JP2884104B2 - Anti-mutagenic agent derived from salamander - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Object of the Invention] The present invention relates to a novel development of an anti-mutagenic agent: bioantimutagen.

More specifically, Zanthoxylum piperitum De Candoll, a citrus plant known as one of the herbal medicines and listed in the Japan Pharmacopoeia,
e) or other novel bioantimutagens obtained by extracting from mature fruits of the same genus plants.

“Industrial application field” According to the extract derived from salmon specified by the present invention, the extract has excellent anti-mutagenic activity and exhibits an efficient DNA repair activity.

So, for example, pharmaceuticals, cosmetics, foods (health foods)
It can be used for the purpose of suppressing genotoxicity, cancer, aging, etc. induced by mutagenic factors, or preventing them.

It can also be used by adding it to veterinary drugs, quasi-drugs, or animal feed.

"Conventional technology" As one of the causes of cancer and genotoxicity, various mutagenic agents existing in the environment have been
There is known a mechanism that causes a change in genetic information in a living body, which is caused by immobilization.

In other words, ultraviolet rays, heat decomposed products of proteins in natural products, and inducible factors such as aromatic nitro compounds, which are known as mutagens, damage DNA and are immobilized as mutations. It has been confirmed that various lesions are caused, and active research is being conducted on the suppression or prevention of those lesions.

In the course of the research, for the purpose of prevention and treatment,
The existence of various anti-mutagenic agents has been identified, and efforts have been made to search and identify them.

This anti-mutagenic substance acts directly on mutagenic factors and inactivates mutagenic activity, and it works on mutagenic cells to repair DNA. And it is roughly classified into those having the property of suppressing the expression of the mutation action, the former is called desmutagen, the latter is proposed as bioantimutagen, and as a substance having the former property, for example, peroxidase, Enzymes such as catalase and vitamin C have been confirmed.
In the latter, active substances extracted from tea, vegetables and the like are known.

"Problems to be Solved by the Invention" The present inventors have sought to find new substances having useful antimutagenic activity in natural products: plants and to develop them. Has been continued.

As a result, the citrus plant, Zant (Zant
hoxylum piperitum De Candolle) or other fruits of the same genus plant (hereinafter simply referred to as “sansho”), a water-soluble extract with anti-mutagenic activity and efficient DNA repair I found that antimutagen exists.

[Configuration of the invention]

The present invention comprises a bioantimutagen containing, as an active ingredient, a water-soluble extract having a molecular weight of 10,000 or less obtained from the fruit of a fruit of the citrus plant Sansho.

"Means for solving the problem" Extraction was performed using various solvents from salamander, and the resulting components were confirmed to have anti-mutagenic activity. As a result, strong activity was found in the water-soluble extract. The indicated material was found to be present.

Therefore, the present inventors, regarding the water-soluble extract,
In order to pursue further details, first, the obtained water-soluble extract was subjected to fractionation by various steps, and the anti-mutagenic activity was measured for the substance collected at each time,
We narrowed down those with high activity.

Specifically, the operation was performed as in the following operation.

Procedure The water-soluble extract obtained from the salamander is freeze-dried, chloroform / ethyl acetate is added to the obtained dried product, and the mixture is thoroughly stirred and then centrifuged. The organic solvent layer <chloroform / ethyl acetate layer> (hereinafter, referred to as A extract for convenience) and the residue (hereinafter, referred to as B extract for convenience) were measured for activity. Both the A extract and the B extract showed activity, but it was confirmed that the B extract showed particularly strong activity.

Operation Therefore, next, cold water (0 to 5 ° C.) was added to the B extract obtained by the operation, and the mixture was thoroughly stirred and centrifuged. Similarly, a soluble portion (cold water dissolving portion) (hereinafter, for convenience, C extract) and residue (hereinafter referred to as D extract for convenience)
The activity of the C extract was confirmed to be particularly strong. In addition, D extract
It was confirmed that there was some activity.

Operation Then, as to the C extract obtained by the operation, an attempt was made to identify what kind of substance it was, and as a result, it was confirmed that the extract was a component having the following physicochemical properties.

[1] Test on physicochemical properties (1) Thin layer chromatogram test Test 1. Thin plate: silica gel 60 F254 Developing solvent: ethanol / purified water = (9: 1) Temperature: room temperature Rf value: 0.33 to 0.66 Test 2 Thin plate: silica gel 60 F254 Developing solvent: methanol / acetic acid = (9: 1) Temperature: room temperature Rf value: 0.88 Test 3. Thin plate: silica gel 60 F254 Developing solvent: chloroform / methanol / purified water = (13:
7: 2) Lower layer of mixed solution Temperature: room temperature Rf value: 0.12 (2) Solubility test in solvent It was confirmed that it was easily soluble in water and insoluble in methanol, ethanol, ethyl acetate and chloroform.

(3) Molecular weight confirmation test It was confirmed that the molecular weight was 10,000 or less by passing through an ultrafiltration membrane (Centrieult I, manufactured by Sartorius) for separating a molecular weight of 10,000.

(4) Color Test When the suspension was suspended in ethanol and a magnesium ribbon and hydrochloric acid were added, the suspension exhibited a red color, confirming that it contained flavonol.

When dissolved in water, a 5% α-naphthol / ethanol solution was added, and H ₂ SO ₄ was gently overlaid, the presence of a saccharide was confirmed by the interface exhibiting a purple-red color. (Morish reaction) Due to the appearance of greenish brown by the ferric chloride reagent,
It was confirmed to contain polyhydric phenol.

When n-butanol was added with hydrochloric acid and heated, it turned pale red.

(5) Infrared absorption spectrum test The infrared absorption spectrum by the KBr method is as shown in FIG.

[2] Examination of the production method The fractionation operation attempted so far and the method of obtaining an extract containing the anti-mutagenic active substance at a high concentration based on the physicochemical properties (solvent and fractionation operation) As a result of various investigations, it has been found that the production method described below can easily obtain a target substance having high activity.

Production Method The salamander is crushed or pulverized, immersed in purified water at room temperature for 2 to 3 days, and the filtrate is collected by filtration. The residue obtained at this time is extracted again according to the above operation, the filtrate is separated, and mixed with the previously obtained filtrate to obtain an extract.

Next, this extract is freeze-dried, and the obtained dried product is washed several times with chloroform and ethyl acetate, respectively, and then centrifuged to separate the residue. Furthermore,
Cold water (0-5 ° C) was added to the residue, and the mixture was stirred well, centrifuged, and insolubles were removed. The resulting solution was dried under reduced pressure to obtain an extract.

Incidentally, the extraction solvent does not need to be specified in purified water, and other known hydrophilic organic solvents, for example, polyol solvents such as 1,3-butylene glycol, propylene glycol and polyethylene glycol, glycerin, acetone, methanol, A mixed solution of ethanol or the like and water can also be used.

[3] Test for action or effect In measuring the effect of the anti-mutagenic agent according to the present invention, the method of Mochizuki and Kada (Mutation Research,
According to I. 95, p. 457, 1982), the ultraviolet irradiation method and the bioantimutagen were added, and the immersion time was partially improved.

 The measuring method is as follows.

(1) Preparation of strain (a) Used strain Escherichia Coli B / r WP2 trp ₋ was used.

(B) Ultraviolet irradiation method The bacteria precultured overnight are washed three times with a 100 mM sodium phosphate buffer (pH 7.0), suspended in the same buffer, and transferred to a petri dish (90 mm in diameter). At this time, the liquid layer is about 4 mm.

Then, while stirring this, 47.8 J / m ² phase equivalent ultraviolet rays were irradiated.

(2) Sample preparation Sample (extract, extraction) obtained by the manufacturing method
Was dissolved in a 50% aqueous solution of dimethylsulfoxide (DMSO), and 60 μl of the solution was collected, placed in a sterilized test tube, and added with 100 mM sodium phosphate buffer (500 μl) to prepare.

As a comparative sample, cinnamaldehyde, which is known as a known anti-mutagenic substance, was used.

(3) Measurement method 100 μl of the bacterial suspension obtained in the above (1) was added to the prepared solution of the above (2), and the mixture was shaken at 37 ° C. for 30 minutes.
After adding ml of soft agar and spreading on a minimum glucose agar medium, culturing is carried out at 37 ° C. for 72 hours, and the grown colonies are judged as mutated bacteria.

The viable cell count was determined by diluting the mixed solution after shaking at 37 ° C. for 30 minutes with 100 mM sodium phosphate buffer to 50 times ³ times (1.25 × 10 ⁵ times) and pre-culture. The liquid medium was spread on a medium prepared by adding agar, and the number of colonies after culturing at 37 ° C. for 24 hours was defined as the viable cell count.

(4) Results The antimutagenic effect of the water-soluble extract derived from salamander is shown in the following table, “Table 1”.

In addition, it was confirmed that the addition of the water-soluble extract derived from salmon obtained in the present invention into the system clearly reduced the mutation rate.

[4] Effects of the invention
It is evaluated as an anti-mutagenic agent (bioantimutagen) as shown in Table.

In particular, the extracted active ingredient has the effect of suppressing DNA mutation induced by ultraviolet irradiation by 53% by adding 100 μg per plate. This also means
It is considered that DNA mutations induced by irradiation with ultraviolet rays are efficiently repaired and returned to normal.

Accordingly, the bioantimutagen derived from salmon obtained in the present invention has genotoxicity,
For the purpose of suppressing or preventing cancer, aging, etc., it can be used, for example, in the development of pharmaceuticals, cosmetics, foods, or pharmaceuticals using the fermentation industry, genetic engineering, and the like.

In addition, in the application to the field of medicines and cosmetics, or food, it is not always necessary to use the purified substance, for example, A
It is also possible to use the extracts from D to D or the extracts shown in the production method.

That is, the present inventors sought a novel anti-mutagenic agent for a natural product: a plant, and as a result of intensive studies, found that a useful anti-mutagenic agent was contained in a water-soluble extract obtained from salamander. He discovered that a sexually active substance was present, and based on this, disclosed a purification method and the like.

As a result, it is possible to promote the effective use of plants (natural products), which is expected to contribute to health promotion and related industries.

[Brief description of the drawings]

FIG. 1 is an infrared absorption spectrum of a water-soluble C extract derived from salamander obtained by the operation.

Claims (1)

(57) [Claims] 1. A bio-antimutagen containing, as an active ingredient, a water-soluble extract having a molecular weight of 10,000 or less obtained from the fruit of the fruit of the citrus plant Sansho.