JP2810220B2 - Active oxygen scavenger - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an in vivo active oxygen scavenger comprising dehydrodieugenol B or coniferyl aldehyde.

[Prior Art] 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, cancer, and aging. Active oxygen in the body is a 1,2,3 electron-reducing molecular species of ³ O ₂ (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, a living body has an enzymatic defense mechanism since it is constantly exposed to oxygen.

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 known. ing.

Vitamin C and Vitamin E have antioxidant properties,
It has been tested as a substance that scavenges active oxygen free radicals.

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, particularly on OH radicals having a strong biological action. .

[Means for Solving the Problems] The present invention is an in vivo active oxygen scavenger comprising dehydrodioigenol B or coniphenylaldehyde.

Dehydrodieugenol B and coniferyl aldehyde have also recently been found in clove oil (Ikem
oto, T. et al: International Cong-ress of Essential
oil Fregrance and Flavors. New Delhi (India), 198
9) One of the phenylpropanoid derivatives.

The method for using the active enzyme scavenger of the present invention may be a method in which an ordinary active oxygen scavenger is used.
It can be applied to cosmetics and the like.

Next, an example for clarifying the effect of dehydrodioigenol B and coniferyl aldehyde on the active oxygen scavenging action will be described.

[Examples] Example 1. Deoxyribose method <Principle> In this method, an OH radical is generated in a Fenton system, and the reaction between the OH radical and deoxinbose (3.
1 × 10 ⁹ M ^-1 S ^-1 ) produced malondialdehyde (MD
The reaction product generated when A) is reacted with thiobarbituric acid is measured (TBA method) to determine the TBA value (Ba
rry Halliwell, John MCGutteridge, O-kezie I. Aruo
ma: Improved Analytical Biochemistry, 165, 215-219, 1987).

That is, if there is a scavenger of OH radicals in this system, TB
Taking advantage of the decrease in the A value, the inhibition rate was calculated from the TBA value after addition of the trapping substance, and the inhibition rate was calculated from the TBA value after addition, and was shown as the OH radical inhibition activity value.

<Method> 28 m in 0.1 ml of 0.2 M KH ₂ PO ₄ -KOH buffer (pH 7.4)
M deoxyribose 0.1 ml, 2 mM dehydrodiogenol B or coniferyl aldehyde 0.1 ml, 1.04 mM EDTA
0.1 ml, 1 mM ascorbic acid 0.1 ml, 1 mM FeCl ₃ 0.1 ml, 1
0.1 ml of an aqueous solution of hydrogen peroxide (mM) is sequentially added, and the reaction solution is adjusted to 1.0 ml with distilled water.

After incubation of 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 rate of dehydrodieugenol B or coniferyl aldehyde was calculated by the following equation and is shown in Table 1.

A ₁ : Absorbance after adding capture substance A ₂ : Absorbance before adding capture substance In addition, since a similar inhibitory effect was observed even at a concentration of 0.2 mM, it can be said that this substance is a substance having an extremely high OH radical inhibitory effect.

Example 2 Inhibition of Peroxidation of Unsaturated Fatty Acid (Methyl Linoleate) Lipid <Principle, Method> 3.0 ml of 1.0 mM hypoxanthine, 2.0 U / ml xanthine oxidase (made by buttermilk, manufactured by Wako)
15 mM, 0.15 ml of distilled water, 0.006 ml of 0.1% Triton X100, and 0.3 mM of methyl linoleate were added to the reaction mixture at 2 mM or
0.4 ml of 20 mM dehydrodioigenol B (or coniferyl aldehyde) is added, and the peroxidation inhibitory action of methyl linoleate is measured. In addition, distilled water was added to the control instead of dehydrodioigenol B and coniferyl aldehyde.

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 solution A ₃ : Absorbance when adding distilled water instead of xanthine oxidase From the results shown in Table 2, it was found that dehydrodioigenol B and coniferyl aldehyde captured peroxy radicals and alkoxy radicals generated by superoxide and lipid peroxidation, and suppressed lipid peroxidation.

Example 3 Inhibition of Peroxidation of Microsomal Membrane Lipids <Principle, Method> 0.1 M phosphate buffer (containing 1.5 mM KCl, pH
7.4) 0.15 ml of 20 mM dehydrodiogenol B (or coniferyl aldehyde) 0.15 ml, 20 mM ADP 0.15 ml, 1
0.15 ml of a microsome (26.32 mg protein / ml) suspension fractionated from rat liver was added to an OH radical generating composition solution composed of 0.15 ml of mM ferrous sulfate and 0.15 ml of 0.15 M hydrogen peroxide solution, A 1.5 ml reaction solution is prepared with distilled water.

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

A ₁ : Absorbance of microsomes and phosphate buffer in autoxidation system A ₂ : Absorbance of complete system with distilled water added instead of capture substance A ₃ : Absorbance of complete system with capture substance added From the results shown in Table 3, dehydrodioigenol B has a high inhibitory effect on lipid peroxidation on the microsomal membrane, which is a biological sample, despite the high protein concentration.
At the minute, the inhibition rate was 67.95%. Coniferyl aldehyde also showed an inhibition rate of 37.39%, though inferior to dehydrodieugenol B.

Example 4. Confirmation of OH radical scavenging action by ESR analysis <Principle and method> 0.1 ml of 0.1 M phosphate buffer (pH 7.4), 0.1 ml of 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 hydrogen peroxide solution
0.1 ml was sequentially added to generate OH radicals.

0.1 ml of 2 mM dehydrodiogenol B or coniferyl aldehyde was added to this reaction system, and 1.0 ml of distilled water was added.
It was adjusted to ml.

As a control, a system to which distilled water was added instead of dehydrodioigenol B or coniferyl aldehyde was used.

The measurement was performed by ESR (Electron Spin Resonator, JEOL JEOL-P
(E-3X type), and performed 1 minute after the start of the reaction.

 The result is shown in FIG.

The inhibition rate of the peak with respect to the control is defined as F, and dehydrodiogenol B or coniferyl aldehyde is used.
The rate constant of the reaction with the OH radical was determined by the following equation, and was defined as the OH radical scavenging ability, and is shown in Table 4.

K _S : reaction rate constant of _{dehydrodioigenol} B (or coniferyl aldehyde) -OH radical K _DMPO : reaction rate constant of DMPO-OH radical (3.4 × 1
⁹ ) [DMPO]: DMPO concentration [S]: Dehydrodioigenol B (or coniferyl aldehyde) concentration [F]: DMPO-OH peak suppression rate [Effects of the Invention] From the results of the examples, it is clear that the active oxygen scavenger of the present invention has inflammation,
Reactive oxygen, which causes carcinogenesis, ischemic injury, radiation injury, aging, cataract, and autoimmune injury, especially OH radical, which is the most reactive and has no defense mechanism in the living body It is clear that this is a very useful drug that directly captures and eliminates. In particular, dehydrodioigenol B and coniferyl aldehyde are excellent in safety and stability, and are more practical than other active oxygen scavengers.

[Brief description of the drawings]

FIG. 1 (A) shows the case where water is put in place of the trapping substance.
1 shows a chromatogram of ESR, wherein (B) shows a chromatogram of ESR when dehydrodiogenol B was added, and (C) shows a chromatogram of ESR when coniferyl aldehyde was added. In addition, divalent manganese ion was used as a standard substance.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 31/11 ADU A61K 31/11 ADU AGZ AGZ // A61K 35/78 35/78 C

Claims (1)

(57) [Claims] 1. Dehydrodieugenol B represented by the following structural formula:
(Dehydrodieugenol B) or Conif-erylaldeh
Active oxygen scavenger composed of yde (coniferyl aldehyde).