JPS6210968B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for isolating and purifying factors that inhibit mutagenicity of mutagenic substances from broccoli waste. N-Butyl-N-acetoxymethylnitrosamine, reaction product of sorbic acid and nitrous acid, 2-
It is known that thermal decomposition products of amino acids such as aminoanthracene, ethidium bromide, tryptophan, ornithine, citrulline, lysine, and arginine are mutagenic substances. 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 broccoli, which is a familiar part of the diet, contains a factor that inhibits the mutagenicity of mutagenic substances. Ivy. The present invention involves treating broccoli youth containing factors that inhibit the mutagenicity of mutagenic substances with an anion exchange resin, eluating the fraction adsorbed to the resin at a low salt concentration, and 280nm and 404nm
A method for isolating and purifying the mutagenic inhibitor, the method comprising obtaining an active fraction containing the mutagenic inhibitor that has a characteristic absorption spectrum, is stable to heat, and is inactivated by proteolytic enzymes. Regarding. According to the present invention, in order to separate and purify the mutagenic inhibitor from broccoli, the broccoli containing the inhibitor is washed with water, put in a juicer to make juice, treated with an anion exchange resin, and the fraction adsorbed on the resin is This is achieved by elution at low salt concentrations. Anion exchange resins that can be used include DEAE-cellulose, TEAE-cellulose, DEAE-cepharose, and DEAE-cephadex. In order to efficiently adsorb and selectively elute active ingredients using anion exchange resin,
Before that, a step of centrifuging the juice to obtain a supernatant, a step of further ultracentrifuging the supernatant obtained by centrifugation to obtain a supernatant, a step of fractionating ammonium sulfate, redissolving the precipitate and desalting, and a step of cation exchange. Preferably, the pretreatment includes a step of treating with a resin to obtain a fraction that is not adsorbed, either alone or in combination. Treatment with a cation exchange resin can also be carried out later on the fraction adsorbed on the anion exchange resin. In addition, the active ingredient fraction obtained by adsorption to anion exchange resin and elution at low salt concentration is subjected to gel filtration and/or
Alternatively, it can be further purified by ultrafiltration. For the purpose of desalting mentioned above, dialysis using cellulose membranes, artificial kidneys, hollow fibers for ultrafiltration with a molecular weight cut-off of 10,000 or less or around 10,000, and low-molecular molecules such as Cephadex G-25 are used. Gel filtration using a sieve gel can be used. Cation exchange resins include CMC, P-cellulose, CM-Sephadex, SP-Sephadex, CM-Sepharose, Amberlite IRC-
50, there is Amber Light CG50. For the purpose of gel filtration of the active fraction eluted after adsorption to an anion exchange resin, Sephadex G-50,
Sefadex G-75, Sefadex G-
100, Cephacryl, and Biogel P-30 can be used. Ultrafiltration is UM-10, PM-10, PM-30
An ultrafiltration membrane that permeates molecules having a molecular weight of 30,000 or less, or a hollow fiber for ultrafiltration can be used. To avoid deterioration of the active ingredient during operation, the entire processing step is preferably carried out at a temperature below 15°C, more preferably below 4°C. The mutagenic inhibitory factor obtained by the method of the present invention has a characteristic absorption spectrum at 280 nm and 404 nm, is stable to heat, and is inactivated by proteolytic enzymes. The molecular weight of this product was determined to be approximately 55,000 using sodium dodecyl sulfate gel electrophoresis and gel filtration using Sephadex G-100. The substance is a histidine-requiring salmonella described below.
In a mutation test of TA100 strain with 2-aminoanthracene, mutation was significantly inhibited. The mole percentages of the constituent amino acids of the factor are as follows. Asp 14.3 Thr 8.1 Ser 7.6 Glu 6.9 pro 4.9 Gly 8.7 Ala 9.1 Val 6.8 Met 1.4 Ileu 6.7 Leu 11.8 Tyr 1.6 Phe 5.6 Lys 2.6 His 1.0 Arg 3.0 The present invention will be explained in more detail with reference to the following examples. Example After washing 2 kg of broccoli with water, it was made into juice using a juicer. This juice was centrifuged at 9000G for 30 minutes to obtain 900 ml of supernatant. This supernatant was salted out with ammonium sulfate, and the fraction with an ammonium sulfate saturation of 0.15 to 0.75 was centrifuged to obtain a precipitate. Dissolve this in 0.02M phosphate buffer (PH7.0) and use a cellulose membrane to
Dialysis and desalting were performed using 0.02M phosphate buffer (PH7.0). The dialysate was diluted with 0.02M phosphate buffer (PH
7.0) to a buffered DEAE-cellulose column (5 cm in diameter x 22.5 cm in height), and after washing the column with the same buffer, apply a linear concentration gradient of 0 to 0.5 M KCl (0.02 M phosphate buffer) in 900 ml increments. It was eluted. The flow rate was 3 ml/min, and 15 ml was collected per test tube.
This elution curve is shown as an accompanying drawing. KCL concentration
A fraction showing strong inhibitory activity was eluted between 0.05 and 0.15M. DEAE-Cellulose chromatography elution fraction 80
ml to 15ml with PM10, and 14ml of concentrated liquid to 0.1M
Gel filtration was performed using a Sephadex G-75 column (2.5 x 90 cm) equilibrated with phosphate buffer (PH7). The flow rate was 0.3 ml/min, and 5.8 ml was collected per test tube to obtain 70 ml of active fraction. The protein was quantified using the Lowry method to obtain a total protein amount of 19 mg. All the above operations were performed at 4°C. The activity is determined by the histidine-requiring Salmonella described below.
It was determined by a mutation test using TA100 strain with 2-aminoanthracene. In order to know the degree of purification at each stage of purification, we calculated the activity ratio of 50% mutation inhibition using the protein amount measured by the Lowry method as a standard.If the original youth is 1, the ammonium sulfate fractionation shows that the activity ratio is 3. Double, DEAE―
350 by cellulose column chromatography
It was found that the activity was increased by 500 times and 500 times by treatment with Cephadex G-75. Although only about 50% of the mutagenic inhibitory factor isolated and purified from this broccoli was inactivated by heating at 100°C for 90 minutes, it was found that it was inactivated by pronase. It was found to be a proteinaceous substance with a molecular weight of 55,000 by sodium dodecyl sulfate electrophoresis and Cephadex G-100 gel filtration. This factor has a characteristic absorption at 404 nm, which shows a Soret band specific to heme proteins, and when sodium bisulfite is added, the Soret band shifts to 436.5 nm.
From phenomena such as a decrease in absorption intensity, it was recognized that this was due to a change from an oxidized form (Fe ³⁺ ) to a reduced form (Fe ²⁺ ), and this revealed that it had the properties of a heme protein. Mutation inhibition effect test on 2-aminoanthracene: 0.5 ml of each fraction separated by the above method was added to 10 μg of 2-aminoanthracene dissolved in 0.01 ml of dimethyl sulfoxide, and reacted at 37°C for 30 minutes.
To then inactivate all inhibitory factors in the system
Heat treatment at 100℃ for 10 minutes. After heat treatment, add 3 ml of the following soft agar and remove salmonella.
Add 0.1ml of TA100 (histidine auxotrophic) bacterial solution. To this was added 0.5 ml of S-9 mix, which was obtained by removing the liver with enhanced enzyme activity with PCB from SD rats and adding liver microsomes obtained by homogenizing and centrifuging and a buffer solution with the following composition. Pour onto base agar, culture at 37°C for 2 days, and count the number of revertant colonies that do not require histidine.

[Table] The inhibitory effect of 2-aminoanthracene, a mutagenic substance, without brotucolliage use treatment was 5,000 to 6,000 revertant colonies;
-The number of aminoanthracene reverting colonies was counted and the extent to which the number of reverting colonies decreased was investigated. Note that even when the above-mentioned mutagenic substance is used in place of 2-aminoanthracene, a similar mutation-inhibiting effect is exhibited.

[Brief explanation of the drawing]

The figure is an elution curve diagram in DEAE-cellulose column chromatography.

Claims (1)

[Scope of Claims] 1. Broccoli waste containing a factor that inhibits the mutagenicity of mutagenic substances is treated with an anion exchange resin, and the fraction adsorbed to the resin is treated at a low salt concentration. Mutagenic inhibition characterized by elution to obtain an active fraction containing the mutagenic inhibitor that has characteristic absorption spectra at 280 nm and 404 nm, is stable to heat, and is inactivated by proteolytic enzymes. A method of separating and purifying substances. 2. The method according to claim 1, which comprises the step of centrifuging the broccoli youth to obtain a supernatant before the treatment with the anion exchange resin. 3. The method according to claim 2, which includes the step of further ultracentrifuging the centrifuged supernatant to obtain a supernatant. 4. The method according to any one of claims 1 to 3, which includes a step of fractionating broccoli youth with ammonium sulfate, desalting, and dispersing the active fraction before treatment with an anion exchange resin. Method. 5. Before treatment with an anion exchange resin, broccoli youth is brought into contact with a cation exchange resin, or the active fraction obtained by treatment with an anion exchange resin is brought into contact with a cation exchange resin, and the unadsorbed fraction is removed. 5. The method according to any one of claims 1 to 4, for collecting a sample. 6 Claims 1 to 5 include the step of further purifying the active ingredient by performing gel filtration and/or ultrafiltration after eluting the active ingredient adsorbed on the anion exchange resin at a low salt concentration. Either way. 7. The method according to any one of claims 1 to 6, wherein all steps are performed at a temperature of 15°C or lower.