JPH0565490B2 - - Google Patents

Description

[Detailed description of the invention]

OBJECT OF THE INVENTION The present invention relates to an anti-ultraviolet-induced mutagenic agent (bioantimutadiene) derived from Saxifrage. More specifically, the source of the raw material is Saxifraga stlonifera Meerb.
The present invention relates to a novel anti-ultraviolet ray-induced mutagenic agent whose active ingredient is a condensed tannin fraction with specific physicochemical properties extracted from the whole plant. "Industrial Application Field" The condensed tannin fraction derived from Saxifrage according to the present invention can be used to treat DNA damaged by ultraviolet rays.
It has excellent repairing and improving effects. Therefore, this drug can be used as, for example, pharmaceuticals, quasi-drugs,
It can be used in cosmetics, foods (health foods), fermentation industry, genetic engineering fields, etc. Specifically, it is used in medicines for the human body, health foods (functional foods), cosmetics, etc. to improve tissue cells mutated by ultraviolet rays.
Alternatively, it can be used with the hope of preventing it. Furthermore, it is of course possible to use it by adding it to veterinary medicines and feeds. ``Prior art'' Research on cell mutations has been carried out by many researchers, and
Various mutagenic factors have been identified. It is widely recognized that these factors play a role in inducing cancerous transformation of cells. On the other hand, recently, such research has led to the discovery of the existence of substances with antimutagenic effects, and research trends in this field have been attracting much attention. For example, past research trends regarding DNA repair are explained in detail in ``DNA Repair'' by Kei Takebe, University of Tokyo Press (March 31, 1987). In this field of research, research using Escherichia coli in particular is progressing, and the repair mechanism at the molecular level has been elucidated. An interesting finding has also been reported, suggesting that E. coli and human cells share some commonalities in mutation and repair mechanisms. As a result of these research results, a simple method for searching for anti-mutagenic factors using microorganisms was proposed, and research on this is still important due to its potential for preventing and suppressing cancer. It has become a popular topic and widely practiced. Regarding the method of measuring mutation frequency, for example, see the research paper by Mochizuki, Kata et al.
Research, 95, P457 (1982)) and the article by Nomi et al. (Toxicology Forum, 9(2) P189
-198 (1986)) etc.
In particular, the study method used for Escherichia coli is shown as one of the representative methods. Therefore, among various mutagenic factors, the present inventors selected ultraviolet light, which is the most widely exposed environmental factor, and developed methods to repair and ameliorate the mutations induced in E. coli. They began searching for substances that had this effect, and worked to develop formulations that would be useful in the fields of application listed above. ``Problem to be solved by the invention'' That is, the present inventors sought a substance with anti-ultraviolet-induced mutagenesis effect (bioantimutadiene) in a natural product: a plant, and developed a formulation related to it. We have continued our research with this as a challenge. As a result, we found that an extract obtained from the whole plant of Saxifraga, a plant of the Saxifrage family, contains highly efficient
They discovered the existence of bioantimutadiene, which has DNA repair effects. So, what is the composition of this substance?
When identifying it, we were able to identify it using the following physical and chemical means, leading to the completion of the present invention. [Structure of the Invention] The present invention provides an anti-ultraviolet ray-induced mutagenic agent containing as an active ingredient a condensed tannin having the following properties (a) to (d) obtained from the whole plant of Saxifrage, a plant belonging to the family Saxifrage family. It will also last. (a) Solubility in solvents Easily soluble in a mixture of water and ethanol, slightly soluble in water and ethanol alone, insoluble in chloroform (b) Molecular weight 10,000 or less (c) Thin layer chromatogram Rf under the following conditions Value is 0.87 Thin layer plate: Fluorescent hydrophilic silica gel plate under 254nm UV light Developing solvent: Ethanol: Chloroform: Water:
Acetic acid (8:4:2:1) mixture Rf value under the following conditions is 0.89 Thin layer plate: Fluorescent hydrophilic silica gel plate under 254 nm UV light Developing solvent: n-butanol: methanol: acetic acid: water (20:20) :5:4) Mixture Rf value under the following conditions is 0.82 Thin layer plate: Fluorescent hydrophilic silica gel plate under 254nm UV light Developing solvent: methanol:chloroform:water (4:2:1) (d) Tannin reaction confirmation Test is positive ``Means for solving the problem'' [1] Starting materials The anti-ultraviolet ray-inducing mutagenic substance according to the present invention is derived from Saxifraga saxifrage, a plant of the Saxifrage family.
stlonifera Meerb.) or crude drug name: tiger ear grass as a starting material, it can be produced by extraction and purification. As the starting material in this case, saxifrage or tiger ear grass obtained by a tissue culture method can also be used. [2] Crude extraction method The whole plant of Saxifrage is crushed and extracted with anhydrous ethanol or the like (it does not need to be specific to anhydrous ethanol, but other known hydrophilic organic solvents, such as 1,
Polyol-based organic solvents such as 3-butylene glycol, propylene glycol, and polyethylene glycol, or glycerin, acetone, and methanol alone or as a mixture thereof;
It is also possible to use a solution in which water and each of the above-mentioned solvents are arbitrarily mixed. ) and soaked for 2 to 3 days, then filtered to separate the filtrate. The residue obtained during filtration is again repeated twice according to the above procedure, and the obtained filtrate is mixed with the filtrate obtained in the above procedure, and concentrated under reduced pressure. (The process is carried out until the various organic solvents used in the process are completely removed, resulting in an aqueous suspension or dry state.) This operation yields a greenish-brown water suspension or dry product. Next, chloroform is added to this water suspension or dried product, and after thorough stirring at room temperature, filtration is performed, and the residue is separated and dried again by vacuum concentration to give a brownish-brown color. A crude extract of is obtained. [3] Purification and separation method The crude extract obtained in the above step was dissolved in an 80% aqueous ethanol solution, 3 parts of ethyl acetate was added thereto, thoroughly stirred at room temperature, and then centrifuged. , the supernatant (upper layer) is separated and dried under reduced pressure. Next, a small amount of anhydrous ethanol was added to the vacuum-dried material obtained here to dissolve it, and then a mixture of chloroform and water in a ratio of 2:1 was added and stirred, followed by centrifugation to remove the upper layer ( The aqueous phase) is separated and dried again under reduced pressure to obtain a saxifrage-derived anti-ultraviolet-induced mutagenic substance (hereinafter, for convenience, this substance will be referred to as a saxifrage substance). The anti-ultraviolet ray-induced mutagenic substance of the present invention has the following physical and chemical properties, but for application, it is not necessarily necessary to purify it, and the crude extract described in the previous section may be used. [4] Test results regarding physical and chemical properties <A> Solvent solubility test It was confirmed that the saxifrage substance is easily soluble in a mixture of water and ethanol, and is slightly soluble in chloroform, which is slightly soluble in water and ethanol alone. It was done. <B> Molecular weight Since the aqueous solution of saxifrage substance passes completely through Centrizard I (an ultrafiltration membrane that separates molecules with a molecular weight of 10,000) manufactured by Zaritorius, its molecular weight is
It was confirmed that it was less than 10,000. <C> Thin layer chromatogram test Fluorescent hydrophilic silica gel plate under 254 nm UV light (product name: Silica Gel 60F ₂₅₄ )
(1) Ethanol: chloroform: water: acetic acid (8:
Rf value when using a mixture of 4:2:1) is 0.87
It was confirmed. (2) n-butanol: methanol: acetic acid: water (20:
When using a mixture of 20:5:4), the Rf value is 0.89
It was confirmed. (3) Methanol:chloroform:water (4:2:1)
It was confirmed that the Rf value when using a mixed solution of was 0.82. (4) When methanol:chloroform:water:ammonia water (8:4:2:1) was used, it was confirmed that the sample remained at the origin and turned red in iodine vapor after the chromatogram was developed. <D> Condensed tannin reaction test Saxifrage material exhibits an indigo-gray color when exposed to ferric chloride test solution. Furthermore, it was confirmed that when hydrochloric acid was added and heated in n-butanol, it exhibited a pale red color similar to anthocyanidins. Therefore, it was confirmed that the substance was a condensed tannin. <E> Infrared absorption spectrum test In the infrared absorption spectrum (shown in Figure 1) of the Saxifrage material obtained by the KBr method, specific peaks possessed by hydroxyl groups, carbonyl groups, and benzene rings were confirmed. [5] Test for action or effect The anti-ultraviolet ray-induced mutation action of the saxifrage substance according to the present invention was measured using the method of Mochizuki and Kata (Mutation Research, vol. 95, p. 457).
(1982)), some modifications were made to the ultraviolet irradiation method and soaking time after testing. (1) Assay method (a) Bacterial strain used Escherichia Coli B/rWP2 trp ^- was used. (b) Ultraviolet irradiation method Bacteria precultured overnight were washed three times with 100mM sodium phosphate buffer (adjusted to pH 7.0), and while stirring in the same buffer, 48.6J/m ² of ultraviolet rays were irradiated. The test was carried out by irradiation (90 mm diameter shear, liquid layer approximately 2 mm). (c) Preparation of sample (saxifrage substance) Place a certain amount of sample in a sterilized test tube and dilute with 30μ
Add DMSO and 500 μl of 100 mM sodium phosphate buffer. (d) Measurement of mutagenicity Add 100 μ of the bacterial suspension obtained in (b) above to the solution prepared in (c) above, shake at 37°C for 30 minutes, and then add 2.5 ml of After adding soft agar and spreading on a minimum glucose agar medium, culture is performed at 37°C for 48 hours, and the colonies grown are determined to be mutated bacteria. To determine the number of viable bacteria, shake the mixture at 37°C for 30 minutes, then add 100mM sodium phosphate buffer to the mixture.
Dilute to 6 times of 10 (10 ⁶ times), spread on a medium made by adding agar to the preculture liquid medium, and incubate at 37℃ for 24 hours.
The number of colonies after culturing for hours was defined as the number of viable bacteria. (2) Test results The following table (Table 1) shows the results regarding the anti-mutagenic effect of saxifrage substances. The survival rates in Table 1 were determined based on the number of viable bacteria in the absence of ultraviolet irradiation and without the addition of saxifrage material, which was taken as 100%. On the other hand, the mutation rate in Table 1 is the mutation frequency when no saxifrage substance is added during ultraviolet irradiation.
It was set as 100% and calculated based on this. That is, the mutation rate of the saxifrage substance obtained in the present invention is clearly reduced by adding it to the system. It was confirmed that the number of viable bacteria increased to the same level as before UV irradiation when the added amount was 100 μg/plate.

[Table] [6] Effects of the invention As shown in Table 1 above, the saxifrage substance obtained by the present invention was evaluated as an antimutagenic substance (bioantimutadiene). Also, the following is a summary of its effects. As a result, it can be stated as follows: (1) Saxifrage substances significantly inhibit mutations induced by ultraviolet irradiation. It markedly increases the survival rate of mutated Escherichia coli. (3) Therefore, this effect was thought to be to efficiently repair DNA mutations induced by ultraviolet rays and return them to normal. In other words, we discovered that a substance with anti-ultraviolet-induced mutagenesis effect (bioantimutadiene) exists in the extract extracted from the whole plant of Saxifragae, a plant belonging to the Saxifrage family. Through pursuit and research, we succeeded in isolation and identification, leading to the completion of the present invention.Until now, it was not known at all that saxifrage plants have anti-UV-induced mutagenic effects, and the success of the present invention It has a great effect on promoting the effective use of plants (natural products), and can be applied to fields such as fermentation industry, development of pharmaceuticals using genetic engineering, quasi-drugs, cosmetics, and food. We also believe that it can be used in various fields such as biotechnology, biology, and biological sciences.

[Brief explanation of the drawing]

FIG. 1 is an infrared absorption spectrum of saxifrage material.

Claims (1)

[Scope of Claims] 1. An anti-ultraviolet ray-induced mutagenic agent containing as an active ingredient a condensed tannin fraction having the following properties (a) to (d) extracted from the whole plant of saxifrage, a plant belonging to the family Saxifrage family: . (a) Solubility in solvents Easily soluble in a mixture of water and ethanol, slightly soluble in water and ethanol alone, insoluble in chloroform (b) Molecular weight 10,000 or less (c) Thin layer chromatogram Rf under the following conditions Value is 0.87 Thin layer plate: Fluorescent hydrophilic silica gel plate under 254nm UV light Developing solvent: Ethanol: Chloroform: Water:
Acetic acid (8:4:2:1) mixture Rf value under the following conditions is 0.89 Thin layer plate: Fluorescent hydrophilic silica gel plate under 254 nm UV light Developing solvent: n-butanol: methanol: acetic acid: water (20:20) :5:4) Mixture Rf value under the following conditions is 0.82 Thin layer plate: Fluorescent hydrophilic silica gel plate under 254nm UV light Developing solvent: methanol:chloroform:water (4:2:1) (d) Tannin reaction confirmation Test positive.