JPH09176075A - Antimicrobial, antifungal and anti-inflammatory active substance and production thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the above polymer exhibiting antimicrobial, antifungal, anti-inflammatory and antioxidizing activity and useful also as a pigment or dye by polymerizing guaiacol using peroxidase in the presence of hydrogen peroxide. SOLUTION: (A) Guaiacol is polymerized using (C) peroxidase in the presence of (B) hydrogen peroxide to provide the objective polymer excluding a compound of formula I, preferably a polymer of formula II, etc. Furthermore, the reaction is carried out in 1×10<-5> to 1×10<-4> mol/mg concentration of the component B in a buffer solution such as sodium phosphate at about 25 deg.C and crude enzyme, etc., prepared from horseradish, etc., is preferably used as the component C.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention disclosed herein relates to an antibacterial / antifungal or anti-inflammatory active substance, a method for producing the same, and an antibacterial / antifungal or anti-inflammatory agent.

[0002]

2. Description of the Related Art It is known that plants show various induced resistance reactions to invasion of microorganisms. This is due to the action of various genetically regulated enzymes. Peroxidase is one of the enzymes that activates the expression of such induced resistance reaction, and is known to have an action of modifying and polymerizing a phenolic compound. However, the structure of the compound produced by the action of peroxidase and the degree of chemical / physiological action of the compound have not yet been sufficiently clarified.

[0003]

Therefore, the present inventors analyzed the compound obtained by allowing peroxidase to act on guaiacol, which is a phenolic compound, to analyze a novel antibacterial / antifungal and antifungal agent which has not been heretofore available. Investigations have been advanced for the purpose of providing an inflammatory active substance, and the invention disclosed in the present specification has been achieved.

[0004]

SUMMARY OF THE INVENTION Disclosed herein is an invention in which guaiacol or a guaiacol polymer is reacted with peroxidase in the presence of hydrogen peroxide.

The guaiacol or guaiacol polymer used as a starting material in this reaction may be commercially available or synthesized.
In addition, the guaiacol polymer may be obtained according to the method disclosed herein or may be obtained by other methods. Usually, dimers and trimers are used. By using such a guaiacol polymer as a starting material, a polymer having a high degree of polymerization can be efficiently obtained by a simple method. For example, when guaiacol trimer is used as a starting material, guaiacol hexamer can be efficiently obtained. The guaiacol or guaiacol polymer as a starting material may be used alone or in combination of two or more kinds. Other substances may coexist during the reaction as long as they do not adversely affect the reaction with peroxidase.

The type of peroxidase used in this reaction is not particularly limited. Therefore, a crude enzyme prepared from horseradish, green beans, or the like or a commercially available enzyme can be used.

The reaction between guaiacol and peroxidase is carried out in the presence of hydrogen peroxide. The concentration of hydrogen peroxide is usually 1 × 10 ⁻⁵ mol to 1 × 10 ⁻⁴ m per 1 mg.
However, the reaction can be carried out at a concentration outside this range. In addition, the reaction may be performed in a buffer solution. As the buffer solution, for example, sodium phosphate buffer solution or Tris-hydrochloric acid buffer solution can be used. The reaction temperature may be a temperature at which peroxidase is active, but it is usually about 25 ° C. By increasing the reaction time or increasing the concentration, a substance with high activity can be produced. The progress of the reaction can be followed by TLC using an appropriate developing solution.

The reaction product is antibacterial even if it is a crude product.
Since it exhibits antifungal, anti-inflammatory or antioxidant activity, it can be used in these applications as a crude product without further purification. However, since compounds having particularly strong activity and compounds not having such activity coexist in the crude product, it is preferable to purify and use the compound having particularly strong activity.
Purification can be performed by methods well known to those skilled in the art, such as column chromatography, reverse phase column chromatography, preparative HPLC and the like. The present inventors discovered a guaiacol polymer having the structure shown below by isolating a plurality of compounds contained in the crude product. All of these compounds are novel compounds.

[0009]

Embedded image All of these compounds exhibit antibacterial / antifungal, anti-inflammatory and antioxidant activities. Excellent activity has been found in guaiacol dimers and guaiacol trimers. In particular, it has been confirmed that guaiacol trimer shows a remarkable urinary mitotic inhibitory activity and completely inhibits urinary mitotic division at 2 μg / ml. Further, some guaiacol polymers have a good color, and can be expected to be used as pigments and dyes.

[0010]

EXAMPLES The present invention will be specifically described below with reference to examples, but the technical scope of the present invention is not limited to these examples.

(Example 1) 8 mg of guaiacol and 80 ml of 0.35% hydrogen peroxide were suspended in 4 liters of 0.1 M Tris-hydrochloric acid buffer (pH 8.0), and peroxidase prepared from horseradish (NAO, PEO). −
131: IC class) 40,000 units (as defined by Toyobo) were added and reacted at 25 ° C. for 15 minutes. Then, it was extracted with ethyl acetate to obtain 7.99 g of an extract. This is an open column (Wakogel C-10
0; 10% H ₂ O; φ3.6 × 30 cm). Using a hexane solution in which the concentration of ethyl acetate was gradually increased as an eluent, 29 mg of a crude product from a fraction of a 20% ethyl acetate solution, 368 mg of a crude product from a fraction of a 23 to 30% ethyl acetate solution, 33 to 37 were used. A crude product of 250 mg was obtained from the fraction of a% ethyl acetate solution. 368 mg of the crude product obtained from the fraction of a 23-30% ethyl acetate solution was applied to a medium pressure column (Millipore Preparative).
e C18; φ1.0 × 100 cm; 0.5 ml / mi
Dimer A was obtained by elution with a methanol / water mixture according to n). The crude product (29 mg) obtained from the fraction of the 20% ethyl acetate solution was a single product, and was designated as dimer B. 33-37% crude product obtained from 37% ethyl acetate solution
0 mg to a medium pressure column (Millipore Prepa
ratio C18; φ1.0x100cm; 0.5
(ml / min) by elution with a methanol / water mixture to give trimer A (8.5 mg) and tetramer A
(2.5 mg) and tetramer B were obtained.

The identification data of these compounds are as follows. The tetramers A and B were found to be tetramers as a result of MS analysis.

[Dimer A] NMR δ _H (500 MHz, acetone-d ₆ ): 3.8
5 (6H, s), 6.83 (2H, d, J = 7.6H
z), 6.84 (2H, d, J = 2.1 Hz), 6.9.
2 (2H, dd, J = 2.1, 7.6 Hz) EI MS m / z: 246 (M ⁺ ).

Embedded image [Dimer B] NMR δ _H (500 MHz, CDCl ₃ ): 3.82
(3H, s), 3.84 (3H, s), 5.39 (1
H, s), 6.46 (1H, dd, J = 2.5, 9.0)
Hz), 6.65 (1H, d, J = 2.5 Hz), 6.
83 (1H, d, J = 9.0 Hz), 6.85-6.9.
0 (2H, m), 6.99 (1H, d, J = 7.9H
z), 7.06 (1H, ddd, J = 2.3, 6.7H
z) EI MS m / z: 246 (M ⁺ ).

Embedded image [Trimer A] NMR δ _H (500 MHz, CDCl ₃ ): 3.86
(3H, s), 3.94 (3H, s), 3.96 (3
H, s), 5.36 (1H, s), 5.65 (1H,
s), 6.49 (1H, dd, J = 2.8, 8.5H
z), 6.68 (1H, d, J = 2.8 Hz), 6.8
5 (1H, d, J = 8.5Hz), 6.90 (1H,
d, J = 8.2 Hz), 6.98 (1H, d, J = 8.
2 Hz), 7.03 (1H, dd, J = 2.1, 8.2
Hz), 7.04 (1H, d, J = 2.1 Hz), 7.
08 (1H, dd, J = 2.1, 8.2Hz), 7.1
3 (1H, d, J = 2.1 Hz) API MS m / z: 369 (M + H) ⁺

[Chemical 6] Example 2 The following formula:

Embedded image Guaiacol trimer represented by (trimer B) 45mg
And 4.0 ml of 0.35% hydrogen peroxide solution were suspended in 450 ml of 0.1 M Tris-HCl buffer (pH 8.0), and peroxidase prepared from horseradish (Nacalai,
PEO-131: I-C grade) 14,000 units (as defined by Toyobo) was added and reacted at 25 ° C for 15 minutes. Methanol was added to the obtained crude product to inactivate the enzyme, and then MALDI TOF MS spectrum was measured. The results were as shown in FIG.

The mixture was extracted with chloroform and the solvent was distilled off to obtain 45 mg of a solid substance. This was reduced in 0.1N hydrochloric acid in the presence of zinc, and extracted with chloroform. The solvent of the chloroform layer was distilled off,
44 mg of solid was obtained. This was further purified by preparative TLC (solvent system: chloroform: methanol = 100: 2), and guaiacol hexamer A4.
8 mg and Rf = 0.50 guaiacol hexamer B2.
7 mg were obtained. These identification data were as follows.

[Hexamer A] NMR δ _H (500 MHz, CDCl ₃ ): 3.92
(6H, s), 3.97 (6H, s), 3.99 (6
H, s), 6.95 (2H, d, J = 8.2Hz),
6.99 (2H, d, J = 2.1Hz), 7.02 (2
H, d, J = 1.8 Hz), 7.05 (2H, dd, J
= 1.8, 8.2 Hz), 7.14 (2H, d, J =
2.1 Hz), 7.26 (2H, d, J = 2.1H
z), 7.29 (2H, d, J = 2.1) API MS m / z: 735 (M + H) ⁺ [hexamer B] API MS m / z: 735 (M + H) ⁺ proton-proton in COZY. It was found that there are three ABX lines.

Example 3 Instead of the starting material guaiacol trimer B of Example 2, the following formula:

Embedded image A similar reaction was performed using guaiacol dimer C represented by

(Example 4) The guaiacol dimers A and C, the trimers A and B, the tetramers A and B, and the hexamers A and B were compared in antifungal activity and embryonic division inhibitory activity, respectively. did.

The antifungal activity was tested using Cladosporium herbarum. The spores were spore germination medium (glucose 0.2%, yeast extract 0.1%, NaH
PO ₄ .12H ₂ O 0.37%, citric acid 0.1%) and suspended at sample concentrations of 1000 μg / ml and 50, respectively.
0 μg / ml, 250 μg / ml, 125 μg / ml,
It was cultured at 27 ° C. as 63 μg / ml, and after 24 hours, it was observed under a microscope whether spore germination was observed.

Eggs and semen used for the sea urchin embryo inhibitory activity test were sexually mature sea urchins ( Hemicentrotus pulcherrim ) collected in Okayama during the breeding season (January-March).
us )). The eggs were stirred and allowed to stand for 3 minutes, and the eggs on the surface and the bottom were removed by the tilting method and the suction method, respectively. The cytotoxicity of the plant extract was evaluated by adding about 100 fertilized eggs to the serially diluted sample solution and observing the morphological change of the embryo.

The results are shown in FIG.

[Brief description of the drawings]

FIG. 1 MAL of a crude product obtained by reacting guaiacol and a guaiacol trimer with peroxidase.
It is a DI TOF MS spectrum.

FIG. 2 is a graph showing the antifungal activity of guaiacol dimers, trimers, tetramers and hexamers, as well as the embryonic division inhibitory activity.

Front page continuation (72) Inventor Shinichiro Kajiyama 1912 Hiyoshidai 4th Town, Takatsuki City, Osaka Prefecture 1212 (72) Inventor Hiroshi Kanzaki 1-17 Tsuwawamachi, Tsushima Okayama City, Okayama Prefecture (72) Ikki Kawazu Okayama Prefecture, Okayama Prefecture Ichijuku Honmachi 8-51-12

Claims (5)

[Claims] 1. A guaiacol polymer obtained by polymerizing guaiacol with peroxidase in the presence of hydrogen peroxide (wherein the formula: Excluding compounds represented by). 2. The following formula: A guaiacol polymer represented by. 3. The method for producing a guaiacol polymer according to claim 2, which comprises a step of polymerizing guaiacol using peroxidase in the presence of hydrogen peroxide. 4. A step of polymerizing a guaiacol polymer with peroxidase in the presence of hydrogen peroxide,
A method for producing an antibacterial / antifungal or anti-inflammatory active substance. 5. An antibacterial / antifungal or anti-inflammatory agent containing the guaiacol polymer of claim 2.