JPH03172155A - Mutagen-carcinogen-resistant agent - Google Patents

Abstract

PURPOSE:To provide a mutagen and carcinogen-resistant agent having an excellent suppressing activity against mutagen substances and added to drinks and foods to eliminate or weaken the mutability thereof by containing alginic acid having a specific weight-average mol.wt. as an active ingredient. CONSTITUTION:For example, a sea alga such as Hizikia fusiforme is ground, extracted with an alkali aqueous solution and mixed with an acid. The precipitates are subjected to a washing process using an organic solvent and other processes to provide a mutagen and carcinogen-resistant agent containing as an active ingredient alginic acid having a (B/A) ratio of >=0.5 between a weight-average mol.wt. (A) determined with a gel permeation chromatography using pullulan as a standard and a weight-average mol.wt. (B) determined by a low angle laser light-scattering method with the gel permeation chromatography.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides an anti-mutagenic and carcinogenic agent that uses a specific alginic acid as an effective ingredient and has an excellent suppressive effect against mutagens, and an anti-carcinogenic agent that uses the same. Concerning food and beverages containing

[Prior art and problems to be solved by the invention] Currently, cancer (malignant neoplasm) is the leading cause of death in Japan, and the majority of human cancers are caused by carcinogenesis in the environment. The food and drinks we consume on a daily basis are considered to be the most important carcinogenic factors that exist in the environment, and there is a wealth of epidemiological data that supports this view. There's no time left.

In recent years, the Ames test, which uses histidine requirement as an index, has been established as a means of predicting the carcinogenicity of environmental factors such as foods and drinks and medicines (Mutation Research).
Mutation Research, Vol. 31, pp. 347-364, 1975). Many research institutions have found a high correlation of 80-90% between the mutagenicity and carcinogenicity of compounds tested using this method (Proceedings of the National Academy of Sciences).
of Sciences (U.S.A.) (P
roceedings of the Nationa
lAcademy of Sciences (U.S.
．． A. )) Volume 72, pages 5135-5139, 197
5).

Therefore, eliminating mutagenicity is expected to have a considerable effect on protecting the body from the risk of carcinogenesis. Also,
Moreover, mutations occur as a result of damage to DNA, which is genes, and even if they do not lead to cancer, the effects of mutagens on human health cannot be underestimated. This is why food and drink products that are not mutagenic are desired.

To date, the mutagens present in food and beverages include:
Nitrosamines produced by the reaction between nitrous acid and secondary amines, heterocyclic amines present in burnt food, and mycotoxins such as aflatoxin are known, and some have been proven to be carcinogenic. many. However, food and drinks are things we ingest on a daily basis, and we often ingest these mutagens without realizing it. In particular, many of the heterocyclic amines present in burnt food exhibit high mutagenicity (see Table 1). H
utagens and carcinogens)
, University of Tokyo Press (U
niVerC i ty of Tokyo Press
, Tokyo), p. 7, 1981>

(Left below) Table 1 Mutagenicity of heterocytalic amines present in burnt food *1) HeIQ: 2-amino-3,4-dimethyl-yodazo [4.5-fl quinoline IQ: 2 -Amino-3-methylimidazo[4.5-
flquinoline}1eIQx: 2-amino-3,8-dimethylimidazo[4.5-flquinoxaline Trp-P-2: 3-amino-1-methyl-50-pyrido[4.3-bl indole Orn-P-1: 4-Amino-6-methyl-IH-
2. 5, 10,101) 1-tetraazafluoranthene GIU-P-1: 2-amino-6-methyl diviride [
1.2-a: 3',2'-d coimidazole Trp-P
-1:3-amino-1,4-dimethyl-50-pyrido[4.3-bl indoleGlu-P-2:2-aminodipyrido[1.2-a3
゜, 2゜-dl imidazole AαC: 2-amino-9H-pyrido [2.3-bl indole HeAαC: 2-amino-3-methyl-90-pyrido [
2.3-bl indole Lys-P-1: 3,4-cyclopentenopyride [3. 2-alcanorebazonorePhe-P-1: 2-amino-5-phenylpyridine *2) Salmonella typhimurium (Sallll
The number of revertant colonies generated per 1μ9 sample as a result of an experiment conducted using the T^98 strain in the presence of rat liver homogenate (39 mix).

An object of the present invention is to provide a novel antimutagenic/carcinogenic agent that can eliminate or attenuate the mutagenicity of foods and drinks, and foods and drinks containing the same. It is.

[Means for Solving the Problems] In order to achieve the above-mentioned object, the present inventors have made extensive research efforts and found that alginic acid having specific properties has been found to be a mutagenic/carcinogenic substance, especially burnt. We have discovered the fact that it has a very strong inhibitory effect on the heterocyclic amines present in the compound.

Although it has long been known that alginic acid adsorbs Trp-P-1 (Journal of the Japanese Society of Agricultural Chemistry, Vol. 63, No. 3, p. 582, 1989), as mentioned above, alginic acid, especially certain alginic acids, Until now, it was completely unknown that alginic acid had a strong inhibitory effect against mutagens, and the present inventors have developed a new anti-mutagenic and carcinogenic agent using this specific alginic acid. This is what I discovered.

That is, the present invention uses gel permeation chromatography to determine the weight average molecular weight (denoted as A) using pullulan as a standard, and gel permeation chromatography to determine the weight average molecular weight (denoted as B) by low-angle laser light scattering. The present invention relates to an antimutagen/carcinogenic agent characterized by containing alginic acid with a ratio (B/A> of 0.5 or more) as an effective ingredient, and to a food/beverage product containing the same.

The structure of the present invention will be described in detail below.

The alginic acid used in the present invention was determined using gel permeation chromatography using pullulan as a standard, and the weight-average molecule Li (denoted as A) was determined using gel permeation chromatography using the low-angle laser beam scattering method. The ratio (B/A) of the determined weight average molecular weight (referred to as B) is 0.5 or more.Argonic acid with a ratio of A and B (B/A) of less than 0.5 is an anti-mutagenic There is no effect of progenic action.

In addition, the weight average molecular weight A determined using pullulan as a standard is calculated by creating a calibration curve (calibration curve) using pullulan, which is a standard substance with a known molecular weight, and using this calibration curve to calculate the chromatogram of the sample. The peak intensity Ni corresponding to each molecular weight Mi is read from , and the weight average molecular weight (MWA') is determined by the following formula.

MWA = ΣMi2Ni/ΣMiNi (i = 1 to (3)) In addition, the weight average molecular weight B determined by the low-angle laser light scattering method is the absolute molecular weight from the ratio of the LS (light scattering) intensity and the RI (differential refraction) intensity. The molecular weight can be calculated using the following procedure.

The device constant (K
).

K = (A””sTD/A”STD) x
(1/ST[). MW) However, A1SSID = LSC photodestruction of the standard material > peak area A'' STD = Rl (differential refraction) peak area of the standard material STD. MW = Instrument constant corrected after measurement of an unknown molecular weight sample of the standard material ( K”).

K'=KX (STD.CONC/SHP.CONC
) X (A”SMP/A”STD) However, ST[). CONC=standard substance concentration SHP. CONC=unknown sample concentration AR1SHP=RI of unknown sample (differential refraction>peak area AR1STD=RI of standard substance (differential refraction)>peak area Next, using this K', determine the MWB by low-angle laser light scattering method.

MWB = (1/K”) X (A1SSH
P/8”SHP) However, A1SSHP = LS (light scattering) peak area of unknown sample AR1SHP = RI (differential refraction) peak area of unknown sample Regardless of the type, the raw material for alginic acid may have the above properties. Although any seaweed can be used, usually seaweed such as hijiki or kelp is crushed, extracted with an alkaline aqueous solution such as sodium hydroxide or potassium hydroxide, and then acidified with an acid such as hydrochloric acid or sulfuric acid. It can be obtained by washing an alkaline extract of seaweed with an organic solvent such as ethanol, methanol, acetone, acetonitrile, diethyl ether, ethyl acetate, or dimethyl sulfoxide.

Details of the case where the method described above is employed as a method for producing alginic acid will be described below.

First, the degree of pulverization of seaweed is not a particular problem, but from the perspective of extraction efficiency, pulverization above a certain level, such as JIS Z
8801-1982 No. 3.5 (largest @) or higher is preferable. The pulverizing means may be any means commonly used in the food industry, such as a stone mill, high-speed pulverizer, hammer mill, roll pulverizer, or the like.

The concentration of the alkaline aqueous solution used for extraction is suitably 0.1 to 2N. The amount of alkaline aqueous solution used is 10 pieces of crushed seaweed
Approximately 0.5 to 2 liters per 0 g is appropriate. The temperature and time for alkaline extraction are from 80°C to the boiling point of the alkaline aqueous solution.
30 minutes or more, for example 30 minutes to 1 hour, is appropriate. It is then sufficient for the acidification to be t) below 82, for example to a pH of 1 to 2. Acidification may normally be carried out at room temperature, but may also be carried out under heating or cooling (for example, at about 1 to 30°C).
.

For washing with an organic solvent, it is appropriate to add 20 to 100% of the organic solvent to 1 g of the alkaline extract, and leave the mixture to stand or shake at 1 to 30°C, usually at room temperature, for 5 minutes to 1 hour. After standing or shaking, the organic solvent is removed by centrifugation, filtration, etc. This operation may be repeated several times.

The alginic acid to be contained in the antimutagenic/carcinogenic agent of the present invention may be a free acid or salts such as sodium salt, potassium salt, magnesium salt, calcium salt, and the like.

The present invention includes the above-mentioned anti-mutagen/carcinogenic agents as well as foods and drinks containing the agents. There is no particular restriction on the food or drink in this case, but specific examples include seasonings such as sauces, soy sauce, and dressings, powdered soups, powdered soft drinks, and snacks such as biscuits. The amount added can be varied, and there is no particular limit, but from 0.1 to
Approximately 10% is appropriate.

According to Wakabayashi, beef and chicken grilled on a gas stove;
and HeIQx detected in beef grilled on an electric hot plate was 0.5/13 grilled meat, respectively.
nQ, 2.1 ng and 0.3 ng (
Environmental Mutagen Research, Vol. 7, No. 1, pp. 35-41, 1
985). Although I'leIQX alone cannot explain all of the mutagenicity of these grilled meats, the antimutagenic alginic acid can reduce the effects of these mutagens (7) 200
By adding 0 to 3000 times the amount, the mutagenicity can be suppressed by 50%.

If 10 times as much mutagen is present in grilled meat, and the amount of yakiniku eaten at one time is 100g, the amount of mutagen ingested would be 2.1 μ9 for chicken grilled on a gas stove, which is the largest amount. Therefore, the amount of antimutagenic alginic acid required for this is 4.2 to 6.3 mg. If 100% inhibition is desired, roughly 10 times this amount, i.e. 42
It is sufficient to use ~ez3mg. The weight ratio to meat is 0. 042~0. It can be said that a very small amount of 0.063% has a sufficient effect.

In summary, the intake amount of the anti-mutagen/carcinogenic agent of the present invention or the anti-mutagen/carcinogenic agent in the food and drink containing it is as follows, based on the amount of the anti-mutagen/carcinogenic agent: It will be as follows.

That is, the antimutagenic alginic acid of the present invention is suitably used in an amount ranging from 2,000 to 3,000 times (W/W) to 20,000 to 30,000 times the amount of the mutagen. Therefore, the resistance of mutagen-containing foods and drinks will vary depending on the content of the mutagen.

For example, for Yakiniku, which is a food that contains a lot of mutagens and is a daily item, 4.2
~6.3mg to 42~63! rtg is suitable.

There is no particular restriction on the timing of ingestion of the anti-mutagen/carcinogenic agent of the present invention or the food or drink containing it;
or during a normal meal, or within a time not too far before or after these times, preferably within 30 minutes.

The anti-mutagenic alginic acid of the present invention can be prepared from edible hijiki, kelp, etc., and can be said to have no toxicological problems.

[Example] Next, the present invention will be roughly explained in detail with reference to Examples and Test Examples. The present invention is not limited thereby.

[Example] ■. Preparation of anti-mutagenic alginic acid Weighed the ground hijiki seaweed 2009, and poured it into a 1N NaO port 2f.
! It was boiled inside and extracted. The supernatant obtained by centrifuging this liquid was stored as an alkaline extract of hijiki, the precipitate was extracted again in the same manner, and the centrifuged supernatant was added to the alkaline extract of hijiki. The alkaline extract of hijiki was diluted with H2 using hydrochloric acid, and the resulting precipitate was collected by centrifugation to obtain an alkaline extract of hijiki. This alkaline extract of hijiki is
IN}-1cJ2, 0. IN HCj! , and ethanol (each of these solutions or solvents was washed with
(7! In addition, repeat the operation of shaking for 10 minutes, centrifuging and discarding the upper groove 4 times), then suspend in water and lyophilize, anti-mutagenic alginic acid (a) and (b)
I got it.

The weight average molecular weights A and B of the obtained alginic acids (a) and (b) were measured by the method described below.The results are shown in Table 2.

In addition, antimutagenic alginic acids (C) and (d) were obtained from ground cat kelp 2009 by the same preparation method as above. The measurement results are also shown in Table 2.

■． Production of powdered vegetable soup containing anti-mutagenic alginic acid A powdered vegetable soup containing anti-mutagenic alginic acid was produced using the recipe shown below.

Skimmed milk powder 101g Cooked powdered peas 30g Soybean powder
209 Oatmeal 10g dried potato (diced) iog beer yeast ff110
g Dried onion 1g Dried carrot (diced > 8g salt)
4g black pepper 2g anti-mutagenic alginic acid 2g m. Powdered soft drink containing antimutagenic alginic acid A powdered soft drink (orange) containing antimutagenic alginic acid was produced according to the formulation shown below.

Wenzhou 1/5 powdered fruit juice 1009 citric acid
26g powdered natural fragrance
11g Antimutagenic Alginic Acid 2g Frost Sugar 8619 Test Example■. Measuring method of mutagen suppression effect ■Method Pre-incubation method (Sugimura, Nagao: Chemical Mutagiens (ChelliCal Mutag)
enS), Vol. 6, p. 41, 1981).

■Bacterial strain used Salmonella typhimurium (S
almonella th murium) T
A98 strain (hereinafter abbreviated as TA9B strain).

■Measurement of mutagenicity The antimutagenic alginic acid was 0.0. Dissolve INNaQ in your mouth,
50μi (anti-mutagenic alginic acid content: 0.
05-1Rg > 39 mix (9000xg supernatant of rat liver homogenate in which the drug-metabolizing enzyme system was induced with phenobarbital and 5.6-penzoflavone, nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphorus) Acid (NADP
I) Added coenzymes such as # and glucose-6-phosphate (GAP), magnesium ions (}fill2''), etc.〉500μl, TA98 strain preculture solution 1
00 μl and 50 ul of an aqueous solution of mutagen (Trp-P-1) (Trp-P-1: 0.054
Add μ9>. After shaking this mixture at 37°C for 20 minutes, dissolved 2rrJ containing trace amounts of histidine and biotin was added.
Mix with R soft agar and spread on a minimum glucose agar medium.

After standing at 37°C for 48 hours, count the number of revertant colonies that do not require histidine on the medium. In addition, the mutagenicity suppressing effect was expressed by the degree to which the number of colonies generated on the medium containing only the mutagen was defined as 100, and how much it decreased when anti-mutagenic alginic acid was added. The calculation formula is shown below.

A= (<B-C)÷(D-C)) xl00A:
Mutagenicity rate (%) with the addition of anti-mutagenic alginic acid B: Number of colonies in the medium supplemented with anti-mutagenic alginic acid C: Number of colonies in the solvent control medium D: Number of colonies in the control medium without the addition of anti-mutagenic alginic acid Number of colonies■. Analysis method of anti-mutagenic alginic acid Determination of weight average molecular weight using pullulan as standard Anti-mutagenic alginic acid was dissolved in the following elution solvent and applied to gel permeation chromatography.

・Analysis conditions column: TOSOH TSKgel G6000PW
XL 7.8x300mmTOSOH TSKgel
G4000PWXL 7.8x300mmTOSO
H TSKgel G3000PWXL 7.8x30
0 mmTOSOH TSKgel G2500PWX
L 7.8x300 mm The above four columns were connected in series in this order and used.

Column temperature: 40°C Elution solvent: 25 mH Na2HPO4, 13.5
mH Nail(1)H 11.6) Flow rate: 1 m/min. Detector: Differential refraction detector RI8000 ■Determination of weight average molecular weight by low-angle laser light scattering method Anti-mutagenic alginic acid was dissolved in the following elution solvent and applied to gel permeation chromatography.

・Analysis condition column: TOSOH TSK! llel G6000
PWXL 7.8X300 mlllTOSOH TS
Kgel G4000PWXL 7.8x300mm
TOSOH TSKgel G3000PWXLγ. 8
X300mlTOSOH TSKgel G2500
PWXL 7.8x300 n The above four columns were connected in series in this order and used.

Column temperature: 40°C Elution solvent: 25 mH Na2HPO4, 13.5
mN NaOH (pH 11.6 > Flow rate: 1 m/min. Detector: Differential refraction detector R18011 Low angle laser light scattering detector LS-8000 ■, Results ■ Mutagen suppressing effect of antimutagenic alginic acid (a) The results are shown in Figure 1.

In Figure 1, the solid line shows the inhibitory effect of antimutagenic alginic acid (a) on the mutagen RP-P-1 0.054μ9, and the horizontal axis shows the antimutagenicity added per plate. The amount of alginic acid (#F2 milligrams), the vertical axis shows the number of reverted mutation colonies that were generated, and the number of colonies on the control plate to which no antimutagenic alginic acid was added (this is 1
00). Also,
The dashed line is sodium alginate 500-600 CPS
(manufactured by Kimitsu Chemical Industry Co., Ltd.) (1), the results of the same experiment were shown, and the mutagenicity rate did not decrease even if the amount of alginic acid added was increased, indicating that this alginic acid has antimutagenicity. It shows that it cannot be done.

On the other hand, mutagen (Trp-P-1: 0.05
The amount of antimutagenic alginic acid required to suppress the mutagenicity of 4μ9> by 50% is approximately 200% of the mutagen used.
It can be seen that the amount is 0 to 3000 times (0.134 Jlfff).

(2) Anti-mutagenic alginic acid The weight average molecular weight and anti-mutagenic/carcinogenic properties of alginic acid are shown in Table 2.

(a) to (d) are antimutagenic alginic acids prepared according to the examples of the present invention, and (e) is alginic acid! ! (manufactured by Tokyo Kasei Kogyo Co., Ltd.), (f) is sodium alginate (ultra low viscosity product) 300-500 CPS, (i7) is sodium alginate 15-25 CPS, (h) is sodium alginate 100-20O CP&, ( ) is sodium alginate 500-600 CPS, all manufactured by Kimitsu Chemical Industry Co., Ltd. Regarding mutagenicity and carcinogenicity, anti-mutagenic alginic acids (a) to (d) are all It had the same level of mutagen suppressing effect as mentioned in ① above.Other algin!!(e)～(
Regarding i), adding 10 mg of alginic acid per plate had no effect.

In this way, alginic acid (a) with B/A of 0.5 or more ~
All of (d) are found to have antimutagenic and carcinogenic properties, but
None of alginic acids (e) to (i) with a B/A of less than 0.5 have antimutagenic or carcinogenic properties.

Figure 2 shows the chromatogram when the weight average molecular weight of antimutagenic alginic acid (a) was determined by the low-angle laser beam scattering method. Figure 3 shows the chromatogram (differential refractive intensity) when determined as a standard, and Figure 4 shows the chromatogram when the weight average molecular weight of alginic acid (g) was determined by low-angle laser light scattering method. Fig. 5 shows a chromatogram when the weight average molecular weight of ) was determined using pullulan as a standard.

In FIGS. 2 and 4, the solid line represents the differential refraction intensity, and the broken line represents the light scattering intensity. In addition, in FIGS. 2 to 5, all horizontal axes indicate retention time (minutes).

[Effect of R-light] As explained above, the anti-mutagenic/carcinogenic agent of the present invention is a novel product containing, as its active ingredient, a specific alginic acid that has a strong inhibitory effect on mutagens. . Furthermore, since the food/drink containing the anti-mutagenic/carcinogenic agent contains the above-mentioned alginic acid as an active ingredient, it is useful as a non-mutagenic health food/drink that meets modern requirements.

[Brief explanation of the drawing]

Figure 1 is a characteristic diagram showing an example of the dependence of the added amount of the anti-mutagenic alginic acid of the present invention on the mutagenicity rate, and Figures 2 and 3 are chromatograms of an example of the anti-mutagenic alginic acid of the present invention. Figures 4 and 5 are diagrams showing chromatograms of an example of alginic acid for comparison.

Claims (2)

[Claims] (1) Weight average molecular weight determined using pullulan as a standard using gel permeation chromatography (referred to as A) and weight average molecular weight determined using gel permeation chromatography and low angle laser light scattering method (referred to as B) The ratio (B/A
) is 0.5 or more, an antimutagenic/carcinogenic agent containing alginic acid as an active ingredient. (2) A food or drink product containing the anti-mutagen/carcinogenic agent according to claim (1).