JP2746453B2 - Active oxygen scavenger - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an in vivo active oxygen scavenger comprising a compound selected from coniferyl benzoate, eugenol, dehydrodiisoeugenol, and isoeugenol.

[Conventional technology]

Recently, there have been many clinical reports showing that active oxygen and free radicals damage biological membranes and tissues, which are associated with various diseases, as well as cancer and aging. Is 1,2,3-electron reduced species of ³ O ₂ in the active oxygen in vivo (Superoxide anion radical), H ₂ O ₂ , OH
(OH radical) and ¹ O _{2 in the} excited state. Further, peroxy radicals and alkoxy radicals generated by peroxidation of lipids are known.

On the other hand, since a living body is constantly exposed to oxygen, it has an enzymatic defense mechanism.

For example, there is a superoxide dismutase (SOD) that performs a disproportionation reaction with respect to a superoxide anion radical, and a prophylactic and therapeutic agent for a disorder derived from in-vivo active oxygen containing this as an active ingredient is also known. ing.

Vitamin C and vitamin E have been tested as substances that scavenge active oxygen free radicals because of their antioxidant properties.

However, the production method of superoxide dismutase is difficult, and the availability of raw materials is limited, so that stability and safety remain problems.

Vitamin E and vitamin C remain difficult in experiments using living organisms, such as insufficient stability and active oxygen scavenging action.

Furthermore, the OH radical has high reactivity as an active oxygen species and does not have a specific defense mechanism in a living body like the above-mentioned enzyme.

In view of the above, there is a strong demand for a scavenger having a direct scavenging effect on active oxygen, especially on OH radicals having a strong biological action.

[Problems to be solved by the invention]

Accordingly, an object of the present invention is to provide an active oxygen scavenger having a direct scavenging effect on active oxygen, especially on OH radicals having a strong biological action.

[Means for solving the problem]

The present invention relates to Coniferylb represented by the following structural formulas A to D.
An active oxygen scavenger consisting of a compound selected from enzoate (coniferyl benzoate), Eugenol (eugenol), Dehydrodiisoeugenol (dehydrodiisoeugenol), and iso-Eugenol (isoeugenol).

(A) Coniferyl benzoate (B) Eugenol (C) Dehydrodiisoeugenol (D) Isoeugenol Coniferyl benzoate used in the present invention is a known compound obtained from a benzoin resin (karl F
reudenberg und Friedrich Bittner: Coniferylalkohol
aus Siambenzoe.Eingegangen.12.Juni.1950).

Eugenol used in the present invention is clove oil or Nu.
A known compound (Ak) obtained by steam distillation of tmeg (nutmeg) essential oil and purifying it by column chromatography
ira Isogai et al .: Isolation from Nutmeg of Growth
Inhibitory Substances to Silkworm Larvae, Agr.Biol.
Chem., 37 (4), 889-895, 1973 / Fujita et al .: Japan Chemical Magazine,
87, No. 9, 110-112, 1966).

The dehydrodiisoeugenol used in the present invention is a known compound obtained by steam distillation of Nutmeg (nutmeg) and purification by column chromatography (Akira Isogai et al .: Isolation from Nutmeg of G
rowth Inhibitory Substances to Silkworm Larvae, Ag
r. Biol. Chem., 37 (4), 889-895, 1973).

Isoeugenol used in the present invention is a known compound obtained by heating and isomerizing eugenol with caustic potash in the presence of methanol or water, and is used as a base preparation for carnation-based compounded flavors, and as various compounded flavors and food flavors. Used.

The method of using the active oxygen scavenger of the present invention may be a method in which an ordinary active oxygen scavenger is used, such as a drug or
It can be applied to cosmetics and the like.

Next, the present invention will be described in more detail with reference to examples.

〔Example〕

Example 1. Deoxyribose method <Principle> In this method, an OH radical is generated in a Fenton system, and the reaction of the OH radical with deoxyribose (3.
1 × 10 ⁹ M ^-1 S ^-1 ) produced malondialdehyde (MD
A) is reacted with thiobarbituric acid to measure a reactant produced (TBA method) to obtain a TBA value (Barry Halliwell, John MCGutteridge, Okezie I. Aru
oma: Improved Analytical Biochemistry. 165.215-219.1987).

In other words, taking advantage of the fact that the TBA value decreases when an OH radical scavenger (coniferyl benzoate, eugenol, dehydrodiisoeugenol, isoeugenol) is present in this system. The inhibition rate was determined from the TBA value after the addition, and was shown as an OH radical inhibition activity value.

<Method> To 0.1 ml of 0.2 M KH ₂ PO ₄ -KOH buffer (pH 7.4), add 2 ml
0.1 ml of 8 mM deoxyribose, 0.1 ml of 20 mM capture substance, 1.0
0.1 ml of 4 mM EDTA, 0.1 ml of 1 mM ascorbic acid, 1 mM FeCl ₃
0.1 ml and 0.1 ml of 1 mM aqueous hydrogen peroxide were added sequentially,
Prepare with distilled water to make 1.0 ml.

After incubating the reaction solution at 37 ° C. for 60 minutes, thiobarbituric acid-MDA adduct (absorbance 532 nm) is obtained by the TBA method.
Was measured.

The OH radical inhibition rates of the trapping substances were calculated by the following equations, and are shown in Table 1.

A ₁ : Absorbance before adding capture substance A ₂ : Absorbance before adding capture substance Further, even when the concentration of the added scavenger is changed from 20 mM to 2 mM, the same level of inhibitory effect is observed, and it can be said that these scavengers are substances having extremely high OH radical inhibitory activity.

Example 2. Inhibitory effect of unsaturated fatty acid (methyl linoleate) lipid on peroxidation <Principle. Method> 3.0 ml of 1.0 mM hypoxanthine, 2.0 U / ml xanthine oxidase (made by buttermilk; manufactured by Wako)
15 ml, 0.15 ml of distilled water, 0.006 ml of 0.1% Triton X100 and 0.3 ml of methyl linoleate were sequentially added to the reaction solution.
0.4 ml of a capture substance of mM was added, and the peroxidation inhibitory action of methyl linoleate was measured.

Note that distilled water was added to the control instead of the trapping substance.

After shaking the reaction solution at 37 ° C. for 24 hours, the peroxide (TBA-MDA adduct) was measured by the TBA method, and the peroxidation inhibition rate was determined by the following formula. The results are shown in Table 2.

A ₁ : Absorbance when adding capture substance A ₂ : Absorbance when adding distilled water instead of capture substance A ₃ : Absorbance when adding distilled water instead of xanthine oxidase From the results shown in Table 2, it was found that these trapping substances trap superoxide and peroxy radicals and alkoxy radicals generated by peroxidation of lipids, and suppress lipid peroxidation.

Example 3. Peroxidation inhibitory action of microsomal membrane lipids <Principle. Method> 0.1 M phosphate buffer (pH 7.5 containing 1.5 mM KCl)
4) 0.15 ml, 20 mM capture substance 0.15 ml, 20 mM ADP 0.15 ml, 1 mM
0.15 ml of a microsome (3.9 mg protein / ml) suspension fractionated from rat liver was added to a 0.15 ml OH radical generating solution composed of 0.15 ml of ferrous sulfate and 0.15 ml of 0.15 M hydrogen peroxide solution, and distilled. Prepare 1.5 ml reaction with water.

The solution was incubated at 37 ° C. for 60 minutes,
Of the peroxidation of microsomal membrane lipids after 60 minutes and 60 minutes
The inhibition rate was measured by the TBA method, and the inhibition rate was determined by the following equation.

A ₁ : Absorbance of the autosomes of microsomes and phosphorus buffer in the autoxidation system A ₂ : Absorbance of the complete system with distilled water added in place of the capture substance A ₃ : Absorbance of the complete system with the capture substance added From the results in Table 3, it was found that these capturing substances also exhibited a high inhibitory effect on peroxidation of microsomal membrane lipids as a biological sample.

Example 4. Confirmation of OH radical scavenging action by ESR analysis <Principle. Method> 0.1 M phosphate buffer (pH 7.4) 0.1 ml, 5 mM diethylenetriaminepentaacetic acid (DETAPAC) 0.1 ml, 0.1
0.1 ml of M DMPO (5,5-dimethyl-1-pyrroline-1-oxide), 0.1 ml of 1 mM ferrous sulfate, 0.15 M aqueous hydrogen peroxide
0.1 ml was sequentially added to generate OH radicals.

To this reaction system, 0.1 ml of a 2 mM capture substance was added, and adjusted to 1.0 ml with distilled water.

As a control, a system to which distilled water was added instead of the capturing substance was used.

The measurement was performed by ESR (Electron Spin Resonator, JEOL JEOL-P
E-3X type), and the first minute after the start of the reaction.

 The result is shown in FIG.

The suppression rate of the peak with respect to the control is F, and the reaction rate constant between the trapping substance and the OH radical is obtained by the following equation.
Table 4 shows the OH radical scavenging ability.

Ks: Reaction rate constant of trapping substance-OH radical _KDMPO : Reaction rate constant of DMPO-OH radical (3.4 × 10 ⁹ ) [DMPO]: DMPO concentration [S]: Trapping substance concentration [F]: DMPO-OH peak Suppression rate [Effects of the Invention] From the results of the examples, it can be seen that the active oxygen scavenger of the present invention comprising a compound selected from coniferyl benzoate, eugenol, dehydrodiisoeugenol, and isoeugenol provides inflammation, carcinogenesis, ischemic injury, Radiation damage, aging,
It is clear that this is a very useful drug that directly captures and eliminates OH radicals, which are the most reactive and have no protective mechanism in the living body, among cataracts and reactive oxygen species that cause autoimmune disorders. is there.

In particular, the active oxygen scavenger of the present invention is excellent in safety and stability, and is more practical than other active oxygen scavengers.

[Brief description of the drawings]

FIG. 1 shows an ESR chromatogram before adding a capture substance (control). 2 to 5 show ESR chromatograms after the addition of a capture substance. * Capturing substance Fig. 2: Coniferyl benzoate Fig. 3: Eugenol Fig. 4: Dehydrodiisoeugenol Fig. 5: Isoeugenol Note that divalent manganese ion was used as a standard substance.

Claims (1)

(57) [Claims] (1) Co represented by the following structural formulas (A) to (D):
niferyl-benzoate, Eugen
An active oxygen scavenger comprising a compound selected from ol (eugenol), Dehydrodiisoeugenol (dehydrodiisoeugenol), and iso-Eugenol (isoeugenol). (A) Coniferyl benzoate (B) Eugenol (C) Dehydrodiisoeugenol (D) isoeugenol