JPH0369891B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a high molecular weight substance that inhibits the mutagenicity of mutagenic substances in edible burdock roots, and a method for isolating and purifying the same. Recently, it is well known that most carcinogenic substances exhibit mutagenicity and that mutagenic substances are highly likely to be carcinogenic substances. Especially the AF-2 (2-(2-Frill)-3-
(5-nitro-2-furyl)acrylamide) and Trp- present in burnt components such as grilled meat and grilled fish.
P-1 (3-amino-1,4-dimethyl-5H-pyrido[4,3-b]-indole) and Trp-
It has been reported that substances such as P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole) exhibit mutagenicity and are carcinogenic to animals. . The present inventors previously proposed a method for separating and purifying factors that inhibit the mutagenicity of mutagenic substances from cabbage use (liquid material obtained by mechanically crushing cabbage). (Unexamined Japanese Patent Publication 1973-
122715 and US Pat. No. 4,191,752). This method consists of (1) centrifuging the used cabbage, (2) ultracentrifuging the supernatant, (3) contacting the supernatant with anion-exchanged cellulose, and (4) adsorption onto the anion-exchanged cellulose. (5) collecting an active fraction eluting at a low salt concentration; (6) further purifying the fraction by adsorbing it to a molecular sieve. This method is characterized by the following. The final fraction thus obtained is a heme protein that has specific absorption spectra at 280 nm and 404 nm, is stable to heat, and has the property of being inactivated by proteolytic enzymes. The inhibitory factors isolated and purified from this use of the cabinet were Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido[4,3-b]-indole),
Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole), N-butyl-N-acetoxynitrosamine, reaction products of sorbic acid and nitrite, 2- It is recognized that it inhibits the mutagenicity of various mutagenic substances that exhibit mutagenicity through the metabolism of aminoanthracene, ethidium bromide, and thermal decomposition products of amino acids such as tryptophan and ornithine. However, as mentioned above, the final fraction from the cabbage use is deactivated in the presence of proteolytic enzymes, and nitroaminobenzene dyes for hair dyes (e.g. 4-nitro-orthophenylenediamine and 2-nitro-paraphenyldiamine) are deactivated in the presence of proteases. Not only is it not possible to inhibit the mutagenicity of mutagenic substances that exhibit mutagenicity without metabolism, such as (enylenediamine), but it also significantly enhances (promotes) the mutagenicity. There is. The present inventor previously reported Agric.Biol.Chem.42(6), 1236~
p. 1238, in 1978, supernatants obtained by centrifuging cabbage, broccoli, shishito pepper, eggplant, apple, burdock, escharot, ginger, pineapple, and mint leaves were found to contain a mutagen of the pyrolysis product of tryptophan. Qualitatively reported that it inhibits sexual activity. All of these vegetables and fruits exhibit the activity of inhibiting mutagenicity caused by substances that are metabolized and exhibit mutagenicity, but according to subsequent research by the present inventor, among these, the Only the purified active fraction exhibits excellent activity to inhibit mutagenicity caused by substances that exhibit mutagenicity without being metabolized, such as nitroaminobenzene dyes, and has extremely specific effects. It was made clear that. For example, among the above-mentioned red juices, only burdock root juices exhibit mutagenicity without being metabolized, such as 2-nitro-p-phenylenediamine, 4-
It can inhibit the mutagenicity of nitroaminobenzene dyes for hair dyes such as nitro-o-phenylenediamine (however, its inhibitory activity is low and not sufficiently satisfactory),
In addition, burdock root does not inhibit the mutagenicity of nitroaminobenzene hair dyes such as 2-nitro-4-aminophenol and 2-amino-5-nitrophenol (as described later). In Example 2, a comparison is made between the high molecular weight material of the present invention and burdock youth). The reason why burdock root juice increases mutagenicity in this way is not clear, but it is thought that certain components (impurities) contained in burdock root juice increase mutagenicity. It will be done. In addition, according to the research of the present inventor, water-soluble substances obtained from plants belonging to the Asteraceae family, such as burdock root, butterbur, and lettuce seedlings, in the same manner as burdock seedlings, can also be used for hair dye. It was also revealed that this product does not exhibit any activity to inhibit mutagenicity caused by substances that exhibit mutagenicity without being metabolized, such as nitroaminobenzene dyes. Incidentally, the above-mentioned document merely discloses a supernatant obtained by centrifuging juices of vegetables and fruits, and does not describe a method for separating and purifying the active ingredient from the supernatant, and thus in a purified state. There is no mention or suggestion of certain active ingredients. Therefore, an object of the present invention is to provide isolated and purified high molecular weight substances that inhibit mutagenicity contained in edible burdock roots. Another object of the present invention is to provide a high molecular weight substance from burdock roots in an isolated and purified state, which has the activity of inhibiting the mutagenicity of substances that exhibit mutagenicity without metabolism. be. Still another object of the present invention is to provide the above-mentioned high molecular weight substance in a purified state, which does not substantially reduce its mutagenicity inhibiting activity even when treated at elevated temperatures. Still another object of the present invention is to provide a method for efficiently separating and purifying the above-mentioned high molecular weight substances from burdock. Further objects and advantages of the invention will become apparent from the description below. These objects and advantages of the present invention are, according to the present invention, a high molecular weight substance which is extracted and separated from the roots of edible burdock and is soluble in water or an aqueous alkali solution, which is adsorbed on anion exchange cellulose but is not oxidized. It is not adsorbed on ion-exchanged cellulose, has an absorption wavelength peak in the range of 280-300 nm, and was tested using 2-nitro-p-phenylenediamine as a mutagenic substance according to the inhibitory effect test described in the text. When measuring the number of revertant colonies from Salmonella TA98, the following formula was used: DM (%) = (ac) - (b-c) / a-c x 100, where a is the number of colonies in the absence of this high molecular weight substance. is the number of revertant colonies in the presence of this high molecular weight substance, b is the number of revertant colonies in the presence of this high molecular weight substance, and c is the number of spontaneous mutant colonies, the mutation inhibition rate (%) defined as has a mutation inhibiting activity such that the concentration of the high molecular weight substance is 4 to 6 parts by weight per 1 part by weight of the 2-nitro-p-phenylenediamine, and the elemental analysis value is H :4.86±0.14(%), C:48.01±0.28(%), N:
5.53±0.41(%) and O: 41.60±0.55(%), the sugar content is 9.55±0.25% by weight, and the ninhydrin color reaction is substantially negative. This is achieved by the above-mentioned high molecular weight substance extracted and separated from the roots of burdock. Further, the above-mentioned high molecular weight substance provided by the present invention can be obtained by (a) centrifuging burdock root;
(b) Add 1-2M phosphate buffer (PH) to the supernatant to remove impurities.
6.5 to about 7.5), add an aqueous metal salt or ammonium salt of a water-soluble inorganic acid to this mixture for salting out, and then separate the precipitate. After dissolving in 400mM phosphate buffer (PH about 6.5 to about 7.5), dialyze the solution; (d) extract the concentrate by ultrafiltration of the dialysate, or freeze-dry the concentrate; It can be separated from burdock and purified by a method that combines the steps of pulverization and the steps described below. In the present invention, burdock belongs to the family Compositae and the genus Arctium.
Arctium Lappa Linne or Arctium Lappa L.
It is said to be something that can be eaten. In the present invention, the burdock root used as a target for separating and purifying the active fraction is a burdock root sown in spring or autumn and harvested within three years thereafter, preferably sown in spring and then harvested within three years. Burdock roots are harvested in the winter of 2009 or sown in the fall and harvested in the spring of the following year. These can be used fresh or dried. According to the research of the present inventors, the high molecular weight substance obtained from burdock provided by the present invention shows that its mutagenicity inhibiting activity can be improved by treatment at an elevated temperature, e.g., about 100°C for 15 minutes. It has been revealed that it has excellent properties such that it does not substantially deteriorate even when it is heated. Similarly, another characteristic of the high molecular weight substance is that the inhibitory activity is greatly reduced by manganese ions, but the inhibitory activity is not substantially reduced by magnesium or calcium ions. It was also made clear that When the mutagenic inhibitor is separated and purified from the roots of burdock according to the method of the present invention, if it contains impurities, the impurities contained therein are first removed by centrifuging the burdock roots. Burdock root use is obtained by washing raw burdock root with water and passing it through a juicer equipped with a filter to remove most of the fibers, or obtaining it as an aqueous solution or aqueous suspension, or by adding dried burdock root powder to water or a phosphate buffer. Suspended in liquid (PH about 6.5 to about 7.5) for a sufficient time at an elevated temperature if necessary or ground in a grinder to remove most of the fibrous material and form an aqueous solution or suspension. can be manufactured. Dried burdock root powder should be as fine as possible, and when using this powder it is usually approx.
Preferably, the juice is prepared as an aqueous solution or suspension by grinding in a mill at a temperature of 20 DEG to about 85 DEG C. for about 2 to 24 hours. If the thus produced burdock juice still contains impurities, it is centrifuged to remove the impurities. The centrifugation may be normal centrifugation at 7,000 to 15,000×G, or may be ultracentrifugation at 100,000 to 200,000×G. Alternatively, ultracentrifugation may be performed after normal centrifugation. Conventional centrifugation is usually carried out for 30 to 60 minutes, and thereby removes fibers, chloroplasts, mitochondria, etc. in the juice. Moreover, it is preferable to carry out ultracentrifugation for usually 1 to 2 hours, thereby further removing microsomes, ribosomes, and the like. After removing impurities from the burdock root as described above, the supernatant is then added to a phosphate buffer solution (about PH6.5).
~7.5) is added. When juice is produced by adding a phosphate buffer solution to burdock powder, the phosphate buffer solution may not be further added depending on the case. It will be understood that this case also falls within the scope of the present invention. As mentioned above, the phosphate buffer solution added to the burdock root is preferably in the range of about 6.5 to about 7.5. These are usually the monosodium or monopotassium salt of phosphoric acid and the dibasic salt of phosphoric acid.
-sodium salt or 2-potassium salt.
It preferably consists of 1-potassium phosphate and 2-potassium phosphate. Further, it is convenient to use a phosphate buffer solution having a concentration of about 1 to 2 molar. The phosphate buffer solution is 1/20 to 1/2 of the supernatant.
Preferably, it is added in an amount of 100 parts by volume. The phosphate buffer solution has a concentration of phosphate ions in the supernatant to which the phosphate buffer solution is added, from about 10 to about
It is best to add it to a concentration of 400mM. The salt that is then added to the resulting solution for salting out is an alkali metal or ammonium salt of a water-soluble inorganic acid. Examples of the inorganic acid include mineral acids such as carbonic acid, sulfuric acid, hydrochloric acid, and phosphoric acid. As the alkali metal salt or ammonium salt of such an inorganic acid, preferably potassium carbonate,
Ammonium sulfate, sodium sulfate, potassium phosphate, etc. are used. The amount of the alkali metal salt or ammonium salt of a water-soluble inorganic acid used is preferably about 30 to 80% by weight, based on the volume of the supernatant-phosphate buffer mixture. The solution containing the precipitate thus salted out is then separated from the precipitate. Various means can be used for separation, such as filtration or centrifugation. The filtration can be carried out under normal pressure, reduced pressure or increased pressure. Preferably, separation is performed by centrifugation. Centrifugation is usually carried out by so-called conventional centrifugation under the conditions described above. The separated and obtained precipitate is then separated from about 10 to about
It is dissolved in 400mM phosphate buffer solution (PH6.5-7.5) and subjected to dialysis as a solution. It is usually desirable to use the phosphate buffer solution in an amount of about 5 to 10 ml per gram of precipitate. Dialysis can be performed using water or a phosphate buffer, but it is usually preferable to use a phosphate buffer. The dialysis membrane used in dialysis is a cellulose membrane or a cellophane membrane for artificial kidneys. The dialysis solution, which has thus been freed from low molecular weight substances by dialysis, is then subjected to ultrafiltration. The dialysis solution is concentrated by ultrafiltration. It is desirable to continue ultrafiltration until dialysis reaches 1/3 to 1/5 volume. Filters that can be used for ultraviolet rays include:
Examples include PM-30, XM-50, XM-100, and XM-300 (manufactured by Amicon), but the molecular weight
XM-, an ultrafiltration membrane that passes less than 300,000 molecules
300 is particularly preferred. The concentrate thus subjected to ultrafiltration constitutes the high molecular weight material of the present invention. According to the present invention, such a concentrated solution can be further subjected to conventional freeze-drying to form a powder, if necessary. When the concentrate contains a phosphate buffer, it is desirable to add about 3 to 5 times the volume of water to the concentrate and subject it to ultrafiltration again. The high molecular weight substance of the present invention extracted and separated from the edible burdock root by the method of the present invention has sufficient mutation inhibitory activity as determined by the mutation inhibition rate measured according to the inhibitory effect test described later. This was appreciated. According to the method of the present invention, in contrast to the above method of the present invention,
By making further partial changes, the high molecular weight substance of the present invention can be produced with even higher purity. Such a modification is a step in which the dialysate is treated with a cation exchanger before being subjected to ultrafiltration, and the treated liquid is subjected to ultrafiltration. Since the high molecular weight compounds of the present invention are not adsorbed on the cation exchanger, this step removes impurities that would be adsorbed on the cation exchanger. Examples of the cation exchanger include cellulose and dextran exchangers having a carboxyl group, sulfonic acid group, or phosphoric acid group as a cation exchange group, such as CM-cellulose, P-cellulose, CM-cephadex, SP-cephadex, or CM- Cephalose or the like (manufactured by Pharmacia or Watmann) is used. Another such change is that the concentrate obtained by ultrafiltration (which may or may not be treated with a cation exchanger before ultrafiltration) is , about 10 to about 400mM
In this step, the sample is diluted with a phosphate buffer (pH about 6.5 to about 7.5) and subjected to ultrafiltration again. This second ultra-pass is preferably carried out 2 to 3 times, each time 1/3
Concentrate to ~1/5 volume and dilute with phosphate buffer to the volume before ultrafiltration. This second ultrafiltration process produces substances ranging from low molecular weight to considerably high molecular weight (approximately
A considerable amount of impurities (up to 300,000) can be removed. When this ultrafiltration is carried out again, it is necessary to dialyze the obtained concentrate against water, thereby obtaining the high molecular weight substance of the present invention as a phosphate-free solution. be able to. Further, another such change is a step of treating the concentrated liquid obtained by ultrafiltration or ultrafiltration again with trichloroacetic acid to cause precipitation. By separating the precipitate from this system, impurities mainly derived from sugar can be removed from the concentrate. The separated precipitate is then suspended in water and dialyzed against water to remove the trichloroacetic acid it contains, giving the high molecular weight substance of the invention in a highly pure state. In the above process, trichloroacetic acid can be used in a proportion of 1 to 10 g per 100 c.c. of the concentrated liquid to which trichloroacetic acid is added. Also,
The generated precipitate can be separated, for example, by centrifugation, filtration, or the like. According to the method of the present invention, by carrying out the method consisting of the above (a) to (d) in combination with any one or more of the above three changes, the present invention can be carried out in an extremely highly pure state. of high molecular weight substances are obtained. The high molecular weight compound of the present invention produced with such changes exhibits a property of being more easily soluble in an alkaline aqueous solution than in water. The purified high molecular weight substances in concentrate or powder form provided by the present invention include:
It is administered to humans or other non-human animals either as such or in combination with other pharmaceutically acceptable carriers or adjuvants as appropriate. According to the invention, there is therefore also provided a pharmaceutical composition comprising the high molecular weight substance of the invention, optionally together with a pharmaceutically acceptable carrier or adjuvant, or a medicament comprising said composition. Examples of carriers or adjuvants commonly known to those skilled in the art include excipients (e.g., starches such as corn starch and potato starch; sugars such as lactose and diosaccharide; inorganic salts such as calcium sulfate and calcium phosphate); lubricants (e.g., stearin); acid magnesium, talc), disintegrants (e.g. carboxymethylcellulose, cellulose, agar, etc.),
Examples include encapsulants for capsules (eg, gelatin). Examples of the drug form include tablets, granules, sugar coatings, powders, syrups, solutions, and capsules. The high molecular weight substance of the present invention in the above form is preferably administered orally. Moreover, the high molecular weight substance of the present invention can also be used for external administration (skin application). In this case, ointments such as ointments with an oil-soluble base such as wax,
It is preferable to use it in the form of a water-containing emulsion, an aqueous solution, or the like. According to the present invention, the high molecular weight substance of the present invention is administered to an animal before or after ingesting a mutagenic substance, alone or in the form of the above-mentioned composition or drug, to prevent mutations from occurring. A method is provided for preventing mutations caused by a mutagenic agent in an animal by administering an amount effective to cause the mutagenic agent to mutagenize the animal. The dosage can be appropriately determined for the target animal by a specialist such as a doctor or pharmacist, but is usually about 100 to 2.5 g/Kg body weight/day. Since the active fraction of the present invention is a component contained in burdock root, which is eaten as food, its toxicity is not a problem. Ingesting a mutagenic substance includes not only ingesting the substance but also contacting the substance. The high molecular weight substance of the present invention is extremely effective in preventing mutations caused by not only substances that exhibit mutagenicity without undergoing metabolism but also substances that exhibit mutagenicity after undergoing metabolism (mutagenic substances). ing. In particular, it is unique in that it has an excellent effect that is completely different from that of burdock root, even though it is a component derived from burdock root, against substances that are mutagenic without being metabolized. This is quite surprising. Examples of mutagenic substances that exhibit mutagenicity without undergoing the above metabolism include 2-nitro-p-
Examples include phenylene diamine, 4-nitro-o-phenylene diamine, 2-amino-4-nitrophenol, 2-amino-5-nitrophenol, and salts thereof such as sulfates. Examples of mutagenic substances that exhibit mutagenicity after being metabolized include p-phenylenediamine, o-phenylenediamine, 2,4-diaminoanisole, and salts thereof, such as sulfates. can. The high molecular weight substance obtained by freeze-drying provided by the present invention is convenient to make into any form of drug, and is an odorless, brown powder that is soluble in water or alkali and is stable. It has particularly excellent storage stability. According to the research of the present inventors, the mutation-inhibiting activity exhibited by the high molecular weight substance of the present invention is such that the high molecular weight compound acts directly on the mutagenic substance expressed by the mutagenic substance. It was thought that the expression was inhibited by the action of Therefore, it is desirable to use the high molecular weight substance of the present invention preventively, especially in animals that may ingest mutagenic substances or have clearly ingested mutagenic substances, to prevent the development of mutagenicity. . EXAMPLES Hereinafter, the method of separating and purifying a high molecular weight substance by the method of the present invention, the mutation inhibiting activity and other properties of the high molecular weight substance of the present invention will be explained in more detail with reference to Examples. Example 1 (comparative example) (1) Separation and purification of active high molecular weight substances from burdock: After washing burdock roots (5000 g) harvested in winter with water,
Crushed with Juicer (Toshiba JC-540A), approximately
Obtained 3400ml of burdock youth. 3400 ml of this juice was centrifuged at 9000×G for 30 minutes to obtain 2200 ml of brown transparent supernatant. This supernatant was added to a 1M phosphate buffer (PH6.8, consisting of 2-potassium phosphate and 1-potassium phosphate).
(same below) was mixed in 1/20 amount (volume). Next, ammonium sulfate was added to 1100 ml of this mixture in an amount of 80% of its weight to perform salting out. This salting out mixture was then centrifuged at 9000xG for 15 minutes to remove the precipitate.
I got 80g. 60g of this precipitate was added to 50mM phosphate buffer (PH6.8).
was used to make the total volume 600 ml. This solution was heated to 4°C using the above phosphate buffer (PH).
6.8) to obtain 696 ml of dialysate. Next, 464 ml of this dialysate was subjected to ultrafiltration using a membrane filter XM-300 (manufactured by Amicon) and concentrated to ⅓ volume. 50mM phosphate buffer (PH6.8) was added to this concentrate to bring the total volume to 200ml, and the concentrate was again concentrated by ultrafiltration using the same filter as above to obtain 140ml of the concentrate. Of this, 70 ml of the ultra-superconcentrated liquid was freeze-dried at -54°C for 24 hours using a freeze dryer (manufactured by Central Kagaku Co., Ltd., FDX-1-54) to obtain 450 mg of high molecular weight substance as a dry powder. Obtained. (2) Ultraviolet absorption characteristics of active high molecular weight substances Figure 1 shows the ultraviolet absorption curve measured for an aqueous solution of freeze-dried powder. In FIG. 1, the vertical axis represents absorbance, and the horizontal axis represents wavelength (nm). As is clear from FIG. 1, the active fraction of the present invention has an absorption wavelength peak in the range of 280 nm to 300 nm (maximum wavelength of about 290 nm). (3) Adsorption of active high molecular weight substances to anion exchange resin and cation exchange resin 27 ml of the concentrate obtained by the method (1) above was
It was sent to a cellulose (anion exchange cellulose) chromatography column. The condition is column size
The size was 2.5 x 12 cm, the elution rate was 36 m/hr, and the elution volume was 10 ml/test tube. The elution buffer was 50 mM potassium chloride dissolved in 50 mM phosphate buffer, and the concentration was gradually increased to 2M potassium chloride to examine the adsorption properties. As a result, no fraction exhibiting inhibitory activity was detected in the fractions eluted without being adsorbed to the DEAE-cellulose column. Similarly, 7 ml of the above concentrated solution was sent to a CM-cellulose (cation exchange cellulose) column chromatograph. The conditions are: column size 2.5 x 7 cm;
The elution rate was 40 mg/hr and the elution volume was 5.8 ml/test tube.
Elution buffer was dissolved in 50mM phosphate buffer
Increasing concentrations starting with 50mM potassium chloride
The adsorption properties were investigated by elution up to 0.1M potassium chloride. As a result, it was not adsorbed to the CM-cellulose column and was completely eluted. The elution curve is shown in Figure 2 of the attached drawings. The horizontal line above the peak in FIG. 2 indicates that a fraction of that width has mutagenicity inhibiting activity. The eluted fraction was subjected to an inhibitory effect test, which will be described later, and the mutation inhibition rate was 88%. This is an aqueous solution of dry powder in Table 1 (described below).
mg/ml) was almost consistent with the mutation inhibition rate (87.4%) of 500 μl/plate. From the results of this mutation inhibition rate, it was confirmed that the active high molecular weight substance of the present invention is a polymer electrolyte having a strong anionic group. (4) Inhibitory effect of active high molecular weight substances (a) Inhibitory effect test was conducted using dimethyl sulfoxide.
Add the fractions obtained in each step of the above method (1) or the final fraction (fraction containing the high molecular weight substance of the present invention) to the mutagenic substance dissolved in 0.02 ml.
0.5 ml was added and reacted at 37°C for 30 minutes. As a control, 0.5% phosphate buffer was added instead of each fraction sample.
ml and the same reaction was carried out. After that, in order to sterilize the reaction system, the temperature was increased to 100°C.
The mixture was heat-treated for 10 minutes. After heat treatment, soft agar (0.6% Difco-
Add 3 ml of Salmonella TA98 (histidine auxotrophic) and 0.1 ml of Salmonella TA98 (histidine auxotrophic) bacterial solution, pour onto the selective agar medium described below, culture at 37°C for 2 days, and count the number of revertant colonies that do not require histidine. In addition, the mutagenic substance is one that expresses mutagenicity only through metabolism (for example, 2-aminoanthracene, ethidium bromide,
Trp-P-1, Trp-P-2, etc.) were investigated in the same manner except that 0.3ml of S-9mix obtained by adjusting as described below was added to the soft agar. . S
-9mix was prepared as follows. Specifically, using SD rats, a liver homogenate with enhanced drug-metabolizing enzyme activity is obtained by PCB, and then the liver microsome fraction (S-9) is obtained by centrifuging this liver homogenate. The S-9mix was prepared by adding an inorganic salt having the following composition to the obtained liver microsomes. Inorganic salts added to S-9: Liver microsomes 3ml 0.25M phosphate buffer 4ml 0.16M MgCl ₂ 0.5ml 0.66M KCl 0.5ml 0.05M G-6-P 1.0ml 0.04M NADP 1.0ml Selective agar medium: MM (X20) 50ml 40% Glucose 10ml 0.8% Difco nutrient broth 10ml Biotin (100μg/ml) 1ml Agar 15g Distilled water 930ml Composition of MM (X20): (NH ₄ ) ₂ SO ₄ 2.0% KH ₂ PO ₄ 20.0% MgSO ₄・7H ₂ O 0.2% Sodium citrate 1.0% Adjusted to pH 7.0 with KOH The activity of the inhibitory effect was determined by varying the concentration of the high molecular weight substance of the present invention against the mutagenic substance in the same manner as above. The amount (mg/plate) of the high molecular weight substance required to inhibit 50% of the mutagenicity determined by the inhibition effect test was determined and expressed. (b) Mutagenicity inhibitory effect on 2-nitro-p-phenylenediamine (80 μg/plate) Table 1 shows the concentration dependence of the mutation inhibitory effect of the dry powder (active high molecular weight substance of the present invention). .

[Table] As is clear from the above results, the high molecular weight substance of the present invention shows concentration dependence (Table 1);
In any case, it showed a good inhibitory effect on 2-nitroparaphenylenediamine. Table 2 shows the inhibitory effect of the high molecular weight substance (final fraction) of the present invention on mutagenicity induced by various mutagenic substances.

[Table] Nylenediamine
40 + 56.9

TABLE The results in Table 2 demonstrate the particularly pronounced inhibitory effect of the high molecular weight substances of the invention. That is, the inhibitory factors present in the juice of cabbage and broccoli, which are plants reported in the above literature, are mutagenic substances (ethidium bromide, 2-aminoanthracene, etc.) that exhibit mutagenicity through metabolism.
Some of the nitroaminobenzene dyes (2-nitroparaphenylenediamine, 4 -nitro-orthophenylenediamine, etc.), whereas the high molecular weight substance of the present invention exhibits a remarkable inhibitory effect against any of the above-mentioned mutagenic substances as shown in Table 2. The specificity of its action and effect is remarkable. (5) Heat resistance of active high molecular weight substance Next, the heat resistance of the high molecular weight substance of the present invention was investigated. That is, 500 μl each of the final fraction (aqueous solution of lyophilized powder) of the present invention was heated under reflux at 100°C for 15 minutes and then cooled to room temperature, and 500 μl of each of the final fraction (aqueous solution) without heat treatment was added to the third It was mixed with each mutagenic substance at the concentration shown in the table and reacted at 37°C for 30 minutes. Next, an inhibitory effect test was conducted in the same manner as in (4) above. The results are shown in Table 3.

[Table] As described above, it was confirmed that the high molecular weight substance of the present invention is stable and does not deactivate even when heated, and also exhibits a good inhibitory effect. (6) Stability of active fraction against polyvalent metal ions Furthermore, the stability of the high molecular weight substance of the present invention (final fraction) against various polyvalent metal ions was investigated. That is, each of magnesium chloride, manganese chloride, and calcium chloride was added to the high molecular weight substance of the present invention (an aqueous solution of freeze-dried powder in the final fraction) so that the concentration in the solution (final concentration) obtained was 10 mM. Mixed and dissolved. Each of these solutions was incubated at 37 °C for 30
Processed for minutes. Thereafter, 500 μl of these reaction mixtures were mixed with each mutagenic substance at the concentrations shown in Table 4, and
The reaction was carried out at 37°C for 30 minutes. After the reaction, an inhibitory effect test was conducted in the same manner as described in (4) above. The results are shown in Table 4.

【table】

[Table] As shown above, the final fraction of the present invention is extremely stable against magnesium chloride and calcium chloride, but has the specificity that its mutagenic inhibitory activity is greatly reduced by manganese chloride. It has been confirmed that it is equipped. Example 2 (1) Separation and purification of active high molecular weight substances from burdock: After washing burdock roots (5000 g) harvested in spring with water,
Crushed with Juicer (Toshiba JC-540A), approximately
Obtained 3400ml of burdock youth. 3400 ml of this juice was centrifuged at 9000×G for 30 minutes to obtain 2600 ml of brown transparent supernatant. To this supernatant, 1/20 volume (volume) of 1M phosphate buffer (PH6.8) was mixed. Then add 1500ml of this mixture to 80% of its weight.
Salting out was carried out by adding the weight of ammonium sulfate. This salting-out mixture was then centrifuged at 9000×g for 15 minutes.
90g of precipitate was obtained. 90g of this precipitate was added to 50mM phosphate buffer (PH6.8).
The total volume was 1000 ml. This solution was heated to 4°C using the above phosphate buffer (PH).
6.8) Dialyzed (cellulose membrane) and dialysate 1162
Got ml. Next, 962 ml of this dialysate was added to CM-cellulose (manufactured by Watmann, wet volume 500 g) that had been equilibrated with 50 mM phosphate buffer (PH6.8).
After stirring thoroughly at room temperature for 5 minutes, the mixture was filtered under reduced pressure. CM−
The liquid as a fraction that did not adsorb to cellulose
880ml was obtained. Next, 800 ml of this liquid was subjected to ultrafiltration using a membrane filter XM-300 (manufactured by Amicon) and concentrated to 1/4 volume. 50mM phosphate buffer (PH6.8) was added to this concentrated solution to make the total volume 800ml, and the mixture was again concentrated by ultrafiltration using the same filter as above, and the same operation was repeated twice. After ultrafiltration, 200 ml of a concentrated solution (hereinafter referred to as final ultrafiltration solution) was obtained. Trichloroacetic acid was gradually added to 130 ml of the ultraconcentrated solution at room temperature under stirring to give a final concentration of 2%. After stirring the solution for 10 minutes at room temperature, the precipitate formed was
Centrifugation was performed at ×g for 15 minutes to obtain 4.5 g of precipitate. This precipitate was resuspended in 130 ml of water. This precipitate was dialyzed against water and desalted to obtain about 160 ml of dialysate. This was freeze-dried at -54℃ for 48 hours using a freeze dryer (manufactured by Central Kagaku Co., Ltd., FDX-1-54) to form a dry powder.
730 mg (high molecular weight substance of the present invention) was obtained. (2) Ultraviolet absorption characteristics of active high molecular weight substance Same as the active high molecular weight substance of Example 1 (1), 280 nm
The peak of absorption wavelength is in the range of ~300 nm (maximum wavelength approx.
290nm). (3) Adsorption properties of active polymeric substances to anion-exchanged cellulose and cation-exchanged cellulose As shown in Example 1 (1), the active polymeric substances are adsorbed to anion-exchanged cellulose, but not to cation-exchanged cellulose. It had a characteristic. (4) Inhibitory effect of active high molecular weight substance (a) The inhibitory effect test was conducted in the same manner as described in Example 1 (4) (a). (b) Mutation inhibition effect on 2-nitro-p-phenylenediamine (200 μg/plate) 50% inhibition of mutagenicity determined for each fraction obtained in each step of the method (1) above. The dry weight of each fraction required for this is shown in Table 5.

[Table] Also, dry powder (active high molecular weight substance of the present invention)
Table 6 shows the concentration dependence of the mutation inhibition effect of .

[Table] As is clear from the above results, the inhibitory activity of the high molecular weight substance (lyophilized powder) of the present invention is 9000×
The inhibitory activity of the G supernatant (control) is increased approximately 15 times and that of the dialysate after salting out (Table 5), and the final fraction of the present invention exhibits its concentration dependence ( Table 6) showed a good inhibitory effect on 2-nitroparaphenylenediamine in any case. Table 2 shows the inhibitory effect of the active high molecular weight substance of the present invention (final fraction) on mutagenicity induced by various mutagenic substances. We considered the case where it is not done. Results similar to those shown in Table 2 were obtained. (5) Active polymeric substance heat resistance and stability against polyvalent metal ions These properties were similar to those of the active polymeric substance of Example 1. (6) Intrinsic viscosity of active high molecular weight substance The final fraction was dissolved in a 1% NaOH aqueous solution,
1mg/ml, 0.8mg/ml, 0.5mg/ml and 0.25mg/ml
Each concentration was prepared in ml. Take 0.5ml of this solution,
The viscosity was determined by measuring the falling time at 25±0.1°C using a Canon Manning semi-micro viscometer No. 100 (Kabuki Kagaku Kikai Kogyo Co., Ltd.). Separately, as a control experiment, the falling time of a 1% NaOH aqueous solution was measured. The results are shown in Table 8. From the viscosity values at each concentration of the final fraction, a straight line showing the relationship between reduced viscosity (ηsp/c) and concentration (C) was determined by the least squares method, and the intrinsic viscosity was determined by extrapolating it to zero concentration. . The intrinsic viscosity [η] of the final fraction is
It was determined to be 37.0ml/g. Table 8 Final fraction concentration (mg/ml) ηsp*/c 1 37.35 0.8 37.23 0.5 37.67 0.25 36.78 (7) Elemental analysis of active high molecular weight substances Table 9 shows the results of elemental analysis of the final fraction. Note that elemental analysis was performed for H, C, and N, and other values were calculated as oxygen atoms.

[Table] (8) Carboxyl group of active high molecular weight substance The ¹³ C-NMR spectrum of the final fraction (solid) was measured using tetramethylsilane (TMS) as an internal standard (Instrument: JEOL FX-100,
Manufactured by JEOL Ltd.; Attachment cloth
polarization/Magic Angle Spinning (CP/
MAS) unit). As a result, a wide range of signals with maximum values of 173.68, 144.64, 127.97, 72.32 and 31.09ppm (δTMS) was observed ( ¹³ C Resonance Frequency =
25.05MHz, Contact Time=1.0ms). The above signal at 173.68 ppm indicates the presence of carboxyl groups. In addition, 20 mg of the final fraction was added to N/100NaOH (=
1000, manufactured by Wako Pure Chemical Industries, Ltd.), and potentiometric titration (back titration) was performed using volumetric analysis N/100H ₂ SO ₄ (=1000, manufactured by Wako Pure Chemical Industries, Ltd.). As a result, 13 ml of N/100H ₂ SO ₄ was required. Based on the above titration based only on carboxyl groups, the final fraction was calculated to have 3.5 eq/10 ³ g of carboxyl groups. (9) Pyrolysis product of active high molecular weight substance 3 mg of the final fraction was pyrolyzed at 590°C using a Curie Point Pylolyzer (manufactured by Nippon Analytical Kogyo, JHP-2 type),
GC-Mass (manufactured by JEOL, JGC-20KP and
JEOL-D300) was used to identify the thermal decomposition products. GC (gas chromatography) is FFAP
A 0.25 mmφ x 80 m capillary column (Wall
coated open tubular capillary column (manufactured by Gascro Industries Co., Ltd.) at a heating rate of 10℃/min.
It was carried out under conditions in the temperature range of 50-180°C. In addition, the ionization voltage of Mass was 70 eV (the same applies hereinafter). As a result, the presence of CO ₂ , phenol, toluene, benzene, cresol, and styrene as main components was confirmed. (10) Reductive decomposition of active high molecular weight substances 1 g of the final fraction and 4 g of Raney Ni in 4% NaOH solution
Dissolve in 100ml _H2 gas 100 in autoclave
The final fraction was reductively decomposed under a pressure of Kg/cm ² at 100° C. for 6 hours. After the reaction, let it cool to room temperature,
The reaction mixture was extracted with dichloromethane for spectrometry, and the product was identified using GC-Mass using the same equipment as above. GC uses silicon SE-30 as the stationary phase liquid.
0.25mmφ×50m capillary column (Wall
coated open tubular capillary column (manufactured by Gascro Industries Co., Ltd.) at a heating rate of 10℃/min.
It was carried out under conditions in the temperature range of 100-250°C. As a result, benzoic acid, phthalide, and 3-phenylpropanol were detected as the main components. (11) Oxidative decomposition of active high molecular weight substances To 500 mg of the final fraction, 5 ml of diazomethane ether solution was added and reacted at room temperature for 30 minutes. After the reaction,
The solid matter was filtered, separated, and washed several times with dichloromethane. The solid was prepared by S. Larsson et al. (Acta.Chem.
Oxidative decomposition was performed with potassium permanganate according to the method of Scad.25647 (1971). The oxidative decomposition product is converted into chloroform:acetone=
Extraction was carried out at a ratio of 1:1 (volume ratio). A residue was obtained by drying with glass salt overnight and removing the solvent. This residue was dissolved in 0.5 ml of methanol. The above methanol solution was GC-GC-
The product was identified by Mass. the result,
Benzoic acid was detected. GC is 15% FFAP (Packed) on Gas-Chrom Q.
Using a 2.0 mmφ x 2 m packed column using Column, Applied Science Co., Ltd.) as a distributing agent,
The temperature range was 50 to 200°C with a heating rate of min. (12) Sugar content of active high molecular weight substance The aqueous solution obtained by heating and dissolving 10 mg of the lyophilized powder of the final fraction in 10 ml of 50 mM phosphate buffer was analyzed using the phenol-sulfuric acid method (M.Dubolis.et.al.Anal.
Chem., 28 , 350 (1956)).
It contained 95.7 μg of sugar per ml of solution. Therefore, the total sugar content in the lyophilized powder was calculated to be 9.55% by weight. (13) Ninhydrin color reaction of active high molecular weight substances The lyophilized powder of the final fraction was dissolved in 50mM phosphate buffer (PH6.8) and added to 0.2ml of a solution (1mg/ml).
0.2 ml of 35% concentrated hydrochloric acid was added and hydrolysis was carried out at 110°C for 2 hours. The solvent was removed under reduced pressure at 50°C to obtain a dry residue. This dry residue was dissolved in 50mM phosphate buffer (PH6.8).
Dissolve in 1 ml of silica gel thin layer plate (Wakopure
Chemical Industries Ltd) with n-propanol:water=64:36 (volume ratio).
An attempt was made to develop color using ninhydrin, but no color was observed on the thin silica gel plate. Ninhydrin color reaction is known as a color reaction of amino acids. Example 3 6 mg of the final fraction (lyophilized powder) obtained in Example 2 (1) was added to 1 ml of 50 mM phosphate buffer (PH6.8).
dissolved in Using this solution, the mutation inhibition effect on various mutagenic substances listed in Table 10 was determined according to the method described in Example 1 (4). The results are shown in Table 10. For comparison, the same test was conducted on the 9000×G supernatant fraction (used at 0.5 ml/plate) in Example 2 (1). The results are shown in Table 10.

[Table] It shows that.
From the above results, o-phenylenediamine, p-phenylenediamine, and 2,4
- For diaminoanisole, the active high molecular weight substance of the present invention and the 9000×G supernatant similarly inhibit the expression of mutagenicity, but mutations that are not metabolized and exhibit mutagenicity 2-amino-4-nitrophenol and 2-amino-
Regarding 5-nitrophenol, the active high molecular weight substance of the present invention inhibits the expression of mutagenicity, but
On the contrary, it can be seen that the supernatant liquid at 9000×G significantly promotes mutagenicity. In short, the high molecular weight substance of the present invention is a high molecular weight substance that has a strong anionic group and has an absorption wavelength peak in the range of 280 nm to 300 nm, as is clear from the above results, and It has the property of greatly reducing the inhibitory activity due to manganese ions, is extremely stable to heat, and has a high ability to inhibit the mutagenicity of many mutagenic substances, especially those that express mutagenicity without undergoing metabolism. Has inhibitory activity. Although the complete structure of the high molecular weight substance of the present invention is not clear, as is clear from the above analysis results, it contains carbon, hydrogen, nitrogen, and oxygen, and has a benzene ring, a sugar structure, and a carboxyl group. It has the structural feature of having.

[Brief explanation of drawings]

FIG. 1 of the accompanying drawings is an ultraviolet absorption curve of the active high molecular weight material of the present invention. FIG. 2 shows an elution curve when a solution containing an active high molecular weight substance obtained by the method of the present invention was passed through a cation exchange cellulose.

Claims (1)

[Scope of Claims] 1 (i) A high molecular weight substance extracted and separated from the roots of edible burdock root and soluble in water or alkaline aqueous solution, (ii) Adsorbed on anion exchange cellulose but not in cation exchange. (iii) has an absorption wavelength peak in the range of 280-300 nm; and (iv) is 2-nitro-p-phenylene as a mutagenic substance according to the inhibitory effect test described in the text. When measuring the number of revertant colonies from Salmonella TA98 using diamine, the following formula DR (%) = (ac) - (b-c) / a-c x 100, where a is this polymeric substance Mutation inhibition defined as: is the number of revertant colonies in the absence of , b is the number of revertant colonies in the presence of this polymeric substance, and c is the number of spontaneous revertant colonies. having mutation inhibiting activity such that the concentration of the high molecular weight substance exhibiting a ratio (%) of 50% is 4 to 8 parts by weight per 1 part by weight of the 2-nitro-p-phenylenediamine; ) Elemental analysis values show H: 4.86 ± 0.14 (%), C: 48.01 ± 0.28 (%), N: 5.53 ± 0.41 (%) and O: 41.60 ± 0.25 (%), (vi) Sugar content is 9.55 ±0.25% by weight, and (vii) the above-mentioned high molecular weight substance extracted and separated from burdock root, which is substantially negative in ninhydrin color reaction. 2. The high molecular weight substance according to claim 1, wherein the mutation inhibition rate is not substantially reduced by heat treatment at about 100°C for 15 minutes. 3. The high molecular weight substance according to claim 1, whose mutation inhibition rate is greatly reduced by manganese ions. 4 (i) A high molecular weight substance extracted and separated from edible burdock roots and soluble in water or alkaline aqueous solution, (ii) Adsorbed on anion-exchanged cellulose but not on cation-exchanged cellulose; (iii) has an absorption wavelength peak in the range of 280-300 nm; (iv) Salmonella TA98 using 2-nitro-p-phenylenediamine as a mutagen according to the inhibitory effect test described in the text; When measuring the number of reversion colonies from is the number of mutant colonies, b is the number of revertant colonies in the presence of this polymeric substance, and c is the number of spontaneous revertant colonies, and the mutation inhibition rate (%) defined as 50 %, the concentration of the high molecular weight substance is 4 to 8 parts by weight per 1 part by weight of the 2-nitro-p-phenylenediamine, and (v) the elemental analysis value is: H: 4.86 ± 0.14 (%), C: 48.01 ± 0.28 (%), N: 5.53 ± 0.41 (%) and O: 41.60 ± 0.55 (%), (vi) Sugar content is 9.55 ± 0.25% by weight. , and (vii) a mutant containing the above-mentioned high molecular weight substance extracted and isolated from burdock root together with a pharmaceutically acceptable carrier or adjuvant, which is substantially negative in ninhydrin color reaction. Compositions used as inhibitors.