JPH0686389B2 - Anti-UV-induced mutagenic agent derived from Yashabushi fruit - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Object of the Invention] The present invention relates to a novel development of an antimutagenic agent: bioantimutadiene.

More specifically, the raw material origin is a nut of Yashabushi (Alnus firma), which is a plant of the family Birchaceae, that is, a condensed tannin fraction having a certain physicochemical property extracted from the fruit of the crude drug name "Yaguruma" as an active ingredient. The present invention relates to a novel anti-ultraviolet-induced mutagenesis agent.

"Industrial field of application" The condensed tannin fraction derived from the pods of the present invention has an excellent repair / improvement effect on DNA damaged by ultraviolet rays.

Therefore, this drug is, for example, medicines, quasi drugs, cosmetics,
It can be used for foods (health foods). Specifically, it can be used in combination with pharmaceuticals for humans or animals, foods (health foods and feeds for livestock), cosmetics, etc. to improve or prevent the tissue cells mutated by ultraviolet rays or prevent them. It can be used as expected.

“Prior Art” Studies on cell mutations have been conducted by many researchers so far, and various mutagenic factors have already been confirmed. It is widely recognized that one of these factors causes canceration of cells.

On the other hand, recently, the existence of a substance having an antimutagenic effect has been discovered by such research, and the research trend in this field has been receiving much attention.

For example, the research trend up to now on DNA repair is written by Kei Takebe, "DNA Repair", The University of Tokyo Press (March 1987).
31st month) and so on.

In this field of research, research using Escherichia coli has been particularly advanced, and the repair mechanism at the molecular level has been elucidated.

And, from various research results, interesting findings that Escherichia coli and human cells were found to have some commonality in mutation and its repair mechanism were reported.

Based on these research results, a simple search method for antimutagenic agents using microorganisms has been proposed, and up to the present, research on this is an important theme because of its potential for prevention and suppression of cancer. It has been widely adopted as

Regarding the method for measuring the mutation frequency, for example, a research paper by Mochizuki, Kada et al. (Mutation Research, 95, P457-474)
(1982)) and an article by Nomi et al. (Toxicology Forum, 9 (2), P189-198 (1986)), etc., and in particular, the examination method using E. coli is shown as a typical example. Has been done.

Therefore, the present inventors, among various mutagenic factors,
We picked up ultraviolet rays, which is the most widely exposed environmental factor, and started to search for substances that have a repairing / improving effect on the mutation of Escherichia coli induced by the ultraviolet rays. We have developed a formulation that is useful for

[Problems to be Solved by the Invention] That is, the present inventors have made an earnest research by finding a substance having an anti-UV-induced mutation in a natural product: a plant, and developing a formulation related thereto. Has continued.

In the process, Birch (Alnu)
s firma), an extract obtained from the fruit of "Yaguruma", which is a nut, has been found to contain bioantimutadiene with an efficient DNA repair action.

Then, when the identification of what component this is, it was possible to identify it by the physicochemical means as shown below, and the present invention was completed.

[Structure of Invention]

The present invention relates to the solidity of Birch of the family Birchaceae: anti-condensation type tannin fraction (hereinafter, referred to as C-1) having the following characteristics (a) to (c), obtained from an arrow wheel as an active ingredient. UV-induced mutagenic agent and (a) readily soluble in water, insoluble in ethanol, ethyl acetate, chloroform.

(B) When made into an acetyl form, its molecular weight is 3,500 to 5,5.
It's at 00.

(C) The Rf value of the thin-layer chromatogram is around 0.35.

Thin layer plate: 254nm UV fluorescent hydrophilic silica gel plate Developing solvent: Chloroform: Methanol: Acetic acid (3: 2: 0.
2) Mixed solution Similarly, an anti-ultraviolet ray induction using a condensed tannin fraction (hereinafter referred to as C-3) having the following characteristics (a) to (c), which is extracted from the seeds of Birchgrass of the family Birchaceae, as an active ingredient Mutagenic agent, (a) slightly soluble in water, easily soluble in ethanol, ethyl acetate,
Insoluble in chloroform.

(B) When made into an acetyl form, its molecular weight is 2,500 to 3,2.
It's at 00.

(C) The Rf value of the thin-layer chromatogram is around 0.54.

Thin layer plate: 254nm UV fluorescent hydrophilic silica gel plate Developing solvent: Chloroform: Methanol: Acetic acid (3: 2: 0.
2) Mixed solution It contains an anti-ultraviolet-induced mutagenizing agent containing a mixture of the above-mentioned two condensed tannin fractions as an active ingredient.

[Means for Solving the Problems] [1] Method for Producing Crude Extract Steady of Birch Spodoptera: Nuts of Yarrows are crushed and immersed in 60% -ethanol overnight at room temperature, and then at 80 ° C.
The mixture is subjected to heat extraction for 1 hour, cooled and filtered to obtain a filtrate.

The residue obtained during filtration is again subjected to the above operation twice, and the filtrates are mixed and concentrated under reduced pressure.

The concentration under reduced pressure is carried out until the ethanol contained in the filtrate is completely removed, and the suspension is made into an aqueous suspension or dried.

Next, to the water-suspended state or dry-solid powder state, chloroform is added and stirred, and the water-soluble layer portion or the residual portion is separated and dried by vacuum concentration to obtain crude tannin. Get a powder.

[2] Purification and Separation Method The crude tannin powder obtained in the above step is dissolved in warm anhydrous ethanol (60 ° C.), cooled, and then centrifuged to separate it into a supernatant portion and a precipitation portion (separation). Drawing).

That is, the sedimentation fraction fractionated here is the crude C according to the present invention.
-1, and the supernatant part corresponds to crude C-3.

After drying the precipitated part, it is completely dissolved with a small amount of water, then 3 times the amount of ethanol is added and left overnight in a cool dark place. In this way, the deposited precipitate is recovered, and C-1
Can be obtained.

The supernatant is dried under reduced pressure, completely dissolved in a small amount of absolute ethanol, added with 3 volumes of chloroform, and left in a cool dark place overnight. The precipitate thus deposited is collected to obtain C-3.

[3] Test results relating to physicochemical properties <A> Color reaction C-1 and C-3 are ferric chloride reagent solutions, C-1 is indigo gray, C-3 is dark green, and n-butanol is used. When heated by adding hydrochloric acid therein, an anthocyanidin-like pale red color is exhibited.

<B> Infrared absorption spectrum From the infrared absorption spectra of C-1 and C-3 by the KBr method (Fig. 1 shows C-1 and Fig. 2 shows C-3), hydroxyl group, carbonyl group, benzene ring specific Is confirmed, and the characteristics of condensed tannin are shown.

<C> Solubility in Solvent C-1 is water-soluble and contains ethanol, ethyl acetate,
Insoluble in chloroform.

C-3 is easily soluble in ethanol, sparingly soluble in water, and insoluble in ethyl acetate and chloroform.

<D> Thin Layer Chromatogram (1) As a developing solvent in a hydrophilic silica gel plate (product name: silica gel 60F ₂₅₄ ) that emits fluorescence under 254 nm UV light,
When chloroform: methanol: acetic acid (3: 2: 0.2) was used, the Rf value of C-1 was around 0.35, and the Rf value of C-3 was around 0.54.

(2) C-3 is a lipophilic alkyl silica gel plate (product name: HPTLC RP-8F ₂₅₄ ) that emits fluorescent light under 254 nm UV light,
When using n-hexane: ethanol (4: 1) as a developing solvent, the Rf value is 0.52 to 0.62.

<E> Molecular weight measurement test Since C-1 and C-3 are condensed tannins, they are adsorbed by many phenolic hydroxyl groups by a usual gel filtration method and cannot be measured.

Therefore, in this test, all phenolic hydroxyl groups were acetylated to measure the molecular weight.

(1) Acetylation method Acetylation of C-1 and C-3 was performed with acetic anhydride and pyridine, and the obtained acetylated product was confirmed by an infrared absorption spectrum by a solution method (chloroform) (Fig. 3). Is an acetylated form of C-1, and FIG. 4 shows C.
-3 shows the acetylated form).

(2) Measurement conditions The acetylated products of C-1 and C-3 were analyzed by high performance liquid chromatography (column: Shimamitsu Shimpack HSG-20, mobile phase: tetrahydrofuran, detection: ultraviolet spectrophotometer 25).
0 nm). The standard substance was measured using polystyrene.

(3) Measurement result of molecular weight The acetylated form of C-1 has a molecular weight of 3,500 to 5,500, while C
It was confirmed that the acetylated form of -3 was 2,500 to 3,200.

As described above, since the characteristics of the anti-ultraviolet-induced mutagenesis composition obtained from the nuts and nuts of the present invention can be specified by physicochemically determining,
After that, in the production of this component, it became possible to produce plants and nuts of Alnus firma Sied et Zucc., A birch plant, by extraction and purification by the tissue culture method.

The following table (Table 1) is a summary of the results of the tests according to the above items.

[4] Test on Action or Effect In evaluating the antimutagenic action of C-1 and C-3 according to the present invention, the method of Mochizuki, Kada (Mutation Reseach, Vo
l.95, P457 (1982)), the irradiation method of ultraviolet rays and the soaking time after adding the specimen were partially improved. The measuring method is as follows.

(1) Measurement method (a) using the strain ^{Escherichia Coli B / r WP2 trp -} (b) ultraviolet irradiation overnight, precultured fungus a 100mM sodium phosphate buffer (p
H7.0) and washed 3 times with 48.6 while stirring in the same buffer.
Irradiate with UV of J / m ² (a Petri dish with a diameter of 90 mm, liquid layer approx.
2 mm).

(C) Preparation of specimens (C-1 and C-3) Put a certain amount of specimen in a sterilized test tube, and add 30μ DMSO.
Add 500 μ of 100 mM sodium phosphate buffer.

(D) Measurement of mutagenicity To the solution prepared in (c) above, 100 μ of the bacterial suspension obtained in (b) above was added, shaken at 37 ° C. for 30 minutes, and then 2.5 ml of soft solution was added. After adding agar and spreading it on the minimal glucose agar medium, incubate at 37 ° C for 72 hours. Then, the grown colony is determined to be a mutated bacterium.

To determine the viable cell count, shake the mixed solution at 37 ° C for 30 minutes,
100 mM sodium phosphate buffer, ⁶ times 10 (10 ⁶ times)
The cells were diluted to 1, spread on a medium containing agar in the preculture liquid medium, and the number of colonies after culturing at 37 ° C. for 24 hours was taken as the viable cell count.

(2) Results Results The results of the antimutagenic effects of C-1 and C-3 are shown in the following tables (Tables 2 to 3).

In each table, the survival rate was determined on the basis of the number of viable cells in the absence of ultraviolet light irradiation and without addition of a sample, which was 100%.

On the other hand, in each table, the mutation rate was calculated based on the mutation frequency of 100% when the sample was not added under UV irradiation, which was used as a standard.

That is, C-1 and C-3 obtained in the present invention
Shows that the mutation rate is obviously reduced by adding this to the system.

The increase in the viable cell count was 0.5 mg / plat in C-1.
It was confirmed that e and C-3 appeared up to 0.5 mg / plate, and in particular, the viable cell count per 1 mg / plate of C-3 was recovered to the same level as before the ultraviolet irradiation.

[5] Safety (1) Primary skin irritation test With respect to C-1 and C-3 of the present invention, rabbits with 5% aqueous solution prepared and hair removed on their backs (1 group, 3 animals, weight: 3.9 k)
(around g).

The evaluation was carried out 24, 48, and 72 hours after application by using the erythema and edema as indexes by the primary irritation scoring method.

As a result, neither erythema nor edema was observed in both C-1 and C-3, and it was determined to be negative.

(2) Acute toxicity test ddY mice (5 males / female, 5 mice / group, body weight about 30g) that had been fasted for 4 hours before the test were used, and both C-1 and C-3 were 2,000 mg / Kg as an aqueous solution. Oral administration was performed, and the onset and degree of toxic symptoms were observed over time.

As a result, all mice showed no abnormalities for 14 days, and the autopsy results were also normal.

The LD ₅₀ was determined to be 2,000 mg / Kg or higher.

[6] Use Due to its excellent effects, C-1 and C-3 according to the present invention can be preferably used as an external preparation such as cosmetics for skin and hair.

For example, in the formulation of various commercially available skin care and hair care products such as lotions, creams and emulsions, it may be blended as it is or as a solution with a commonly used base.

Further, it is of course applicable to foods such as health foods, processed foods, confectionery, beverages, etc., and in that case, it may be prepared by mixing with other raw materials according to a conventional method.

In addition, the agent may be solidified with an excipient or the like to give an appropriate granule or tablet.

"Effect of the invention" C-1 and C-3 obtained by the present invention are the above
As shown in Table 2, each was evaluated as an anti-UV-induced mutagen.

That is, the action can be summarized as follows.

(1) Both C-1 and C-3 remarkably suppress the mutation induced by irradiation with ultraviolet rays.

(2) C-1 or C-3 remarkably increases the survival rate of Escherichia coli irradiated with ultraviolet rays even at a very low concentration, as can be seen from the results of the above-mentioned test for action or effect.

(3) It is considered that this action is for efficiently repairing the DNA damage induced by ultraviolet light and returning it to the normal state.

And, this effect is particularly strong in C-3.

Therefore, the application field of C-1 or C-3 according to the present invention is
For example, it can be used for the development of pharmaceuticals, cosmetics, foods, or pharmaceuticals using the fermentation industry, genetic engineering and the like.

Further, in application to the fields of medicines, cosmetics or foods, it is not always necessary to use the purified product of C-1 and C-3. It is quite possible to use -1 crude C-3.

That is, the present inventors' study found that there was a component having an anti-ultraviolet-induced mutagenesis effect in the extract extracted from the nuts of Yasbushi, and to identify what kind of substance it was. Although it succeeded and came to complete the present invention, conventionally, it was not known at all that such C-1 or C-3 has an antimutagenic effect, and the success of the present invention was It is thought that the effect that it brings is great as it promotes the effective use of plants (natural products).

[Brief description of drawings]

FIG. 1 is an infrared absorption spectrum of C-1. FIG. 2 is an infrared absorption spectrum of C-3. FIG. 3 is an infrared absorption spectrum of the acetylated form of C-1. FIG. 4 is an infrared absorption spectrum of the acetylated form of C-3.

Claims (3)

[Claims] 1. An anti-ultraviolet ray-inducing mutagenizing agent, which comprises, as an active ingredient, a condensed tannin fraction having the following properties (a) to (c), which is extracted from the seeds of Yabushiboshi, a birch plant. (A) Easily soluble in water, insoluble in ethanol, ethyl acetate and chloroform. (B) When made into an acetyl form, its molecular weight is 3,500 to 5,5.
It's at 00. (C) The Rf value of the thin-layer chromatogram is around 0.35. Thin layer plate: 254nm UV fluorescent hydrophilic silica gel plate Developing solvent: Chloroform: Methanol: Acetic acid (3: 2: 0.
2) Mixed liquid 2. An anti-ultraviolet ray-inducing mutagenizing agent, which comprises, as an active ingredient, a condensed tannin fraction having the following properties (a) to (c), which is extracted from the scabbard fruit of the Birchaceae plant. (A) Slightly insoluble in water, easily soluble in ethanol, ethyl acetate,
Insoluble in chloroform. (B) When made into an acetyl form, its molecular weight is 2,500 to 3,2.
It's at 00. (C) The Rf value of the thin-layer chromatogram is around 0.54. Thin layer plate: 254nm UV fluorescent hydrophilic silica gel plate Developing solvent: Chloroform: Methanol: Acetic acid (3: 2: 0.
2) Mixed liquid 3. An antibacterial composition comprising a mixture of the condensed tannin fraction according to claim 1 and the condensed tannin fraction according to claim 2, which is extracted from the seeds of the genus Yabushi of the Birchaceae plant. UV-induced mutagen.