JPH07304678A - Active oxygen-catching composition - Google Patents

Abstract

PURPOSE:To prepare an active oxygen-catching composition containing vanillin and a specific glucoside as active ingredients, having an excellent oxidation injury-preventing action, and useful for preventing and treating cancers, cataract, ischemic diseases, etc. CONSTITUTION:This composition contains vanillin and a glucoside of the formula as active ingredients. The active ingredient of the composition is extracted from a highly preservative food, especially a spice. For example, the active ingredient is isolated by extracting allspice with methanol and further subjecting the obtained crude extract to an adsorption chromatography and a high performance liquid chromatography. The extract has an excellent oxidation injury- preventing action even in the state of the methanol crude extract.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an active oxygen scavenging composition that traps and eliminates active oxygen in the body to protect against oxidative damage in the body.

[0002]

2. Description of the Related Art Oxygen is indispensable to humans, and humans use oxygen to efficiently obtain energy. On the other hand, on the other hand, oxygen produces free radicals called active oxygen in the living body, and when the scavenging / scavenging activity of this active oxygen is reduced, its toxicity accumulates, causing various diseases. .

Active oxygen reacts with biological components such as proteins, lipids, nucleic acids and sugars to cause oxidative damage,
It is known to reduce biological function. Further, it is considered that these reactions in the living body are highly likely to be involved in many diseases such as aging, carcinogenesis, cataract, and ischemic disease. In addition, oxygen in food is a major cause of chemical deterioration, which is represented by lipid peroxidation and the generation of rancidity and toxicity caused by it.

[0004]

In order to suppress the above-mentioned oxidative damage to the living body due to active oxygen, supplementary active oxygen is added.
It would be beneficial to find substances in food that could be eliminated. In particular, focusing on spices, which are said to have a high preservation effect among foods, is considered to be significant from the viewpoint of safety, and is also interesting from the viewpoint of new functionality of foods.

The inventor of the present invention arrived at the present invention by finding a composition having an excellent active oxygen scavenging ability as a result of extensive studies on antioxidant substances contained in various spices.

An object of the present invention is to provide a composition that effectively captures and eliminates active oxygen in the body.

[0007]

The present invention is an active oxygen scavenging composition containing vanillin as an active ingredient.

Further, the present invention is an active oxygen scavenging composition containing a glycoside represented by the following formula as an active ingredient.

[0009]

[Chemical 2]

[0010]

EXAMPLES Examples of the present invention will be described in detail below.

I. Experimental method [1-1] Reagents All reagents used are commercially available special grade reagents, and the special types are shown below. Further, the water used was distilled water of ion-exchanged water once, and the organic solvent used was distilled water of a commercially available primary reagent once. However, when extracting with methanol,
When the methanol extract was roughly fractionated by Amberlite XAD-2 gel, the primary reagent was used as it was.

Ferrous Sulfate Wako Pure Chemical Industries, Ltd. EDTA Wako Pure Chemical Industries, Ltd. 2-Deoxy-D-ribose Wako Pure Chemical Industries, Ltd. Hydrogen Peroxide Mitsubishi Gas Chemical Co., Ltd. TBA (Thiobarbitur) Acid) Wako Pure Chemical Industries, Ltd. DTPA Wako Pure Chemical Industries, Ltd. β-Glucosidase Amano Pharmaceutical Co., Ltd. [1-2] Reaction Conditions for Measurement of Active Oxygen and Preparation of Reaction Reagents The reaction reagents are prepared as follows. did. (6) was stored refrigerated, and the others were prepared immediately before use.

(1) 100 μM ferrous sulfate / EDTA solution FeSO ₄ .7H ₂ O (mw278.03) and EDTA (mw372.24) were weighed in the required amounts and mixed well as a solid to give 1 mM. So that it was dissolved in water. This was diluted to 100 μM with 0.1 M phosphate buffer (pH 7.4).

(2) 10 mM 2-deoxyribose solution 2-deoxyribose (2-deoxyribose; mw134.13)
It was dissolved in 0.1 M phosphate buffer (pH 7.4) so that the concentration became 0 mM.

(3) 10 mM sodium benzoate solution Sodium benzoate (mw144.11) was adjusted to 10 mM.
It was dissolved in 0.1 M phosphate buffer (pH 7.4).

(4) 10 mM hydrogen peroxide solution 30% hydrogen peroxide (mw34.02) to 100 mM
It was diluted with 0.1 M phosphate buffer (pH 7.4).

(5) 1% (W / V) TBA (thiobarbituric acid) solution TBA (mw144.15) was adjusted to 1% (W / V) with 50 mM NaO.
Dissolved in H 2 solution.

(6) 2.8% (W / V) TCA solution TCA (mw163.39) was dissolved in water to a concentration of 2.8% (W / V).

(7) 0.1 mM DTPA solution DTPA (mw393.35) was dissolved in 0.1 M phosphate buffer (pH 7.4) to a concentration of 10 mM.

TBA method (oxidation reaction of 2-deoxyribose) In the TBA method, malondialdehyde (MDA) and a malondialdehyde-like substance produced by the decomposition of lipid peroxide are condensed with two molecules of TBA under acidic conditions. It is a method for evaluating the degree of lipid peroxidation by colorimetrically quantifying the red substance produced. In this embodiment, as shown in FIGS. 1 (a) and 1 (b),
Hydroxy radical (.OH) generated chemically by the Fenton system abstracts the hydrogen bonded to the 4-position carbon of 2-deoxyribose to initiate oxidative decomposition, resulting in malondialdehyde. Was measured by the TBA method, and the inhibitory effect of known inhibitory substances or natural substances was examined. The method is as follows.

100μ in test tubes with screw caps
M FeSO ₄ / EDTA solution 200 μl, 10 mM 2-deoxyribose solution 200 μl, 0.1 M phosphate buffer (pH 7.4) 1.2 m
1, 200 μl of a sample having an appropriate concentration was sequentially added, 200 μl of 10 mM hydrogen peroxide solution was added, and the reaction was initiated by stirring, followed by incubation at 37 ° C. for 4 hours. The control without addition of the sample was set to 0 hour, and the control immediately after the start of reaction was set to 0 hour, and 0 hour was stored in an ice bath for 4 hours.

After completion of the reaction, 1 ml each of 1.0% TBA solution and 2.8% TCA solution was added to the reaction solution according to the method of Gutteridge (translated by Toshio Goto, Fizer, Williamson "Organic Chemistry" Maruzen). After stirring well, it was heated at 100 ° C. for 10 minutes. The reaction was stopped by quenching in an ice bath and the absorbance at 532 nm was measured. However, if turbidity occurs when adding the sample, centrifuge (3000 rpm,
Then, the supernatant was measured.

Fluorescence Method [Hydroxylation Reaction of Benzoic Acid] As shown in FIG. 2, when a hydroxy radical is generated in the vicinity of benzoic acid, it is substituted with hydrogen at the 2-position of the benzene ring, and 2-
Since hydroxybenzoic acid is produced, the inhibitory effect of known inhibitors or natural substances was examined by measuring the fluorescence derived from hydroxybenzoic acid. The method is as follows.

100μ in a test tube with a screw cap
200 μl M FeSO ₄ / EDTA solution, 200 μl 10 mM sodium benzoate solution, 1.2 m 0.1 M phosphate buffer (pH 7.4)
Then, 200 μl of a sample having an appropriate concentration was sequentially added, 200 μl of 10 mM hydrogen peroxide solution was added, and the mixture was stirred to start the reaction, and incubated at 37 ° C. for 2 hours. The control without addition of the sample was set to 0 hour immediately after the start of the reaction of the control.

After completion of the reaction, 0.1 mM DTP as a reaction terminator
After adding 2.0 ml of solution A and stirring, the fluorescence intensity derived from hydroxybenzoic acid was measured. However, if turbidity occurred when the sample was added, centrifugation (3000 rpm, 10 minutes) was performed and the supernatant was measured. The reaction terminator was added after the start of the reaction for 0 hours.

<Instrument> Hitachi F-2000 type spectrophotometer <Conditions> Excitation 305 nm Emission 407 nm Response 0.1 sec Excitation 10 nm Emission 10 nm How to obtain suppression effect 2-Deoxyribose oxidation reaction or water of benzoic acid A substance that suppresses the oxidation reaction was used as an active oxygen scavenging substance, and the suppression effect was determined by the following formula.

A. Oxidation reaction of 2-deoxyribose Inhibition (%) = 100- [OD532nm (sample) -OD
532nm (0time)] × 100 / (OD532nm (control) −OD532n
m (0time)] OD532nm (sample): Absorbance when sample is added OD532nm (control): Absorbance when sample is not added OD532nm (0time): Absorbance immediately after the reaction is started without sample Specimen: Inhibition effect Substances added for consideration (known inhibitors or natural products) B. Benzoic acid hydroxylation reaction Inhibition (%) = 100- [fluorescence intensity (sample) -fluorescence intensity (0time)] x 100 / [fluorescence intensity (control) -fluorescence intensity (0
time)] Fluorescence intensity (sample): Fluorescence intensity when sample is added Fluorescence intensity (control): Fluorescence intensity when sample is not added Fluorescence intensity (0time): Absorbance immediately after the start of reaction when no sample is added [1 -3] Extraction of Allspice 500g of Allspice was added to 5.
Extract 3 times with 0 L. The extract was filtered through filter paper (No. 2) and then dried under reduced pressure by a rotary evaporator to obtain a crude methanol extract.

[1-4] Isolation / Production of Active Substances in Allspice Extract (FIG. 3) Crude Fractionation by XAD-2 Amberlite (Amberlite XAD), which is a crude chromatographic extract of allspice in methanol, is subjected to adsorption chromatography. -2) was used to perform crude fractionation in batch mode. Amberlite XAD-2 is a synthetic adsorbent of a porous polymer having a huge network structure. There are polystyrene-based and acrylic-based adsorbents, and they have a very wide range of adsorption performance due to the difference in surface characteristics.
It has the same appearance as ion exchange, but does not have an ion exchange function. Elution solvent from water every 25% 1
The methanol content was increased to 00%, and finally elution was carried out with acetone, and each elution section was concentrated to dryness by a rotary evaporator.

<Fractionation conditions> Column: Amberlite XAD-2 Column size: 3.1 x 42.0 cm Fraction: Each elution fraction 3.0 L Each dry elution fraction was dissolved in methanol or acetone to obtain 2-deoxyribose. The inhibitory effect on the oxidative reaction of benzene and the hydroxylation reaction of benzoic acid was examined.

Analysis by HPLC The methanol extract of allspice roughly fractionated by adsorption chromatography was analyzed by HPLC under the following conditions.

<HPLC Condition> Column: Develosil ODS-HG-5 4.6φ ×
150mm Solvent A: H ₂ O Solvent B: MeOH Gradient Program: 0min (A 100%) → 30min (A 0
%) → 40min (A 0%) → 50min (A 100%) Flow Rate: 1.0ml / min Detection: UV 280nm injection: 1 μl Range: 0.08 Pump: TOYO SODA CCPE UV Detector: TOYO SODA UV-8000 Recorder: TOA Fractionation by FBR-252A HPLC In order to isolate the methanol extract of allspice roughly fractionated by adsorption chromatography, it was fractionated by HPLC under the following conditions.

<HPLC Condition 1> Column: Develosil ODS-HG-5 8.0φ ×
250mm Solvent: MeOH / H ₂ O = 4/6 Flow Rate: 2.0ml / min Detection: UV 280nm <HPLC Condition 2> Column: Develosil ODS-HG-5 8.0φ ×
250mm Solvent: MeOH / H ₂ O = 35/65 Flow Rate: 2.0ml / min Detection: UV 280nm <HPLC Condition 3> Column: Develosil ODS-HG-5 8.0φ ×
250mm Solvent: MeOH / H ₂ O = 3/7 Flow Rate: 2.0ml / min Detection: UV 280nm <HPLC Condition 4> Column: Develosil ODS-HG-5 8.0φ ×
250mm Solvent: MeOH / H ₂ O = 25/75 Flow Rate: 2.0ml / min Detection: UV 280nm Pump: TOYO SODA HCL-803D UV Detector: TOYO SODA UV-8000 Recorder: TOA FBR-252A Purity measurement Peaks obtained In order to perform the purity test of 1., HPLC analysis or TLC analysis was performed under the following conditions.

<HPLC Condition 1> Column: Develosil ODS-HG-5 4.6φ ×
150mm Solvent: MeOH / H ₂ O = 35/65 Flow Rate: 2.0ml / min Detection: UV 280nm <HPLC Condition 2> Column: Develosil ODS-HG-5 4.6φ ×
250mm Solvent: MeOH / H ₂ O = 25/75 Flow Rate: 2.0ml / min Detection: UV 280nm <TLC Condition 1> Silicagel: 60F ₂₅₄ 20 × 20cm 0.25mm Solvent: CHCl ₃ / MeOH / H ₂ O = 6.5 / 3.5 / 1 <TLC Condition 2> Silicagel: 60F ₂₅₄ 20 × 20cm 0.25mm Solvent: CHCl ₃ / MeOH + Acetic acid = 3/1 [1-5] Hydrolysis by β-glucosidase β-glucosidase (Glucosidase) was added to about 20 mg of sample. 2 ml of a solution (5 units / ml, 20 mM acetate buffer pH 5.0) was added and mixed, and the mixture was incubated for 24 hours while shaking in a 37 ° C. incubator. Next, the mixture was extracted with ethyl acetate three times, and the ethyl acetate layer was dehydrated with anhydrous sodium sulfate.
It was concentrated under reduced pressure and used as a sample for aglycone analysis. The aqueous layer was concentrated under reduced pressure, freeze-dried, and then subjected to gas chromatography (G
The sample was used for sugar analysis according to C).

[1-6] Preparation of TMS Compound The aqueous layer section obtained in [1-5] above was TM-treated under the following conditions.
It became S.

Sample: 1 to 2 mg Anhydrous Pyridine: 0.5 ml HMDS: 0.3 ml TMCS: 0.1 ml Anhydrous pyridine, HMDS, TMCS was added to a sample from which water was completely removed.
Was added in this order, and after mixing well, they were reacted for 2 hours in a 37 ° C ventilated incubator to prepare a sample for gas chromatography.

[1-7] Analysis of aglycone The sample obtained for β-glucosidase treatment for aglycone analysis was analyzed by HPLC under the following conditions.

Column: Develosil ODS-HG
-5 4.6φ × 150mm Solvent: MeOH / H ₂ O = 35/65 Flow Rate: 1.0ml / min Detection: UV 280nm [1-8] Analysis of constituent sugars Aqueous component of β-glucosidase treatment under the following conditions. It was analyzed by gas chromatography.

Aparatus: Simadzu Gaschrom
atograph GC-9A Recorder: Simadzu CHROMATOPAC C-R3A Column: Silicon GE SE-52 Carrier gass: N ₂ 40ml / min (H ₂ 0.5kg / cm ² ) (Air 0.5kg / cm ² ) Detection: FID Injection temp.: 230 ° C Column temp .: 140 to 210 ° C (2 ° C / min) [1-9] Instrument analysis The following instruments were used for instrument analysis.

NMR: Bruker ARX400 UV Spectrometer: HITACHI U-BEST 50 EI-MS: JASCO JMS-DX-705L FAB-MS: JEOL JMS-DX-705L IR: JASCO FT / IR-8300 (α) _D : JASCO DIP -370 II. Search for active oxygen trapping element contained in allspice [2-1] Time-dependent change of reaction in active oxygen generation system In order to understand the reaction involving active oxygen, Fe ²⁺ / EDTA /
The oxidation reaction system of 2-deoxyribose by H ₂ O ₂ system was measured with time using TBA method. The measurement results are shown in FIG. The amount of TBA-positive substances (TBARS) produced increased almost linearly until 8 hours later. From these results, the reaction time was set to 4 hours so that a TBRS value sufficient for studying the effect of capturing and eliminating active oxygen was obtained. Also, the hydroxylation reaction of sodium benzoate using the fluorescence method was performed by the method of Tanigawa et al. (Mitsuru Tanikawa, Master's thesis in the Graduate School of Agriculture, Nagoya University (199
2)) was followed.

[2-2] Inhibitory Effect of Known Antioxidants In order to examine the difference in inhibitory degree between 2-deoxyribose oxidation reaction system and benzoic acid hydroxylation reaction system, Fe ²⁺ / EDT
The inhibitory effect of known antioxidants on both systems by the A / H ₂ O ₂ system was investigated. Results are shown in FIG. Known antioxidants include SOD (Super Oxide Dismutase), mannitol,
Histidine, ethanol, sodium formate and catalase were used.

SOD is an enzyme that catalyzes a disproportionation reaction of superoxide (formula (1)) 2O ₂ ⁻ + 2H ⁺ → O ₂ + H ₂ O ₂ (1), and is composed of superoxide and other active oxygen generated from it. Thought to protect the organism from toxicity,
It is widespread in aerobes and is also found in some facultative and obligate anaerobes.

Mannitol is a kind of sugar alcohol derived from hexose and has been found as a scavenger of hydroxy radicals, and is naturally present in free form in plants such as onions and dried persimmons, seaweeds, fungi and mushrooms. To do.

Histidine, ethanol and sodium formate are also scavengers of hydroxy radicals. Catalase is
An enzyme that catalyzes the decomposition reaction of hydrogen peroxide (formula (2)) H ₂ O ₂ + H ₂ O ₂ → O ₂ + 2H ₂ O (2), which is widely distributed in aerobic cells of animals, plants, and microorganisms. It is often found in the liver, red blood cells, and kidneys in animals, and in the chloroplast in plants.

The inhibitory effects of these antioxidants were high, and especially SOD, ethanol and catalase showed high inhibitory effects.

It was also found that the inhibitory effect on the 2-deoxyribose oxidation reaction system was higher than that on the benzoic acid hydroxylation reaction system (FIG. 5). The difference in the inhibitory effect between the system in the oxidation reaction system of 2-deoxyribose and the system in the hydroxylation reaction of benzoic acid is that 2-deoxyribose (1.9 × 10 ⁹ M ^-1 S ^-1 ) for hydroxyl radical
It is considered that this is due to the difference in reaction rate between benzoate and sodium benzoate (4.3 × 10 ⁹ M ^-1 S ^-1 ). In other words, since 2-deoxyribose has a smaller reaction rate with hydroxyl radical than benzoic acid, it is considered that the oxidation reaction of benzoic acid is less likely to be suppressed than the oxidation reaction of 2-deoxyribose.

Next, since methanol is also a scavenger of hydroxyl radicals, the concentration of methanol becomes a problem when the methanol extract is dissolved in methanol and added to the reaction system. Therefore, the degree of suppression by methanol was investigated. Results are shown in FIG. If the final concentration was 0.25 μM or less, the effect of methanol was considered to be less of a problem.

[2-3] Suppressive effect of crude extract The methanol extract of allspice obtained in [1-3] above was adjusted to a final concentration of 10 μg / ml, 1.0 μg / ml, 0.1 μg / ml. It was dissolved in an acid buffer and the inhibitory effect was examined in the oxidation reaction system of 2-deoxyribose. As a result, as shown in FIG. 7, the inhibitory effect of the methanol extract increased depending on the concentration, and the inhibitory rate exceeded 80% at the final concentrations of 10 μg / ml and 1.0 μg / ml.

[2-4] Fractionation by XAD-2 Crude methanol extract (64.0) obtained in [1-4] above
8g) under the conditions shown in [1-4] Amberlite XAD-
Fractionation was carried out according to 2, and each elution fraction was dried to dryness and then dissolved in methanol or acetone to a concentration of 100 mg / ml. Next, it was diluted with a phosphate buffer to a final concentration of 1.0 μg / ml and 0.1 μg / ml, and the inhibitory effect on the oxidation reaction of 2-deoxyribose or the hydroxylation reaction of benzoic acid was examined. The results are shown in FIGS. 8 and 9. Final concentration 1.
At 0 μg / ml, a considerable inhibitory effect was observed in all except water and acetone elution categories. Also, the final concentration of 0.1 μg / ml
, A strong activity was observed in the 75% methanol elution category and the 50% methanol elution category.

[2-5] Analysis by HPLC Fractionation by Amberlite XAD-2 showed strong activity in 75% methanol elution category and 50% methanol elution category as described above. An attempt was made to isolate and generate an active oxygen scavenger.

Each of the eluted components was dissolved in methanol or acetone to a concentration of 10 mg / ml, and analyzed by HPLC under the conditions shown in [1-4]. The result is shown in FIG.
Shown in. As shown, 50% methanol elution category (a)
In the elution time of about 15 minutes (A1) and 19 minutes (A2) in the 75% methanol elution category (b), peaks which are considered to be derived from the active oxygen trapping substance were detected. It is considered that the overlapping peaks were detected because the fractionation was performed in batch with Amberlite XAD-2. Therefore, the active oxygen scavenging substance was searched from the 75% methanol elution category in which the yield was high during fractionation.

[2-6] Fractionation by HPLC Dissolving the 75% methanol elution fraction in a small amount of methanol, adsorbing it on C18 cartridges (SEP-PAK, Waters), eluting with methanol, and drying under reduced pressure with a rotary evaporator. . This was dissolved in a small amount of 40% methanol, and the peaks A1 and A2 shown in FIG. 11 were fractionated under the conditions shown in <HPLC Condition 2> of [1-4]. Since these contain impurities, A2 is further <HP of [2-4]
Purification was performed under the conditions shown in LC Condition 2>. Also, A
1 is the <2-4] <HPLC Condition 3>, <HP
Purification was carried out under the conditions shown in LC Condition 4>. The yield is
A1 was 68.4 mg and A2 was 101.2 mg.

[2-7] Purity test [2-6] The purity test of the peak A1 obtained in [2-6] was carried out by [1-
[4] <HPLC Condition 1>, a pure peak was detected at an elution time of 11 minutes.
Similarly, for peak A2, <1-4] <HPLC Condi
When it was carried out under the conditions shown in section 2>, a pure peak was detected at an elution time of 20 minutes. Moreover, when it performed on condition shown in <HPLC Condition 1> of [1-4], it was detected as a pure spot ( _Rf = 0.49).

[2-8] Inhibitory effect of A1 and A2 In the same manner as in [2-4], the inhibitory effect of A1 and A2 on the oxidation reaction of 2-deoxyribose or the oxidation reaction of benzoic acid was examined. The results are shown in FIGS. 12 and 13. As a result, both A1 and A2 showed activity.

Structural analysis of III.A1 [3-1] Result of instrumental analysis (a) EI-MS [M] ⁺ 152 was confirmed as a peak (Fig. 1).
4).

(B) NMR A1 was dissolved in CD ₃ OD and ¹ H-NMR (400 MHz), ¹³ C-NMR (10
0 MHz) was measured. Tetramethylsilane as a reference substance
(TMS) was used. The results are shown in Table 1.

[0056]

[Table 1]

From ¹ H-NMR, 2, 5, and 6 were considered to be derived from an aromatic ring. Since the coupling constants of 5 and 6 were 7.9 Hz, the H environment was considered to be in the ortho position. Also,
Since the coupling constants of 6 and 2 were 1.9 Hz, metacoupling was considered. 1-CH from ¹³ C-NMR
It was assumed that O was bound to O, 3 to -OMe, and 4 to -OH.

[Identification of Structure] As a result of various instrumental analyzes, it was presumed to be valine (Vanillin). Therefore, when compared with the standard product, both ¹ H-NMR and ¹³ C-NMR coincided with each other. 1)).

IV. Structural analysis of A2 [4-1] Structural analysis of A2 Results of instrumental analysis (a) FAB-MS Glycerol was used as a matrix. The result is shown in FIG. As shown in the figure, the peak of [M + H] ⁺ is 495 and the peak of [M + Na] ⁺ is 517.
Was observed. In addition, a peak of 315 predicted to be a fragment ion of aglycone was observed.

(B) UV absorption A2 was dissolved in an appropriate amount of spectral methanol and measured. As a result, as shown in FIG. 16, since absorption was observed near 280 nm, phenol was considered.

Λ _max = 276.6 nm log ε _max = 3.59 (c) As a result of measurement by the FT-IR KBr method according to Lambert-Beer's law, the following information was obtained.

-3358 cm ^-1 OH stretching intermolecular hydrogen bond 1613 cm ^-1 aromatic 1516 cm ^-1 aromatic 1334 cm ^-1 OH 1204 cm ^-1 OH (d) [α] _D ²⁴ A2 was dissolved in 3 ml of spectral methanol, and optical rotation was obtained. The degree was measured. As a result, the optical rotation is -22.8 (Conc. 0.075
%) Was confirmed.

(E) NMR A2 was dissolved in CD ₃ OD and ¹ H-NMR (400 MHz), ¹³ C-NMR (10
(0 MHz) DEPT, ¹ H- ¹ H COSY, HMQC and HMBC were measured.
Tetramethylsilane (TMS) was used as a reference substance. The results are shown in FIGS. 17 to 21 and Table 2.

[0064]

[Table 2]

Although there is a part where the ¹ H-NMR chemical shift is missing, this is 3.5-4.
This is because at 0 ppm, it has a complicated appearance and cannot be analyzed.

From ¹ H-NMR (see FIG. 17), it is considered that 3 and 5 are derived from an aromatic ring, and their coupling constants are 1.6 Hz,
Is 1.5 Hz, the correlation from ¹ H- ¹ H COSY (see FIG. 20) is observed, meta coupling was estimated.

From ¹ H- ¹ H COZY, correlations were observed in 7, 8 and 9, and ¹ H-NMR (see FIG. 17) of 8 was m, so an aryl group was considered.

From ¹ H- ¹ H COSY (see FIG. 20), G1-G6
Correlation was observed in HMQC (see Fig. 21): 1 at 90-110ppm (G1), 1 at 60-70ppm (G6), 4 at 70-80ppm.
The presence of sugar was suggested from the fact that the correlation of the books (G2, G3, G4, G5) was observed. In addition, a chiral effect was observed in G6, in which C at the 5-position of the sugar was an asymmetric carbon, and two chemical shifts of H at the 6-position occurred due to the effect of optical activity. Furthermore, G1 is considered to be the anomeric position from ¹³ C-NMR (see FIG. 21), and the coupling constant is 7.3 Hz. Therefore, if the sugar is glucose, the β form in which H is in the axial form is suggested. Was done.

Estimation of Structure From the results of various analytical instruments, A2 was estimated to be a glycoside.

[4-2] Hydrolysis with β-Glucosidase [4-1] Since A2 was predicted to be a glycoside having one glucose bonded thereto, β was prepared according to the method of [1-5] above. -Hydrolysis with glucosidase was performed.

[4-3] Identification of constituent sugars The aqueous layer obtained in [4-2] was converted into TMS by the method shown in [1-6] and analyzed by gas chromatography.
As shown in FIG. 22, sugar-derived peaks were confirmed at elution times of 20 minutes and 25 minutes. From the comparison with the standard, the former was identified as the α-type of glucose and the latter was identified as the β-type.

[4-4] Analysis and fractionation of aglycone The ethyl acetate layer obtained in [4-2] was used in the above [2-4].
The analysis was performed under the conditions shown in <HPLC Condition2>. As a result, as shown in FIG. 23, two aglycones were detected. Therefore, Aglycon 1 is <HPLC Condition
Under the conditions shown in 2>, Aglycon 2 is <HPLC Condition 4
The isolation and purification were performed under the conditions shown in <>.

[4-5] Structural Analysis of A2-Aglycone 1 As a result of instrumental analysis (a), 180 was confirmed as a peak of EI-MS [M] ⁺ .

(B) UV Absorption A2-Aglycone 1 was dissolved in an appropriate amount of spectral methanol and measured. As a result, as shown in FIG. 24, since absorption was observed at around 280 nm, phenol was considered.

Λ _max = 272.4 nm According to the Lambert-Beer's law, log ε _max = 2.91 (c) [α] _D ²⁴ A2-aglycone 1 was dissolved in 3 ml of methanol for spectra, and the optical rotation was measured. As a result, the optical rotation is 9.82 (C
onc. 0.11%) was confirmed.

(D) NMR A2-Aglycone 1 was dissolved in CD ₃ OD, ¹ H-NMR, ¹³ C-
NMR, DEPT, ¹ H- ¹ HCOSY, HMQC and HMBC were also measured. Tetramethylsilane (TMS) was used as a reference substance. The results are shown in FIGS. 25 to 29 and Table 3.

[0077]

[Table 3]

From ¹ H-NMR (see Table 3), 3 and 5 were d and d, and the chemical shift was around 6 ppm, so it was considered to be derived from the aromatic ring. Also, the coupling constant is 2.8Hz
Therefore, metacoupling was considered.

From ¹ H- ¹ H COSY (see FIG. 27) 7, 8, 9
A correlation was observed with, and since 8 and 9 were m, an aryl group was considered. Also, 7 is 3 from HMBC (see FIG. 29),
The binding position of the aryl group was deduced from the fact that a correlation was found between 4 and 5.

Since -OMe was found to correlate with 2 from HMBC (see FIG. 29), and at the same time, 2 was also seen with 3, suggesting that -OMe is bound to the meta position with respect to the aryl group. .

Estimation of Structure As a result of various instrumental analyzes, A2-aglycone 1 was identified as 4-allyl-6-hydroxy-2-methoxyphenol (4-allyl-6).
-hydroxy-2-methoxyphenol). This compound is an analog in which OH is bound to the 6-position of Eugenol, and as a result of a literature search, it was found to be a novel compound.

[4-6] Structural analysis of A2-aglycone 2 As a result of instrumental analysis (a) 170 was confirmed as a peak of EI-MS [M] ⁺ .

(B) NMR A2-Aglycone 2 was dissolved in CD ₃ OD, ¹ H-NMR, ¹³ C-
NMR was measured. Tetramethylsilane (T
MS) was used. The results are shown in FIGS. 30 and 31.

Identification of Structure As a result of TLC analysis under the conditions shown in <HPLC Condition 2> in [1-4] above, gallic acid was compared with that of a standard product.
(Gallic Acid). Also, A2-aglycone 2 was identified as gallic acid by comparison with the NMR of the standard product.

[4-7] Estimation of A2 Structure As a result of various instrumental analyzes, it was suggested that A2 is a glycoside in which two aglycones are bound to glucose. It was presumed that aglycone 1 is an eugenol analogue and aglycone 2 is gallic acid.

When the eugenol analog was released by hydrolysis with β-glucosidase, ¹ H-NMR of 3 and 5 (Table 2,
Since the difference in chemical shift in (see Table 3) is small, it is indicated that the non-equivalent H approaches the equivalence, and the OH at the 1-position of the sugar and the OH at the 6-position of the eugenol analog are It was suggested that β-glucoside bonds are present.
If the OH bond at the 1-position of the eugenol analog was bound, it would be expected that no such change would be seen when released.

On the other hand, as for the binding mode between gallic acid and sugar, the HMBC with A2 (see FIG. 21) showed a correlation between the 7'carbonyl and H at the 6-position of the sugar, suggesting an ester bond. . Usually, the ester bond is not hydrolyzed by β-glucosidase, but the solvent conditions are
Since it had a pH of 5.0, it appears to have been hydrolyzed during the 24 hour incubation with shaking.

From the above, A2 is 1 of glucose
OH at the 6-position of 4-allyl-6-hydroxy-2-methoxyphenol, which is an aglycone, is β-glucosidic bonded to the OH at the 6-position, and carbonyl of gallic acid is ester-bonded to the OH at the 6-position of glucose. It is said to be a glycoside.

[0089]

[Chemical 3]

V. Inhibitory effect of each methanol extract of allspice The active oxygen scavenging ability of each isolated substance was investigated, and the activity expression mechanism was also examined. Here, comparative evaluation and examination were performed by comparison with eugenol, which is a known antioxidant.

[5-1] Inhibitory effect of each methanol extract of allspice Methanol of each allspice extract of allspice obtained in [2-6] or [4-2] is adjusted to 2.02 mM with methanol. Dissolved. Then, the final concentration 2.02 × 10 ^-3 mM, 2.02
It was diluted with a phosphate buffer to a concentration of × 10 ^-4 mM, and the inhibitory effect on the oxidation reaction of 2-deoxyribose or the hydroxylation reaction of benzoic acid was examined. The results are shown in FIGS. 32 and 33.

As a result, at the final concentration of 2.02 × 10 ^-3 mM, the strongest inhibitory effect on eugenol was observed, followed by A2.
-The inhibitory effect was observed in the order of A2-aglycone 1 which is a glycoside and eugenol analogue.

On the other hand, at the final concentration of 2.02 × 10 ^-4 mM, the strongest inhibitory effect was observed for A2-aglycone 1 which is an analog of eugenol, and then the inhibitory effect was observed for A2-glycoside and eugenol in this order. . Also, A1 (vanillin)
The inhibitory effect was also recognized.

[5-2] Conclusion Eugenol is a phenolic compound that accounts for 80% of the essential oil component of allspice, and is studied by Fujio et al. (Hideji Fujio, Akira Hiyoshi, Takayasu Asari, Kinyuki Sumie, “Japanese Foods”. It has been reported by Kogyo Kogyo (Journal of Japan), 18241, (1969), that it has a considerably strong oxidation inhibitory effect on lard. In the above experiment, it was shown that among antioxidants, it also has a function of capturing active oxygen as a mechanism of activity expression.

Although A1 (vanillin) has not been recognized as a scavenger until now, the inhibitory effect was observed in the above-mentioned experiments, and thus it was revealed that it has an active oxygen scavenging ability. The expression mechanism is that the hydroxyl radical is trapped by the hydrogen abstraction reaction of the phenolic hydroxyl group (the following formula (a)) or by the substitution with H bonded to the aromatic ring (the following formula (b)). Conceivable.

[0096]

[Chemical 4]

On the other hand, A2- which is an analog of eugenol
Regarding the mechanism of activity expression of aglycone 1 (4-allyl-6-hydroxy-2-methoxyphenol), it is considered that the phenolic hydroxyl group and H bonded to the aromatic ring are involved as in A1. Further, since this substance has OH bound to the 6-position of eugenol and has an o-dihydroxy mechanism, it is considered that it acts as a metal chelating agent and suppresses the oxidation reaction.

[0098]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, various diseases caused by active oxygen having excellent active oxygen scavenging ability,
For example, it is possible to provide a composition expected to be used as a therapeutic agent for carcinogenesis, cataract, ischemic disease and the like.

[Brief description of drawings]

1A and 1B are explanatory diagrams of a TBA method.

FIG. 2 is an explanatory diagram of a fluorescence method using a hydroxylation reaction of benzoic acid.

FIG. 3 is a flow chart illustrating a method for isolating and producing an active substance in an allspice extract.

FIG. 4 is a graph showing the reaction of 2-deoxyribose with the Fe ²⁺ / EDTA / H ₂ O ₂ system over time.

FIG. 5 is a graph showing the inhibitory effect of the system in the oxidation reaction system of 2-deoxyribose and the system in the hydroxylation reaction system of benzoic acid.

FIG. 6 is a graph showing the inhibitory effect of methanol in a 2-deoxyribose oxidation reaction system and a benzoic acid hydroxylation reaction system.

FIG. 7 is a graph showing the inhibitory effect of a methanol extract in a 2-deoxyribose oxidation reaction system.

FIG. 8 is a graph showing the inhibitory effect of a methanol extract fractionated with Amberlite XAD-2 on the 2-deoxyribose oxidation reaction system.

FIG. 9 is a graph showing the inhibitory effect of a methanol extract fractionated with Amberlite XAD-2 on the benzoic acid hydroxylation reaction system.

Fig. 10 HP of 50% methanol elution category (a) and 75% methanol elution category (b) by Amberlite XAD-2
It is a graph which shows the analysis result by LC.

FIG. 11 is a graph showing the results of HPLC analysis of the 75% methanol elution fraction.

FIG. 12 is a graph showing the inhibitory effect of A1 and A2 on the oxidation reaction of 2-deoxyribose.

FIG. 13 is a graph showing the inhibitory effect of A1 and A2 on the hydroxylation reaction of benzoic acid.

FIG. 14 is a graph showing an EI-MS spectrum of A1.

FIG. 15 is a graph showing a FAB-MS spectrum of A1.

FIG. 16 is a graph showing a UV spectrum of A2.

FIG. 17 is a graph showing ¹ H-NMR spectrum of A2.

FIG. 18 is a graph showing a ¹³ C-NMR spectrum of A2.

FIG. 19 is a graph showing ¹ H- ¹ HCOSY spectrum of A2.

FIG. 20 is a graph showing an HMQC spectrum of A2.

FIG. 21 is a graph showing an HMBC spectrum of A2.

FIG. 22 is a graph showing a gas chromatograph of a sugar component using β-glucosidase.

FIG. 23 is a graph showing the results of analysis of glycoside hydrolysis by β-glucosidase by HPLC chromatography.

FIG. 24 is a graph showing a UV spectrum of A2-aglycone 1.

FIG. 25 is a graph showing ¹ H-NMR spectrum of A2-aglycone 1.

FIG. 26 is a graph showing a ¹³ C-NMR spectrum of A2-aglycone 1.

FIG. 27 is a graph showing ¹ H- ¹ HCOSY spectrum of A2-aglycone 1.

FIG. 28 is a graph showing an HMQC spectrum of A2-aglycone 1.

FIG. 29 is a graph showing an HMBC spectrum of A2-aglycone 1.

FIG. 30 is a graph showing ¹ H-NMR spectrum of A2-aglycone 2.

FIG. 31 is a graph showing a ¹³ C-NMR spectrum of A2-aglycone 2.

FIG. 32: A1, eugenol, A2-aglycone 1,
It is a graph which shows the inhibitory effect on the oxidation reaction of 2-deoxyribose of 2 and A2-glucoside.

FIG. 33: A1, eugenol, A2-aglycone 1,
It is a graph which shows the inhibitory effect on the hydroxylation reaction of the benzoic acid of 2 and A2-glucoside.

Claims (2)

[Claims] 1. An active oxygen scavenging composition containing vanillin as an active ingredient. 2. An active oxygen scavenging composition containing a glycoside represented by the following formula as an active ingredient. [Chemical 1]