JP4916106B2 - Antimutagenic agent - Google Patents

Description

  The present invention relates to an antimutagenic agent comprising an extract obtained from Yellow batai OLE_LINK4 (Peltophorum dasyrachis) OLE_LINK4 as an active ingredient, and a food additive and a cosmetic additive comprising the antimutagenic agent. .

  In recent years, the importance of preventive measures for preventing cancer has been widely called for, and many studies have been conducted on mutagenic substances that cause carcinogenesis. For example, it has been reported that 2-chloro-4-methylthiobutyric acid (CMBA), which is a substance produced from methionine present in sanma, is treated with salt by mutagenicity. Patent Documents 1 and 2). Furthermore, according to the results of animal experiments, it has also been shown that CMBA may cause gastric cancer (Non-patent Documents 3 and 4).

  In this way, the fact that there are substances that have mutagenic properties in fish components such as Sanma is a serious problem for Japanese people who eat a lot of fish. In addition, many mutagenic substances exist in various living environments including foods.

For this reason, the present condition is that the importance of the substance which can suppress the activity of the substance which has mutagenicity as a preventive measure of onset of cancer is increasing.
Chen, W., Et al., Nature, 374, 599 (1995) Chen, W., Et al., Chem. Res. Toxicol., 9, 58-66 (1996) Weisburger, JH, et al., J. Natl. Cancer Inst., 64, 163-167 (1980) Furihata, C. Et al., Cance Lett., 108, 129-135 (1996)

  The main object of the present invention is to provide a novel antimutagenic agent capable of suppressing mutagen activity. More specifically, a novel antimutagenic agent having a high safety and an excellent inhibitory effect on mutagens, and food additives and cosmetics comprising the antimutagenic agent, which are made from ingredients that can be used as foods. It is to provide an additive.

  As a result of intensive studies to achieve the above-mentioned object, the present inventor has found that an extract of yellow butterfly has an excellent inhibitory action against mutagens, and has completed the present invention.

  That is, this invention is providing the following antimutagenic agent.

  Item 1. An antimutagenic agent that contains an extract of yellow batai (Peltophorum dasyrachis) as an active ingredient.

  Item 2. Item 2. The antimutagenic agent according to Item 1, wherein the extract is extracted using at least one extraction solvent selected from the group consisting of water and a polar organic solvent.

  Item 3. Item 3. The antimutagenic agent according to Item 2, wherein the extraction solvent is water, alcohol, or a mixed solvent of water and alcohol.

  Item 4. The extract is of formula (I):

Item 4. The antimutagenic agent according to any one of Items 1 to 3, comprising Thranthine C represented by

  Item 5. Formula (I):

The antimutagenic agent which contains the slangin C shown by this as an active ingredient.

  Item 6. Formula (Ia):

The antimutagenic agent which contains (+)-(9S) -thrangin C shown by these as an active ingredient.

  Item 7. Item 7. A food additive comprising the antimutagenic agent according to any one of Items 1 to 6.

  Item 8. Item 7. A cosmetic additive comprising the antimutagenic agent according to any one of Items 1 to 6.

  Item 9. Formula (Ia):

(+)-(9S) -Thrazine C represented by

  Hereinafter, the present invention will be described in detail.

  The antimutagenic composition of the present invention comprises an extract from yellow batie as an active ingredient.

  In the present invention, yellow batai (Peltophorum dasyrachis) is a plant planted in Southeast Asian countries such as Thailand, Cambodia, Laos, Vietnam, Malaysia and Sumatra. The yellow batie extract may be an extract of the tree, and an extract of the bark is particularly preferable.

  The above yellow batie can be used for extraction as it is, but may be subjected to extraction after being pulverized more finely. Moreover, after powdering, it can be further dried and extracted, or it can be pulverized in water to form a slurry.

  Examples of the extraction solvent include alcohols such as methanol, ethanol, propanol and butanol, glycols such as 1,3-butylene glycol, glycerin and propylene glycol, esters such as ethyl acetate and butyl acetate, ethyl ether and propyl ether. , Ethers such as isopropyl ether, tetrahydrofuran and dioxane, polar organic solvents such as halogenated hydrocarbons such as dichloromethane and chloroform, nonpolar organic solvents such as hexane, cyclohexane and petroleum ether, water and the like can be used. These extraction solvents can be used singly or in combination of two or more.

  Among these, at least one solvent selected from the group consisting of water and a polar organic solvent is preferable in that an extract having excellent antimutagenic activity can be efficiently obtained. In particular, when an alcohol such as methanol or ethanol or water is used alone, or when a mixed solution of water and alcohol is used, an extract that is easy to handle and has excellent activity can be obtained. This is preferable. In this case, in particular, when the extract is used for oral intake such as food additives, it is preferable to use ethanol as the alcohol.

  In the case of using a mixture of solvents, the mixing ratio of each solvent may be appropriately adjusted according to the type of the solvent. For example, when used as a mixed solution of water and alcohol, water: alcohol (weight) Ratio) = 1: 100 to about 100: 1, preferably about 1:50 to 50: 1, and more preferably used at approximately equal weight.

  The extraction method is not particularly limited, and after adding a solvent to the yellow batie, a heating extraction method in which the extract is heated to such an extent that the antimutagenic activity of the extract is not deactivated, a supercritical extraction method, etc. Can be applied as appropriate. In addition, various known extraction methods such as an immersion extraction method in which at least one selected from the group consisting of rice bran and brown rice is immersed in a certain amount of solvent and batch processing, and a continuous extraction method in which the solvent is continuously fed are applied. it can.

  An example of a specific extraction method is, for example, adding an extraction solvent of about 0.5 to 5 times by weight, preferably about 0.8 to 1.2 times by weight with respect to the dry weight of yellow batie. The components having antimutagenic activity can be extracted by immersing and heating and refluxing the solvent for about 30 to 60 minutes. Alternatively, an extraction solvent of about 0.5 to 5 times by weight, preferably about 0.8 to 1.2 times by weight, is added to the dry weight of the yellow butterfly, and immersed for about 1 to 14 days at room temperature. Alternatively, it is possible to extract the active ingredient by heating to about 40 to 60 ° C. and heating for about 10 to 20 hours. Of course, the solvent amount, heating temperature, heating time, etc. are excellent in resistance. What is necessary is just to adjust suitably so that the component which has mutagenic activity can be extracted.

  An extract can be obtained by obtaining an extract from yellow batie by the above-mentioned method and then solid-liquid separating the residue by a conventional method such as filtration or centrifugation. In the present invention, it is possible to use the obtained extract as it is as an antimutagenic agent, but since the activity may be low, it may be used as an extract or powder by appropriately concentrating or distilling off the solvent. You can also.

  Further, the active fraction is separated from the extracted fraction obtained by solvent fractionation operation using one or more organic solvents such as methanol, ethanol, propanol, butanol, chloroform, ethyl acetate, toluene, hexane, benzene and the like. It is also possible to use what was taken as an antimutagenic agent.

  Furthermore, if necessary, one or more suitable separation and purification means such as alumina column chromatography, silica gel chromatography, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, and high performance liquid chromatography may be combined. A fraction or a compound having an activity of inhibiting mutagen activity can be taken out and used as an antimutagenic agent. Thereby, the outstanding activity can be exhibited with a small amount of ingestion.

  Next, referring to FIG. 1, a procedure for purification and identification of an active compound from yellow batie is specifically shown.

  The yellow batie is refluxed with an alcoholic solvent (for example, methanol, ethanol) to obtain an alcohol extract. This extract was suspended in water and re-extracted with chloroform, ethyl acetate, t-butanol and water, and the fractions were concentrated under reduced pressure to obtain a chloroform fraction, an ethyl acetate fraction, a butanol fraction and Obtain the water fraction. The chloroform fraction having the activity of inhibiting mutagen activity is fractionated by silica gel column chromatography. The fraction having mutagen activity-inhibiting action is further fractionated by silica gel column chromatography to obtain active compound 1 having mutagen activity-inhibiting action.

  By various spectral analyses, the active compound 1 is obtained from the formula (I):

It was identified as Surangin C (Surangin C). Furthermore, when the absolute configuration of the 9th position of this slangin C was scrutinized, the formula (Ia):

(+)-(9S) -thrundin C represented by The relative arrangement of this slangin C has been known so far, but for the first time, the absolute arrangement of the 9th position could be clarified.

  The above-mentioned extract from yellow batie and thrangin C (especially (+)-(9S) -thrangin C) have an excellent antimutagenic effect. Therefore, it can be used as an antimutagenic agent for humans and animals. In particular, it can be effectively used for the prevention and treatment of various diseases based on mutation, such as cancer. Moreover, it can be used not only for this, but also in the field of biochemistry, when it is necessary to suppress bacterial mutation, for example, in the case of culture, biochemical analysis and the like.

  The usage form of the antimutagenic agent of the present invention is not particularly limited and can be either oral or parenteral. For example, when taken orally, it can be added to food as a food additive. Can be taken. Moreover, by adding to cosmetics etc. and apply | coating to skin etc., it can be used effectively also for prevention of skin cancer etc.

  When used as a food additive, the amount added is not particularly limited, and may be appropriately determined according to the type of food. For example, it can be used by adding to liquid foods such as soft drinks and carbonated drinks and solid foods such as confectionery and other various foods, but the amount added in these cases is appropriately determined according to the type of food. For example, the dry weight of the above-described extract may be added so that the content is in the range of about 0.005 wt% to 5 wt%.

  Moreover, when adding to cosmetics, what is necessary is just to determine the addition amount suitably in the range which does not inhibit the original function of cosmetics.

  In addition, examples of the administration method and dosage when administering the antimutagenic agent of the present invention to the human body are as follows.

  Administration can be performed by various methods, for example, oral administration using tablets, capsules, granules, syrups and the like. As for the dosage, in the case of oral administration, the amount of active ingredient is usually about 0.01 to 1000 mg / kg for adults, and this may be administered once to several times a day. . The oral preparation can be produced according to a normal production method. For example, excipients such as starch, lactose and mannitol, binders such as sodium carboxymethylcellulose and hydroxypropylcellulose, disintegrants such as crystalline cellulose and carboxymethylcellulose calcium, lubricants such as talc and magnesium stearate, light anhydrous It can be produced as a tablet, capsule, granule, etc. by appropriately combining and formulating a fluidity improver such as silicic acid.

  In recent years, malignant tumors of animals (dogs, cats, etc.) bred as pets tend to increase, but the antimutagenic agent of the present application is added to the food of these animals or the Administration of a mutagenic agent as a drug makes it possible to prevent and treat malignant tumors.

  The anti-mutagenic agent of the present invention comprises an extract of a yellow plant that is a natural plant as an active ingredient, and has an excellent mutagen inhibitory action.

  In addition, the present inventors have succeeded in purifying and identifying the active compound in the above extract, and have found that thrangin C is its active ingredient.

  The antimutagenic agent of the present invention is used as a food additive, cosmetic additive and the like.

  Hereinafter, the present invention will be described in more detail with reference to examples.

Example 1
A mutagen detection test was performed on the extract of yellow butterfly.
(1) Mutagen detection test The mutagen inhibitory effect of each extract of yellow and batie is a mutagen detection method using the induction of SOS reaction as an index (UMU test; Science and Industry, Vol. 62, No. 4, 142, 1988). Here, the “UMU test” is a mutagen detection test using the SOS reaction observed at the time of DNA damage of Escherichia coli, and has many advantages such as a result being obtained in a short time.

  In addition, 2-amino-3,4-dimethylimidazo [4,5-f] quinoline (MeIQ) was used as a mutagen, and Salmonella typhimurium OY1001 / 1A2 was used as a strain. An outline of the test method will be described below.

  That is, a test bacterial solution cultured overnight at 37 ° C. in LB medium (tryptone 1%, yeast extract 0.5%, salt 0.5%) was added to Tgly medium (tryptone 1%, salt 0.5%, glycerol 0). 1 added to .002 mL / mL at a ratio of 1.0 μg / mL tetracycline, 1.0 mM isopropyl-β-D-thiogalactoside, 0.5 μM δ-aminolevric acid, and 0.25 μL / mL trace element) / 50 amount of the inoculum was inoculated and cultured with shaking at 37 ° C.

When the bacterial concentration reached the logarithmic growth phase (A ₆₀₀ was 0.25 to 0.30), 1.0 ml of the bacterial solution was taken in a test tube, and this was shown in the graph of FIG. Extracts of various concentrations were added and cultured at 37 ° C. for 3 hours. The amount of mutagen added was 10 μl of 10 μg / ml DMSO solution for MeIQ.

  After culturing, the bacterial solution was spun down and collected, then the bacteria were resuspended in physiological saline, the amount of bacteria was measured with a part of this bacterial solution, and the β-galactosidase activity was measured with the other part. Here, the logarithmic growth phase is a time when the number of bacteria and cells increases logarithmically and is also called an exponential growth phase.

β-galactosidase activity was measured according to Miller's method (Miller, JH: Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, New York, P352-355 (1972)). That is, after adding 0.25 ml of the test bacterial solution to 2.25 ml of Z buffer, 50 μl of 0.1% sodium dodecyl sulfate aqueous solution and 10 μl of chloroform were added and stirred vigorously. 0.25 ml of a substrate (o-nitrophenyl-β-D-galactopyranoside 4 mg / ml) was added to the solution and reacted at 28 ° C. Then, after 15 minutes, 1.25 ml of 1M Na ₂ CO ₃ was added to stop the reaction, and A ₄₂₀ , A ₅₅₀ and A ₆₀₀ (absorbance) were measured with a spectrophotometer.

  Here, the β-galactosidase activity value was calculated by the following formula.

β-galactosidase activity (unit)
= 1000 (A ₄₂₀ -1.75 × A ₅₅₀ ) /1.5×A ₆₀₀
Moreover, the SOS reaction suppression rate was computed by the following formula.

SOS reaction inhibition rate (%)
= [1- (AC) / (BC)] × 100
In the above formula, A is the β-galactosidase activity value when each extract is added to the mutagen, B is the β-galactosidase activity value induced only by the mutagen, and C is the control β-galactosidase. Each activity value is shown. The same amount of DMSO was used for control. In addition, each test was performed as a set of trials, and the average was taken.
(2) Extraction and separation of yellow batie Yellow batie was extracted and separated according to the procedure of FIG. 1, and the mutagen detection test of (1) above was performed for each fraction.

  50% ethanol (5 liters) was added to the yellow barley bark (5 kg), heated and refluxed for 5 hours, and then ethanol was distilled off to obtain 452.29 g of an ethanol extract.

  Next, water (1.5 liters) was added to the extract, and then re-extraction was performed sequentially using each solvent of chloroform, ethyl acetate and butanol, and the solvent was distilled off to obtain an extract of each solvent. It was. The amount of the extract was 9.44 g of chloroform extract, 30.01 g of ethyl acetate extract, 231.26 g of butanol extract and 181.58 g of water extract. Furthermore, active compound 1 was obtained by fractionating the active chloroform fraction by column chromatography (FIG. 1).

  About the above test result, the beta-galactosidase activity value of each fraction is shown in Table 1, and the SOS reaction inhibition rate is shown as a graph in FIG.

From each test result of Table 1 and FIG. 2, a strong SOS reaction inhibitory effect was observed in the chloroform fraction and Compound 1. The ID _{50 of} Compound 1 was 26 μg / mL.

  The active compound 1 in FIG. 1 was found to be thrangin C from various spectral data. The structural formula and spectrum data are shown in FIG.

  Thrazine C has been reported only in the relative configuration so far (Mahandru, MM et al. (1986) Phytochemistry, 25, 555-556). It was revealed that it has the original activity. The absolute configuration at the 9th position was determined by the Mosher method. The data is shown in Table 2.

In the (R)-and (S) -MTPA esters, the difference in chemical shift value at the 3rd position is positive and the difference in chemical shift value at the 11th position is negative. By applying these to the Mosher method, the 9th position The absolute configuration was determined to be S configuration. From the above results, the structure of Compound 1 was determined to be (+)-(9S) -Surangin C. Reference: Lee, K.-H. et al. (2003) Phytochemistry, 64, 535-541.
As described above, yellow batai extract and thrangin C have a strong mutagen inhibitory action and are useful as antimutagenic agents.

It is a figure which shows the extraction separation process of a yellow batie. The graph which shows the inhibitory effect with respect to MeIQ in Example 1. FIG. 1 shows the structural formula and spectral data of Llangin C.

Claims (6)

An extract of yellow batai (Peltophorum dasyrachis) is used as an active ingredient, and the extract is represented by the formula (I):

The antimutagenic agent containing the thrangin C shown by these. The antimutagenic agent according to claim 1, wherein the extract is extracted using at least one extraction solvent selected from the group consisting of water and a polar organic solvent. The antimutagenic agent according to claim 2, wherein the extraction solvent is water, alcohol, or a mixed solvent of water and alcohol. Formula (I):

The antimutagenic agent which contains the slangin C shown by this as an active ingredient. Formula (Ia):

The antimutagenic agent which contains (+)-(9S) -thrangin C shown by these as an active ingredient. An antimutagenic cosmetic additive comprising the antimutagenic agent according to any one of claims 1 to 5.

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