JPH0567610B2 - - 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 a plant of the Hypericaceae family. More specifically, the raw material originates from Hypericum erectum (Hypericum erectum), a plant of the Hypericum family.
The present invention relates to a novel anti-ultraviolet ray-induced mutagenic agent whose active ingredient is a tannin fraction with certain physicochemical properties extracted from the whole plant of Hypericum perforatum L.) or Hypericum perforatum L. "Industrial Application Field" According to the present invention, the tannin fraction native to Hypericumaceae plants can be
It has excellent repair and improvement effects on DNA. 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 in hopes 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 Shikibe, published by the University of Tokyo Press (March 31, 1987). In this field, 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 mutations and their 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, P457-474 (1982)) and an article by Nomi et al. (Toxicology Forum, 9(2),
P189-198 (1986)) etc., and in particular, the method used for E. 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, they discovered that bioantimutadiene, which has an efficient DNA repair effect, is present in extracts obtained from the whole plant of Hypericum perforatum or Hypericum perforatum, a member of the Hypericum family. . So, what is the composition of this substance?
When we attempted to identify it, we were able to identify it using scientific and scientific means as shown below, leading to the completion of the present invention. [Structure of the Invention] The present invention provides an anti-ultraviolet ray induced tannin fraction having the following properties (a) to (d) extracted from the whole plant of Hypericum perforatum or Hypericum perforatum of the Hypericumaceae family and having the following properties (a) to (d). Contains mutagenic agents. (a) Solubility in solvents Easily soluble in water, methanol, ethanol, and pyridine, insoluble in ethyl acetate and chloroform (b) Molecular weight 10,000 or less (c) Thin layer chromatogram Rf value under the following conditions is 0.88 Thin layer Plate: Fluorescent hydrophilic silica gel plate under 254nm UV light Developing solvent: Methanol: 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 tails around the Rf value of 0.87 under the following conditions Thin layer plate: Fluorescent hydrophilic silica gel plate under 254 nm 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-induced mutagenic substance according to the present invention is a plant of the Hypericaceae family, Hypericum perforatum, Hypericum perforatum, or Herbal medicine name: It can be produced by extracting and purifying Xiaolianxiang as a starting material. As starting materials in this case, these plants obtained by tissue culture can also be used. [2] Crude extraction method: Grind the whole plant of Hypericum perforatum or Hypericum perforatum and add it at room temperature to anhydrous ethanol (it is not necessary to specify absolute ethanol, but other known hydrophilic organic solvents, such as 1,3-butylene). It is also possible to use polyol-based organic solvents such as glycol, propylene glycol, and polyethylene glycol, glycerin, acetone, and methanol alone or a mixture thereof, or a solution in which water is arbitrarily mixed with each of the above-mentioned solvents. ) is added and soaked for 2 to 3 days, then filtered and the filtrate is collected. 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 continued until the various organic solvents contained in the suspension are completely removed, and the aqueous suspension is dried again). This operation yields a reddish-brown water suspension or dry product. Next, for this water suspension or dry matter,
After adding chloroform and stirring thoroughly at room temperature,
Filtration is performed, and the residue is separated and dried again by vacuum concentration to obtain a reddish-brown crude extract. [3] Purification and separation method To the crude extract obtained in the above step, add a mixture of water and ethyl acetate in a ratio of 1:1, thoroughly dry at room temperature, and then centrifuge to remove the aqueous layer. Separate (lower layer). Use the solution obtained here as is, or
After washing with ethyl acetate, the product is dried under reduced pressure to obtain an anti-ultraviolet-induced mutation agent derived from Hypericum perforatum or Hypericum perforatum (hereinafter, for convenience, this will be referred to as Hypericum perforatum material). The anti-ultraviolet-induced mutagenic substance in the present invention has the following physical and chemical properties, but it does not necessarily need to be purified for application, and may be a crude extract as described in the previous section. . [4] Tests on physicochemical properties <A> Solubility test in solvents Hypericum perforatum was confirmed to be easily soluble in water, methanol, ethanol, and pyridine, and insoluble in ethyl acetate and chloroform. <B> Molecular Weight The aqueous solution of the Hypericum perforatum substance was completely passed through Centrizard I (an ultrafiltration membrane that separates molecules with a molecular weight of 10,000) manufactured by Salitorius, and its molecular weight was confirmed to be 10,000 or less. <C> Thin layer chromatogram test A fluorescent hydrophilic silica gel plate (product name: silica gel) under 254 nm UV light was used as a thin layer plate.
60F ₂₅₄ ) and a mixture of (1) ethanol:chloroform:water:acetic acid (8:4:2:1) was confirmed to be 0.88 as the developing solvent. (2) When a mixture of n-butanol:methanol:acetic acid:water (20:20:5:4) was used, the Rf value was confirmed to be 0.89. (3) Methanol: Chloroform: Water (4:
The Rf value when using a 2:1) mixture is
It was confirmed that tailing is centered around 0.87. (4) It was confirmed that when methanol:chloroform:water:ammonia water (8:4:2:1) was used, it remained at the origin. <D> Tannin reaction test Hypericum perforatum exhibits a dark green color when exposed to ferric chloride test solution. <E> Infrared absorption spectrum test The infrared absorption spectrum (shown in Figure 1) of the Hypericum perforatum substance by the KBr method confirmed unique peaks possessed by hydroxyl groups, carbonyl groups, and benzene rings. [5] Test for action or effect The anti-ultraviolet-induced mutation action of the Hypericum perforatum substance according to the present invention was measured by Mochizuki,
Kata's method (Mutation Research, vol.95,
P457 (1982)), some modifications were made to the ultraviolet irradiation method and the soaking time after adding the specimen. (1) Measurement method (a) Bacterial strain used Escherichia Coli B/r WP2 trp ^- was used. (b) Ultraviolet irradiation method The bacteria pre-cultured overnight was washed three times with 100mM sodium phosphate buffer (adjusted to pH 7.0), and while stirring in the same buffer,
Irradiated with 48.6J/ ^m2 ultraviolet rays (diameter 90mm)
The liquid layer was approximately 2 mm). (c) Preparation of sample (Hypericum perforatum substance) Put a certain amount of sample into a sterilized test tube,
Add 30μ of DMSO and 500μ of 100mM sodium phosphate buffer. (d) Measurement of mutagenicity Add 100μ of the bacterial suspension obtained in (b) above to the solution prepared in (c) above, shake at 37°C for 30 minutes, and then After adding soft agar and spreading on a minimum glucose agar medium, culture is performed at 37°C for 48 hours, and the colonies that grow are determined to be mutated bacteria. To determine the number of viable bacteria, shake the mixture at 37°C for 30 minutes, dilute the mixture to 10:6 culture (10: ⁶ culture) with 100mM sodium phosphate buffer, and add to the liquid medium for preculture. The cells were spread on a medium made by adding agar, and the number of colonies after culturing at 37°C for 24 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 Hypericum perforatum substances. In addition, the survival rate in Table 1 is the number of viable bacteria in the absence of ultraviolet irradiation and without the addition of Hypericum perforatum substances.
It was set as 100% and calculated based on this. On the other hand, the mutation rate in Table 1 was determined based on the mutation frequency when no Hypericum perforatum substance was added during ultraviolet irradiation as 100%. That is, when the Hypericum perforatum substance obtained in the present invention is added to the system, the mutation rate is clearly reduced.

[Table] [6] Effects of the invention The Hypericum perforatum substance obtained by the present invention is
As shown in Table 1 above, it was evaluated as an anti-ultraviolet radiation-induced mutagenic substance (bioantimutadiene). In addition, the effects are summarized as follows. (1) Hypericum perforatum substances significantly inhibit mutations induced by ultraviolet irradiation. (2) Hypericum perforatum significantly increases the survival rate of E. coli that has been mutated by ultraviolet irradiation. (3) Therefore, this action efficiently repairs DNA mutations induced by UV radiation,
It was thought that this was to restore normalcy. In other words, we discovered that a substance with anti-ultraviolet-induced mutagenesis effect (bioantimutadiene) exists in an extract extracted from the whole plant of Hypericum perforatum or Hypericum perforatum, a plant belonging to the Hypericum family. In pursuit and research, we succeeded in isolation and identification, leading to the completion of the present invention. Until now, it was not known that Hypericum perforatum or Hypericum perforatum has anti-ultraviolet-induced mutagenic effects, and the success of the present invention will promote the effective use of plants (natural products), and its effects will be significant. It can be applied to fields such as the fermentation industry, the development of pharmaceuticals using genetic engineering, quasi-drugs, cosmetics, and food, and can also be used in various fields such as biotechnology, biology, and biology. I think it is possible.

[Brief explanation of the drawing]

FIG. 1 is an infrared absorption spectrum of Hypericum perforatum material.

Claims (1)

[Scope of Claims] 1. The following (a) extracted from the whole plant of Hypericum perforatum or Hypericum perforatum of the family Hypericumaceae:
An anti-ultraviolet ray-induced mutagenic agent comprising a tannin fraction having the characteristics of ~(d) as an active ingredient. (a) Solubility in solvents Easily soluble in water, methanol, ethanol, and pyridine, insoluble in ethyl acetate and chloroform (b) Molecular weight 10,000 or less (c) Thin layer chromatogram Rf value under the following conditions is 0.88 Thin layer Plate: Fluorescent hydrophilic silica gel plate under 254nm UV light Developing solvent: Methanol: 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 tails around the Rf value of 0.87 under the following conditions Thin layer plate: Fluorescent hydrophilic silica gel plate under 254 nm UV light Developing solvent: methanol: chloroform: water ( 4:2:1) (d) Tannin reaction confirmation test is positive.