. Note that silica gel manufactured by Merck, Germany was used.

[Table 2]

The fractions A to D were subjected to an antimutagenic test. As a result, fractions A and B were found to have strong antimutagenic properties.
The mixture was subjected to silica gel chromatography using a methanol-based solvent, and fractionated into fractions A-1 to A-5 and fractions B-1 to B-5 shown in Table 3.

[Table 3]

Fractions A-1 to A-5, B-1 to B
-5 was subjected to an anti-mutagenic test. The number of viable bacteria was also measured. The results are shown in Tables 4 and 5.

[Table 4]

[Table 5] In Tables 4 and 5, inactive means control (DMS
The number of His ⁺ colonies was larger than that of O), indicating that no anti-mutagenicity was observed.

[Identification of anti-mutagen] As a result of the anti-mutagen test, fraction A-
2, A-4, B-2, B-3 and B-4 were subjected to column chromatography using Sephadex LH-20 (Sweden; Pharmacia Biotech) using methanol as a developing solvent, and fraction A- 3
Was subjected to recrystallization, and as a result, four types of compounds 1-4
was gotten. More specifically, 4 mg of compound 1 and 144 mg of compound 2 from fraction A-2, compound 2 from fraction A-3, and fraction A-4
From 42 mg of compound 2 and 141 mg of compound 3
But 302 mg of compound 3 from fraction B-2
However, 313 mg of compound 3 and 67 mg of compound 4 were obtained from fraction B-3, and 1
03 mg of compound 3 and 24 mg of compound 4 were obtained, respectively. All four compounds are pale yellow crystals and have a melting point of 296-298 ° C for Compound 1 and 25 for Compound 2
Compound 5 had a melting point of 290 to 292 ° C, and compound 4 had a temperature of 300 ° C or higher.

Compounds 1 to 4 were subjected to ultraviolet absorption spectrum (UV) analysis, infrared absorption spectrum (IR) analysis, mass spectrometry (EI-MS), ¹ H nuclear magnetic resonance analysis ( ¹ H-NMR), and ¹³ C nuclear magnetic field. Resonance analysis ( ¹³ C-NM
R). Tables 6 and 7 summarize the analysis results
And FIGS. 2 to 17. 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, and FIG.
Of ¹³ C- ¹ H-NMR spectrum, Figure 6 is an ultraviolet absorption spectrum of compound 2, FIG. 7 is an infrared absorption spectrum of Compound 2, 8 mass spectrum of compound 2, Figure 9 is the Compound 2 ^{13 C-1} H FIG. 10 is an ultraviolet absorption spectrum of compound 3, FIG. 11 is an infrared absorption spectrum of compound 3, FIG. 12 is a mass spectrum of compound 3,
13 is a ¹³ C- ¹ H-NMR spectrum of compound 3, FIG. 14 is an ultraviolet absorption spectrum of compound 4, FIG. 15 is an infrared absorption spectrum of compound 4, FIG. 16 is a mass spectrum of compound 4, and FIG. ¹³ is a ^C-1 H-NMR spectrum.

[Table 6]

[Table 7]

As a result of the analysis test, compound 1 was represented by the following formula 9
5,4′-dihydroxy-7-methoxyflavone (genkwanin) represented by the following formula
Is a 5,4′-dihydroxy-6,7-dimethoxyflavone represented by the following formula 10
Itin).

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Compound 3 is 5,7,4'-trihydroxy-6-methoxyflavone (hispidulin) represented by the following formula 11
Was identified to be 5,7,4'-trihydroxyflavone (apigenin) represented by the following formula (Formula 12).

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An anti-mutagenic test and a viable cell count were performed on the four types of isolated flavones of Compounds 1 to 4. Table 8 shows the results.

[Table 8]

From the results shown in Table 8, the calcifier (Baccharis
gencanin, silcymaritin, hispidalin and apigenin contained in the extract of Trimera Less.)
It can be seen that all kinds of flavones have excellent antimutagenicity and high safety.

[0032]

Industrial Applicability As described in detail above, the invention according to claim 1 relates to an anti-mutagenic agent characterized by comprising bacillus (Baccharis trimera Less.) And / or an extract thereof as an active ingredient. The invention according to claim 2 relates to an anti-mutagenic agent characterized by comprising at least one of gencanin, cilsimaritin, hispidalin, and apigenin, so that Trp-P-
It has excellent anti-mutagenicity against mutagens such as No. 2 and can be effectively used as a drug etc. for the purpose of preventing carcinogenesis, and is highly safe because it is derived from natural products 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 bacillus (Baccharis trimera Less.).

FIG. 2 is an ultraviolet absorption spectrum of compound 1 contained in an extract of bacillus (Baccharis trimera Less.).

FIG. 3 is an infrared absorption spectrum of Compound 1 contained in an extract of Baccharis trimera Less.

FIG. 4 is a mass spectrum of Compound 1 contained in an extract of bacillus (Baccharis trimera Less.).

FIG. 5 is a ¹³ C- ¹ H-NMR spectrum of Compound 1 contained in an extract of Baccharis trimera Less.

FIG. 6 is an ultraviolet absorption spectrum of Compound 2 contained in an extract of Baccharis trimera Less.

FIG. 7 is an infrared absorption spectrum of Compound 2 contained in an extract of Baccharis trimera Less.

FIG. 8 is a mass spectrum of compound 2 contained in an extract of bacillus (Baccharis trimera Less.).

FIG. 9 is a ¹³ C- ¹ H-NMR spectrum of Compound 2 contained in an extract of Baccharis trimera Less.

FIG. 10 is an ultraviolet absorption spectrum of Compound 3 contained in an extract of Baccharis trimera Less.

FIG. 11 is an infrared absorption spectrum of Compound 3 contained in an extract of Baccharis trimera Less.

FIG. 12 is a mass spectrum of compound 3 contained in an extract of bacillus (Baccharis trimera Less.).

FIG. 13 is a ¹³ C- ¹ H-NMR spectrum of Compound 3 contained in an extract of Baccharis trimera Less.

FIG. 14 is an ultraviolet absorption spectrum of Compound 4 contained in an extract of Baccharis trimera Less.

FIG. 15 is an infrared absorption spectrum of Compound 4 contained in an extract of Baccharis trimera Less.

FIG. 16 is a mass spectrum of the compound 4 contained in the extract of Baccharis trimera Less.

FIG. 17 is a ¹³ C- ¹ H-NMR spectrum of Compound 4 contained in an extract of Baccharis trimera Less.

Claims (2)

[Claims] [Claim 1] Baccharis trimera Les
s.) and / or an extract thereof is compounded as an active ingredient. 2. Gencanine represented by the following formula 1 (formula 1):
It is characterized by comprising at least one of cilsimaritin represented by the following formula 2 (formula 2), hispidalin represented by the following formula 3 (formula 3), and apigenin represented by the following formula 4 (formula 4). Anti-mutagenic agent. Embedded image Embedded image Embedded image Embedded image