JPS603287B2 - Method for isolating and purifying factors that inhibit mutagenicity of mutagenic substances from cabbage use - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for separating and purifying factors that inhibit mutagenicity of mutagenic substances from cabbage juice.

N-butyl-N-acetoxymethylnitrone amine, the reaction product of sorbic acid and Nishi-nitric acid, 2-aminoanthracene, ethidium bromide, and thermal decomposition products of amino acids such as tryptophan and ornithine are mutagenic substances. Are known.

In the natural world, there are a number of mushrooms that are said to have anticancer effects, such as oyster mushrooms, oyster mushrooms, and shiitake mushrooms, and it is thought that there are other things that have unknown anticancer effects.

As a result of conducting research to search for substances with anticancer effects from nature, the present inventors discovered that cabbage, which is a familiar part of the diet, contains a factor that inhibits the mutagenicity of the thermal decomposition products of tryptophan. Ta.

According to the present invention, in order to separate and purify the above-mentioned mutagenic inhibitor, the cabbage containing the inhibitor is washed with water, kept at a low temperature, for example, 0°C, and run through a juicer to obtain cabbage juice, which is then centrifuged to extract tissue pieces. The supernatant was further ultracentrifuged to collect the supernatant, and the supernatant was contacted with anion exchange cellulose, and the fraction that did not adsorb to the anion exchange cellulose was applied to the intestinal ion exchange cellulose to obtain a low salt concentration. This is achieved by collecting the active fraction eluted in the water, optionally performing ultrafiltration, and then adsorbing the fraction onto a molecular sieve.

Between the step of ultracentrifuging the supernatant and the step of contacting with the anion exchange cellulose, a step of removing the adsorbed low molecules by dialysis or adsorption on a molecular sieve may be included.

Further, throughout the entire process, the operation is preferably carried out at 5°C or lower. Dialysis is carried out using, for example, a cellophane membrane and 50mM H
EPES (N-2-hydroxyethylpiverazine-N'
-2-ethanesulfonic acid) or phosphate buffer.

Further, as the molecular sieve, one that adsorbs low molecules having a molecular weight of 1000 or less is selected. Anion exchange celluloses that can be used include DEAE-cellulose, TEAE-cellulose, and AE-cellulose.

Examples of cation exchange cellulose include CMC, P-cellulose, and PPM-cellulose.

Molecular sieves that can be used in the final step include Cephacryl and Cephadex G-7i Biogel P-30 Kagi. (Cefacryl and Cefadex are from Phahadacia in Sweden, and biogel is from Bio-Ra in the US.
This is the product name of company d. same as below. ) Ultrafiltration membrane is UM
-10, UM-2 eave, PM-30 etc. molecular weight 30000
Ultrafiltration membranes that are permeable to the following molecules can be used: (U.M.
-10, UM-2 plate, and PM-30 are trade names of Amicon, USA. same as below. ) The mutagenic inhibitory factor obtained by the method of the present invention has a characteristic absorption spectrum of 28 Kagawa and 404n, is stable to heat, and is inactivated by proteolytic enzymes. When measured by sodium dodecyl sulfate gel electrophoresis, it was found to be a proteinaceous substance with a molecular weight of about 43,000. This substance significantly inhibited mutation in the mutation test using tryptophan thermal decomposition products of histidine-requiring Salmonella TA9 Kusu described below. The mole percentages of the constituent amino acids of the factor are as follows.

Asp 1
3.22 Thr
843Ser
6.70GIu
6.38Pro
6.56GIy
7. thing AIa
9.89C$
1.73Va1
5.7
7Met l.
29lieu
3.44 Leu
ll. 64 Tyr
l. 31Phe
5.36Lys
2.59High
l. 07Arg
7.40 The method of the present invention will be explained in more detail with the following examples.

1,000 to 2,000 pieces of cabbage were sufficiently washed with water, kept at 0°C, and run through a juicer to obtain cabbage juice.

This juice was centrifuged at 900°C for 30 minutes at 4°C to remove tissue residue such as chloroplasts and mitochondrial nuclei, and the supernatant was ultracentrifuged at 4°C and 20,000°C for 2 hours to remove microsomes, ribbon bands, etc. did. Using cellophane dialysis membrane, we applied 528' results obtained here.
Dialysis was performed against 0mM HEPES buffer (pH 6.8). The dialysate was exchanged twice. The molecular weight is approximately 100 by dialysis.
Low molecular weight compounds of 0 or less are excluded. The dialyzed cabbage juice was then subjected to DEAE-cellulose force ram chromatography. The column size was 4 x 37 cells, the flow rate was 122/hour, and the elution volume was 15/hour. The eluate was a 50 M HEPES buffer (pH 6).
．． 8), a solution in which the potassium chloride concentration was changed stepwise from 0 to 500 mM was used. Elution is performed at 4°C. When the protein absorption of each of the obtained fractions was examined in 28 skins, five separated peaks were observed. When each of these fractions was examined for its inhibitory effect on thermal decomposition products of tryptophan using the test method described below, strong activity was observed only in the fraction that did not adsorb to DEAE-cellulose, that is, the fraction with a potassium chloride concentration of 0. This elution curve is shown in FIG. Fraction 110, in which a strong inhibitory effect was observed
0M, which was subjected to force lamb chromatography on carboxymethyl cellulose. Column size is 2.5
x 8 tubes, flow rate 69 m/hr, elution volume 3' per test tube.
I took one by one. The elution buffer consisted of 50mM HEPES buffer and 120mM KC1 (Na
CI may be used) to create a concentration gradient for KCI, and K
Elution was performed by continuously changing the CI concentration.

All operations were performed at 4°C. When the protein absorption of 28 Kagawa was examined for each of the obtained fractions, four separated peaks were observed. When the inhibitory effect of each of these fractions was examined using the test method described below, it was found that the KCI concentration was 0.
The activity was observed in the fraction having the maximum elution peak of 09M'. When the fractions from 7 test tubes eluted at this low concentration of KCI were collected, 21M was obtained. This melting curve is shown in FIG. This highly active fraction was subjected to ultraviolet filtration using a UM-10 filter and further concentrated. Concentrated fraction 2.0
was further separated and purified using a Sephacryl S-200 column. Column size is 1 x 92 skin, eluent is 50
The elution rate was 40 drops per tube in mM Tris 1 day CI (pH 8.0) buffer. All operations were performed at 4°C. When the protein absorption of each of these fractions was examined in the same manner as described above, four major separated peaks were observed. When the inhibitory effect of each of these fractions was examined using the test method described below, strong inhibitory activity was observed in the second molten fraction. In order to know the degree of purification at each stage of purification, the amount of protein measured by the Lowry method is used as the standard.
When the activity ratio of 50% mutation inhibition was determined, if the original cabbage juice was 1, DEAE cellulose treatment increased the activity ratio to about 2 times, CMC column chromatography increased it to about 20 pm, and CMC column treatment increased it to about 45 pm. It was found that the sound activity was increased.

The mutagenic inhibitory factor isolated and purified from this cabbage is
loo. Although only 50% of C9 powder was inactivated by heating, it was found that pronase inactivated it.
Molecular weight approximately 4 by sodium dodecyl sulfate electrophoresis
It was found to be 3000 proteinaceous substances. This factor has a characteristic absorption of 404n, which shows the Soretband characteristic of heme proteins, and the factor has been determined by atomic absorption spectrometry to have a protein amount of 1.
per 2, iron 0. Molecular weight of this factor is 43.
000, iron content was calculated to be 0.4 mol per mol of protein.

Also, if sodium bisulfite is added, the Soret port
is 436. Based on phenomena such as a shift to normal skin and a decrease in absorption strength, it is recognized that this is due to a change from an oxidized type (Fe30) to a reduced type (Fe20), which indicates that it has the properties of heme protein. did. Mutation inhibition effect test on tryptophan thermal decomposition products: 10% of tryptophan thermal decomposition products dissolved in 0.02% of dimethyl sulfoxide, 0.5% of each fraction separated by the above method was added, and 30% of tryptophan was dissolved in 37q0 to inactivate all inhibitory factors in the system.
Heat for 1 minute.

After heat treatment, soft agar (0.5% DjfcoA saddle
(manufactured by oLaboratories, Inc., USA) 3'
and 0.1 skin of Salmonella TA moth (histidine auxotrophic) bacterial solution. To this, liver microsomes obtained by extracting the liver with increased enzyme activity using PCB from SD rats and homogenizing and centrifuging were added to S-9 mix 0.3 with a buffer consisting of the following composition. ' was poured onto the agar plate shown below, cultured at 37o0 for 2 days, and the number of histidine-independent revertant colonies was determined. Inorganic salts added to S-9: Liver microsomes 3 I〇. 28MNa2HP.

4 4'〇. laMMgC
12 0.5'0.68
MKCI O-5 kite [〇. 〇8M G-61p l. 〇Secretion〇． 〇Yama MNADP I. Agar base for Z selection of 〇: MN (X20)
50 [40% glucose
10 shouts D o NuUient Brotho. 8
% (manufactured by Difco Laboratories, USA)
10 [biotin (10 ratio' secretion)
1m [Agar
15th evening distilled water 930m
Composition of N (×20): (N ratio) 2S04
2.0%KH2P04
20.0%MgS04・7days 20
0.2% sodium citrate
The effect of inhibiting regulation at pH 7.0 with 1.0% KOH is that the number of re-mutated colonies of the thermal decomposition product of tryptophan, a mutagen, without cabbage juice treatment is 300 to 40, compared with this. The number of returning colonies of tryptophan thermal decomposition products treated with cabbage juice was counted, and the extent to which the number of returning colonies decreased was investigated.

[Brief explanation of drawings]

FIG. 1 is an elution curve diagram in DEAE-cellulose column chromatography, and FIG. 2 is an elution curve diagram in CMC column chromatography. Figure 1 Shrimp Figure N Sachifune

Claims (1)

[Scope of Claims] 1. A step of centrifuging a cartridge containing a factor that inhibits the mutagenicity of a mutagenic substance. A step of ultracentrifuging the supernatant. A step of contacting the supernatant with anion exchange cellulose. a step of applying cation-exchanged cellulose to the fraction that does not adsorb to anion-exchanged cellulose, a step of collecting an active fraction eluting at a low salt concentration, and a step of further adsorbing the fraction to a molecular sieve to purify it. A method for isolating and purifying the mutagenic inhibitory factor as described above, which has a characteristic absorption spectrum at 280 nm and 404 nm, is stable to heat, and is inactivated by proteolytic enzymes. 2. Between the step of ultracentrifuging the supernatant and the step of contacting the supernatant with the anion exchange cellulose, the claim includes a step of performing dialysis or removing adsorbed low molecules by adsorption to a molecular sieve. Method of scope 1. 3 Claim 1 in which the entire process is carried out at a temperature of 5°C or less
Section or method of Section 2.