JP3031844B2 - Hydroxy radical scavenger - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a hydroxy radical scavenger containing a lignan glycoside as an active ingredient. More specifically, the present invention relates to a hydroxy radical scavenger containing a lignan glycoside having a specific structure obtained from sesame seed as a raw material. The hydroxy radical scavenger of the present invention is widely used in the fields of pharmaceuticals, agricultural chemicals, cosmetics, and the like in addition to the food field.

[0002]

2. Description of the Related Art Living organisms efficiently obtain the energy necessary for survival by utilizing oxygen. However, in such energy metabolism, in the process of converting oxygen to water, active oxygen species are generated as intermediates. In general, as reactive oxygen species, superoxide anions released by stimulation of macrophages and the like, hydroxy radicals generated by radiation exposure and the like, and organic radicals generated by chain peroxidation of lipids and the like are known. These reactive oxygen species have high chemical reactivity and react with lipids, nucleic acids, proteins, and the like, causing oxidative disorders leading to various diseases. Excessive irradiation of radiation or ultraviolet rays, ingestion of chemical substances or tobacco, and the like are external triggers. Among these, for example, an important cause of a biological disorder caused by irradiation with radiation is a hydroxyl radical produced from water molecules in a living body by irradiation with radiation.

[0003] The hydroxyl radical is one of the most reactive oxygen species, and immediately reacts chemically with lipids, proteins, nucleic acids or carbohydrates present in the living body, and oxidizes, denatures or decomposes them. And so on. This causes significant damage to genes, biological membranes, tissues, etc.
It is thought to cause various diseases such as carcinogenesis, arteriosclerosis, heart disease, inflammation or cell aging (Halliwell
B. and Gutteridge MC, Biochem. J., Volume 219,
1-14, 1984).

Accordingly, a substance having a function of efficiently eliminating such toxic active oxygen species is useful as a protective agent against oxidative deterioration of components contained in living bodies or foods, pharmaceuticals, agricultural chemicals and the like. It is expected to be practically used in the food field, in particular, health foods and nutritional foods, as well as in the fields of pharmaceuticals, agricultural chemicals and cosmetics. In addition, known antioxidants such as tocopherol and ascorbic acid,
Speaking of hydroxy radical scavenging ability, vitamin E
Some of them are active, but many are still unknown.

[0005] In recent years, as the toxicity of such reactive oxygen species, particularly hydroxyl radicals, to living organisms has become clear, the usefulness of substances having an activity of efficiently eliminating them has been attracting attention. It is being considered as a component derived from a product. As representatives having hydroxy radical scavenging activity, mannitol, tryptophan,
Formic acid and the like have been examined, and their hydroxyl radical scavenging activity has been examined (for example, Yoshihiko Oyanagi, "SOD and Reactive Oxygen Species Regulators-Their Pharmacological Actions and Clinical Applications", No. 2)
24 to 228, The Japan Medical Museum, 1989).

However, there are still very few substances having an extremely small amount of hydroxyl radical scavenging activity which are practically effective, and it is currently difficult to obtain a large amount of this substance in a stable manner industrially. As described above, although there is a demand for a stable supply of an active ingredient having an activity to scavenge hydroxy radicals, there has been almost no practical application to date.

[0007] Sesame seeds are one of the natural products commonly used as raw materials for food. Sesame seed is a kind of oil seed that has been used for food since ancient times, and its pharmacological effects have been handed down. Sesame seeds are still cultivated in the tropics and other parts of the world, and are eaten favorably because of the unique flavor of their oils and seeds. In other words, sesame is a plant material that can be stably obtained in a relatively large amount and is a safe raw material for the human body. In addition, sesame seeds contain lignans as characteristic compounds, and studies on various physiologically active functions including their antioxidant activity have been made (for example, edited by Mitsuo Namiki and Sadasaku Kobayashi, “Sesame seeds” Science, Asakura Shoten, 1989).

In sesame seeds, lignans having excellent antioxidant activity, ie, sesaminol: tetrahydro-
1- [6-hydroxy-3,4- (methylenedioxy)
Phenyl] -4- [3,4- (methylenedioxy) phenyl] -1H, 3H-furo [3,4-C] furan, P-
1: tetrahydro-1- (3-methoxy-4-hydroxyphenyl) -4- [3,4- (methylenedioxy) phenyl] -1H, 3H-furo [3,4-C] furan, sesamolinol: tetrahydro- 1- [3-methoxy-4
-Hydroxyphenoxy] -4- [3,4- (methylenedioxy) phenyl] -1H, 3H-furo [3,4-
C] Furan, pinoresinol: tetrahydro-1,4-
Di (3-methoxy-4-hydroxyphenyl) -1H,
Phenolic lignans such as 3H-furo [3,4-C] furan are included, and many of them have been found to exist as sugar compounds (lignan glycosides) in sesame seeds or their defatted cakes. (Biosci. Biotech. Biochem.,
56, 2087-2088, 1992).

It is also known that a pinoresinol glycoside can be obtained from ground sesame seeds, and that the glycoside has an antioxidant effect on lipid oxidation (Japanese Patent Laid-Open No. 6-1162).
No. 82). However, the publication does not mention lignan glycosides other than pinoresinol glycosides. On the other hand, it has been reported that when sesame seeds are germinated, phenolic antioxidants other than tocopherol and sesamol are produced in the germinated product (Journal of the Japan Society of Food Industry, Vol. 32, 407-). 412, 1985
Year). It is also known to extract an antioxidant or an antioxidant using cultured cells derived from an adult plant of sesame seeds (Abstracts of the 1991 Annual Meeting of the Japanese Society of Agricultural Chemistry, 236, 1991). , Tokuhei 4-214
No. 75, JP-A-5-124949). However, all of the substances disclosed therein are different from the above-mentioned phenolic lignans.

[0010]

Accordingly, the present invention provides
An object of the present invention is to provide a novel hydroxyl radical scavenger, particularly a hydroxy radical scavenger utilizing a novel component.

[0011]

Means for Solving the Problems The present inventors have made intensive studies to achieve the above object, and as a result, have found that a novel lignan glycoside having a specific chemical structural formula has an activity capable of effectively scavenging hydroxy radicals. Have been found, and the present invention has been completed.

That is, the gist of the present invention is that the following structural formula (I)

Embedded image (In the formula (I), R represents one kind of glucosyl residue selected from the group consisting of glucose, galactose and fructose, m represents any one of integers from 1 to 3, and n represents an integer of 0 or 1) A lignan glycoside represented by the following formula (1):

The lignan glycoside according to the present invention is represented by the above-mentioned structural formula (I), and has an aglycone portion having two methylenedioxyphenyl groups and glucose, galactose or fructose at its hydroxyl group. The saccharide moiety is composed of 1 to 3 saccharide residues. The lignan glycoside targeted in the present invention is preferably a glucoside lignan in which the sugar residue in the structural formula (I) is a diglucoside residue and / or a triglucoside residue, and more preferably the following structural formula ( II−
a) one of those represented by (II-b) or (II-c)
Species or two or more species.

[0014]

Embedded image (In the formula (II-a), Glc represents a glucose residue.)

[0015]

Embedded image (In the formula (II-b), Glc represents a glucose residue.)

[0016]

Embedded image (In the formula (II-c), Glc represents a glucose residue.)

The lignan glycoside is a substance isolated from the germinated product using, for example, sesame seed as a raw material,
The structural formulas (II-a) (hereinafter sometimes abbreviated as SG-1), (II-b) (hereinafter sometimes abbreviated as SG-3) and (II-c) (hereinafter abbreviated as SG-3) , SG-5) have physicochemical property values by instrumental analysis as shown below.

The data of the ultraviolet absorption spectrum and the infrared absorption spectrum are as follows. SG-1; UV
λmax (methanol solution; hereinafter abbreviated as MeOH) nm
(Log ε) 230 (4.10), 281 (3.74) and 311 (3.66), IRν (cm ⁻¹ ) (assignment) 34
00 (OH), 2950 (CH), 1670, 162
0, 1505, 1500, 1450 (aromatic ring.
Hereinafter, it is abbreviated as Ar. ), 1260 (Ar-OC) and 1040 (COC). SG-3; UVλmax (Me
OH) nm (log ε) 230 (4.00), 280 (3.
60) and 310 (3.50), IRν (cm ⁻¹ ) (assignment) 3400 (OH), 2900 (CH), 1660,
1610, 1510, 1490, 1450 (Ar), 1
260 (Ar-OC) and 1040 (CO-
C). SG-5; UVλmax (MeOH) nm (log ε)
231 (4.22), 280 (3.81) and 299
(3.83), IRν (cm ⁻¹ ) (attribution) 3400 (O
H), 2900 (CH), 1670, 1510, 149
0, 1450 (Ar), 1260 (Ar-OC) and 1040 (COC).

The molecular weights of SG-1, SG-3 and SG-5 determined by mass spectrometry were SG-1: 856, SG-3: 6.
94, SG-5: 710.

The spectral values of the nuclear magnetic resonance spectra ( ¹³ C-NMR) of SG-1, SG-3 and SG-5 are shown below. SG-1; 198.4, 151.9, 14
8.0, 147.6, 147.0, 135.2, 13
1.9, 124.8, 119.6, 107.4, 10
7.3, 107.2, 106.2, 103.7, 10
3.2, 101.7, 100.6, 100.9, 82.
7, 81.5, 76.3, 76.1, 76.1, 75.
7, 75.5, 74.8, 74.4, 73.2, 69.
7, 69.6, 69.5, 69.4, 68.3, 65.
7, 60.9, 60.7, 51.3 and 46.7. S
G-3; 198.3, 152.0, 148.1, 14
7.6, 147.0, 135.2, 131.8, 12
4.8, 119.6, 107.3, 107.2, 10
7.1, 106.2, 103.8, 101.1, 10
1.7, 100.6, 82.8, 81.6, 76.3,
76.3, 75.7, 75.7, 74.4, 69.6,
69.5, 69.4, 65.7, 60.8, 60.8,
51.3 and 46.7. SG-5; 195.9, 15
1.8, 151.2, 148.2, 147.8, 14
2.4, 131.3, 124.5, 108.5, 10
7.4, 107.1, 107.1, 105.4, 10
3.5, 101.8, 100.7, 100.6, 99.
3, 81.3, 76.3, 76.3, 75.9, 75.
9, 74.0, 69.3, 69.3, 67.9, 65.
0, 60.7, 60.7, 48.7 and 45.1.

The lignan glycoside according to the present invention has an amphoteric portion of a lipophilic aglycone portion and a hydrophilic saccharide portion in its molecule, and has a solubility between water-soluble and fat-soluble. Things. This lignan glycoside is a novel compound that has not been described in the known literature, and can be isolated from sesame seeds as a raw material, but hardly exists in sesame seeds themselves, and particularly by humidifying or germinating sesame seeds. It is a substance that increases significantly in the early stages of germination, and its existence and phenomenon were not known before. Furthermore, the lignan glycoside according to the present invention has biochemical stability such that it is not affected by sugar chain hydrolases such as β-glucosidase and cellulase at all due to its steric structure. This means that known sesaminol glycosides and sterol glycosides, which have almost the same polarity and molecular weight as the lignan glycoside and have conventionally been difficult to mutually separate from the glycoside, This is in contrast to the fact that other glycolipids, oligosaccharides and the like are easily hydrolyzed under the action of the sugar chain hydrolase.

The lignan glycoside according to the present invention can be produced by a chemical synthesis method. For example, it is convenient to prepare the lignan glycoside by the method described below using germinated sesame seeds.

First, sesame seeds that have not been subjected to high-temperature treatment such as roasting or the like, types of white sesame, black sesame, etc., domestic, Chinese, Indian, African, etc., cultivation or oil pressing. Can be used regardless of This is uniformly spread on a suitable medium capable of containing water or water, for example, a preferably sterilized medium such as agar, quartz sand, sea sand, absorbent cotton, sand, or soil, and is 10 to 50 ° C, preferably 30 to 40 ° C. Cultivation is performed at 5 ° C. for 5 to 100 hours, preferably for 24 to 72 hours, while appropriately supplementing the water. The culture may be performed under light or dark conditions. By such a treatment, the novel lignan glycoside according to the present invention can be produced and accumulated in a humidified or germinated product of sesame seeds.

After the sesame seeds swollen or germinated with water are separated from the medium, they are pulverized in a pulverizer such as a food mixer, a blender or a homogenizer. The obtained ground product is n
-It may be a defatted lees obtained by extracting an oil component with a fat-soluble organic solvent such as hexane. Next, a lower alcohol capable of extracting a lignan glycoside or a hydrated product thereof is 1 to 10 times (v / wt) (v: capacity, wt: weight) the pulverized product or the defatted lees thereof. The same applies hereinafter.)
After crushing and extracting operations are repeated as necessary, solids are removed by a conventional method such as decantation, centrifugation, or filtration, and then water and alcohol are removed at normal pressure or reduced pressure by heating or non-heating to remove water. Obtain a lower alcohol extract. The extract is a mixture containing the novel lignan glycoside and various sugar chain compounds.

Here, the hydrated lower alcohol includes:
A linear or side chain lower alcohol having 1 to 4 carbon atoms,
For example, water is mixed with methanol, ethanol, n-propanol, isopropanol, n-butanol and the like, and the alcohol concentration is 30 to 100% (v / v), preferably 5 to 100%.
0 to 100% (v / v), more preferably 50 to 80%
(V / v), most preferably 70-80% (v / v). An alcohol concentration of less than 30% (v / v) is not preferable because a large amount of the water-soluble polysaccharide containing no lignan glycoside according to the present invention is extracted.

In order to remove impurities (fat-soluble substances and water-soluble substances) other than the lignan glycosides according to the present invention in the above hydrated lower alcohol extract, (i) a solvent extraction treatment or (ii) a sugar chain It is desirable to perform a solvent extraction treatment or the like after the hydrolysis enzyme treatment. That is, in the treatment (i), first, in order to remove fat-soluble impurities, the water-soluble lower alcohol extract is mixed with a water-insoluble organic solvent 2 to 10 times (v / wt), for example, chloroform or n-hexane. The mixture is extracted by adding water, and separated into two phases by centrifugation or the like. The organic phase is removed and the aqueous phase is concentrated to dryness. At this time, the target lignan glycoside is concentrated on the aqueous phase side.

Next, in order to remove water-soluble impurities, a small amount, preferably 1 to 5 times (v / wt) of aqueous alcohol (alcohol concentration of 30 to 100% (v / wt)
v)), and this is added dropwise to a relatively large amount, preferably 10 to 200 times (v / wt) of alcohol, which is slowly stirred. After standing, the precipitate is removed by centrifugation or fractional filtration, and then concentrated to dryness to obtain a crude lignan glycoside. Alternatively, an organic solvent having an intermediate polarity and being insoluble or hardly soluble in water, for example, n-butanol, ethyl acetate, methyl ethyl ketone, etc. is 1 to 100 times (v / v).
The extraction method may be used. These operations are repeated if necessary. The alcohol used in this treatment may be the same as the lower alcohols used in extracting the ground sesame seeds.

In the treatment (ii), the water-containing lower alcohol extract is dispersed or dissolved in 1 to 100 times (v / wt) water or a buffer solution (pH 2 to 6). 1 to 30% (wt / wt), preferably 1 to 10%
(Wt / wt) sugar chain hydrolase is added, and the sugar chain is hydrolyzed at 10 to 50 ° C for 1 to 50 hours, preferably 5 to 15 hours, preferably with gentle stirring. At this time, an aqueous extract (having the enzyme activity) of a pulverized non-heated sesame seed may be used instead of the sugar chain hydrolase.

By this enzymatic reaction, sterol glycosides, glycolipids, sesaminol glycosides, flavonoid glycosides, carbohydrates and the like having the same solvent partitioning properties as the novel lignan glycoside of the present invention are obtained. The part is hydrolyzed and is further divided into saccharides (monosaccharides, oligosaccharides and the like) with higher water solubility and aglycones (lignans). On the other hand, the novel lignan glycoside according to the present invention is not affected by such an enzyme and is not hydrolyzed due to the specificity of its steric structure.

The sugar chain hydrolase is an enzyme that hydrolyzes a glucosyl group or a galactosyl group, for example, commercially available β-glucosidase, α-glucosidase, β-glucosidase,
-In addition to glycosidases such as galactosidase and α-galactosidase, at least one or more enzyme agents such as cellulase and amylase are used, or the activity of β-glucosidase or α-galactosidase or cellulase originally contained in sesame seeds is used. You can also. Further, these enzyme agents may be immobilized on a suitable carrier such as activated carbon, celite, a synthetic resin, an ion exchange resin, or a gel to enable continuous use and recovery and reuse.

After the completion of the enzymatic reaction, the reaction solution was subjected to the above (i).
The solvent-extracting treatment is carried out in the same manner as described above to remove the fat-soluble substance and the water-soluble substance, and the non-hydrolysate obtained by the enzymatic reaction is concentrated. That is, a 1 to 100-fold (v / v) low-polarity and water-insoluble or hardly soluble organic solvent such as n-hexane, chloroform, diethyl ether, petroleum ether or the like is added to the enzyme reaction solution, and the mixture is extracted, and fatty acid glycerides, A fat-soluble substance composed of aglycone components derived from glycosides other than the novel lignan glycoside according to the present invention, such as phospholipids, lignans, sterols, etc., produced by the enzymatic reaction is removed as an organic solvent phase.

Then, the remaining liquid (aqueous phase) was added 1 to 100 times (v
/ V) an organic solvent having an intermediate polarity such as n-butanol, ethyl acetate, methyl ethyl ketone and the like, which is insoluble or insoluble in water, is extracted again, and water-soluble substances such as saccharides, proteins, and fibrous substances are extracted with water. Remove as phase. The components extracted into the organic solvent phase at this time are those which are higher in polarity than fat-soluble substances such as sterols and lignans and are not water-soluble substances such as monosaccharides and oligosaccharides (the non-hydrolyzed substances by the enzymatic reaction). If this is dried under reduced pressure and concentrated, an extract fraction (crude lignan glycoside) containing a large amount of the novel lignan glycoside according to the present invention can be obtained.

Thus, the crude lignan glycosides obtained by each of the treatments (i) and (ii) are mixtures of those represented by the structural formula (I), the main component of which is the mixture represented by the structural formula (II) -A), containing at least one or more of the novel lignan glycosides represented by (II-b) and (II-c).

The hydrated lower alcohol extract and the crude lignan glycoside are fractionated into individual lignan glycoside components using an adsorbent such as silica gel or octadecyl silica (ODS), if necessary. It can be purified. That is, for example, a column filled with ODS is prepared, and after equilibrating with water, the hydrated lower alcohol extract or crude lignan glycoside is loaded at a load rate of 0.1 to 5%.
(Wt / v), and using a hydroalcoholic solvent (methanol, ethanol, n-propanol, isopropanol, n-butanol, etc. as an alcohol), elute a predetermined fraction by a step elution method in which the alcohol concentration is sequentially increased. . The eluted fraction obtained here may be further subjected to high-performance liquid chromatography (HPLC) using the adsorbent, preparative liquid chromatography, or the like, if necessary, to further purify each component to higher purity. it can.

The chemical structure of the lignan glycoside is as follows. Each component purified to a single substance with high purity is separated into a lignan (aglycone) portion and a sugar portion by, for example, acid hydrolysis, and these are trimethylsilylated to form a gas. It can be confirmed by subjecting to chromatography or analyzing by nuclear magnetic resonance spectroscopy, mass spectroscopy, or the like.

Next, a method for measuring the erasing activity of active oxygen species will be described below. The hydroxyl radical scavenging activity is measured by a spin trap method (for example, Gow-Chin Yen) using 5,5'-dimethyl-1-pyrroline-N-oxide (hereinafter abbreviated as DMPO) using an electron spin resonance (ESR) apparatus.
and Pin-Der Cuh, J. Agric. Food Chem., Volume 42,
629-632, 1994). That is, in the presence of the ferrous sulfate solution, hydrogen peroxide generates a hydroxy radical and a hydroxy anion by the Fenton reaction. Among them, the hydroxy radical is captured by the coexisting DMPO to obtain a DMPO-OH adduct. This adduct is relatively stable and shows a characteristic quartet in the ESR spectrum. At this time, when a substance having an activity to scavenge hydroxy radicals is present in the reaction solution, the ESR spectrum of the DMPO-OH adduct decreases. The hydroxyl radical scavenging activity of the sample can be measured from the decrease in the integrated value of the spectrum.

Examples of substances having an activity of scavenging hydroxy radicals include mannitol, tryptophan, formic acid and the like in the literature ("SOD and active oxygen species regulators-their pharmacological actions and clinical applications"). The activity was examined, but the activity was 1
It was measured at 0 μmol / mL, 20 μmol / mL for tryptophan, and 100 μmol / mL for formic acid. On the other hand, the test amount of the novel lignan glycoside according to the present invention is measured at 1 μmol / mL or less, and sufficient hydroxy radical scavenging activity is recognized even at such a small concentration. Therefore, the novel lignan glycoside according to the present invention has an extremely high activity as an active ingredient of a hydroxy radical scavenger.

The hydroxy radical scavenger of the present invention comprises a lignan glycoside represented by the above structural formula (I), diglucoside lignan and / or triglucoside lignan among those represented by the above structural formula (I), (II-
Any of the lignan glycosides containing at least one of a), (II-b) and (II-c) can be the active ingredient. These may be chemically synthesized, but may be a water-containing lower alcohol extract obtained from the humidified or germinated sesame seeds described above, and may be liposoluble after or without treating the extract with a sugar chain hydrolase. It is desirable to use crude lignan glycosides obtained by removing substances and water-soluble substances, and high-purity lignan glycosides obtained by fractionating these by column chromatography or HPLC.

[0039]

The present invention will be specifically described below with reference to examples and reference examples. REFERENCE EXAMPLE 1 Preliminarily sterilized quartz sand was spread on a stainless steel vat of 300 cm ² , and 10 g of Chinese sesame seeds were spread thereon, and cultured for 2 days in a constant temperature bath at 40 ° C. while sufficiently spraying distilled water. Germinated. The germination rate was 89% or more. A certain amount of germinated material having the same germination state was ground with a blender together with 100 ml of hydrated methanol (80% (v / v)). The residue was filtered, and the filtrate was concentrated to dryness to obtain a water-containing methanol extract. The extract was then extracted and washed with n-hexane to remove fat-soluble substances, and then extracted and washed with water-saturated n-butanol to remove water-soluble substances to obtain crude lignan glycosides. The crude lignan glycoside was redissolved in 100 ml of aqueous methanol (80% (v / v)) and subjected to high performance liquid chromatography (HPLC) to analyze the composition.

The HPLC conditions were as follows: a pump (CCPM, manufactured by Tosoh Corporation) was connected to a column (Soken Pak ODS-W 5 μm, 10 mm
φ × 250mm), UV absorption detector (UV-8000,
(Manufactured by Tosoh Corporation) and elution was performed with 9 parts of water: methanol.
Start at 0:10 (v: v) and 10: 9 after 60 minutes
Using a linear gradient of 0 (v: v),
ml / min and the detection wavelength was 280 nm.

As a result of HPLC analysis, the composition and content of the novel lignan glycoside in the crude lignan glycoside were determined using sesamin as an external standard.
a)), SG-3 (Structural Formula (II-b)) and SG-5
(Structural formula (II-c)) are the main components, and 115 mg of these are present in the crude lignan glycoside, and the composition is SG
-1 was 30%, SG-3 was 40%, and SG-5 was 30%.

The chemical structure of each lignan glycoside component was confirmed by the following method using each purified product purified to a single component by preparative HPLC under the same conditions as described above. That is, 1N hydrochloric acid was added to each purified product, hydrolyzed at 100 ° C. for 30 minutes, extracted with ethyl acetate, and separated into an ethyl acetate layer and an aqueous layer. The ethyl acetate layer was concentrated to dryness at 40 ° C. or lower, subjected to trimethylsilylation treatment with TMS-PZ (manufactured by Tokyo Chemical Industry Co., Ltd.), and subjected to gas chromatography (GLC) to quantitatively analyze lignan (external standard: sesamin).

The GLC conditions are as follows. GLC apparatus: Hewlett-Packard 5890, column: D
B-17HT (15m × 0.319mm, film th
kickness: 0.15 μm, J & W SCIENT
IFIC), injection method: split method (split ratio 1/10), column temperature: 270 ° C., carrier gas: helium.

The aqueous layer was subjected to a pretreatment filter for HPLC (pore size: 0.2 μm, Meishoridisk W-13).
2, Tosoh Corporation), add 5 ml of acetone to the filtrate, concentrate under reduced pressure to dryness, then perform trimethylsilylation treatment with TMS-PZ (same as above), and subject it to GLC for quantitative analysis of sugar. (External standard: glucose, galactose, fructose).

The GLC conditions are as follows: Column: DB-170
1 (15m x 0.25mm, filmthicknes
s: 1.0 μm: manufactured by J & W SCIENTIFIC, injection method: split method (split ratio 1/5)
0), except that the column temperature was set to 180 ° C.

Reference Example 2 A hydrous methanol extract obtained in the same manner as in Example 1
Disperse in 100 mL of 0 mM acetate buffer (pH 5.0)
-200 mg of glucosidase (Funakoshi), 1 g of cellulase (Boehringer Mannheim) and 1 g of amylase (Wako Pure Chemical Industries) were added and shaken at 50 ° C for 15 hours. The same volume of n-hexane was added to the reaction solution and vigorously shaken. This extraction operation was repeated three times to remove fat-soluble substances. To the remaining liquid from which the n-hexane phase was completely removed, the same volume of n-butanol which had been saturated with water in advance was added and shaken vigorously. This extraction operation was repeated twice to remove water-soluble substances. The n-butanol phase was washed twice with the same volume of distilled water and then concentrated to dryness under reduced pressure to obtain a crude lignan glycoside.

The HPLC analysis by the method described in Reference Example 1 revealed that the novel lignan glycoside in the crude lignan glycoside was S
G-1, SG-3, and SG-5 are the main components, and these are present in the crude lignan glycoside in about 150 mg, the composition of which is SG-1 24%, SG-3 43%, SG-3 5 is 3
3%.

Reference Example 3 The crude lignan glycoside obtained by the method described in Reference Example 1 was
The resultant was subjected to partition chromatography using ODS as a carrier.
YMC-GEL ODS-A (Yamamura Chemical Co., Ltd.) 60
g was packed in a glass column 3 cm in diameter and 50 cm in length and equilibrated with flowing water. To this, 1 g of the crude lignan glycoside was loaded on the top of the column. The fraction components were eluted by a step elution method in which the methanol concentration was sequentially increased from water. Fractions eluted with 30-60% (v / v) methanol were collected and concentrated under reduced pressure to obtain about 100 mg of a column fraction.

This was repeatedly subjected to preparative HPLC, and purification was performed until each lignan glycoside component became single. As a result, each of 5 to 10 mg of a purified product of each of the novel lignan glycosides of SG-1, SG-3 and SG-5 was obtained. The content of these total lignan glycoside components was 2.5% (wt / wt) per dried germinated product and 5.0% (wt / wt) per hydrated methanol extract.

Example 1 The elimination activity of a lignan glycoside component on a hydroxy radical was measured. That is, electron spin resonance (ESR)
Using a device, the hydroxyl radical scavenging activity was measured by a spin trap method using DMPO. 75 μl of 0.1 mM ferrous sulfate solution containing 0.55 mM diethylenetriamine N, N, N ′, N ″, N ″ pentaacetic acid, 75 μl of 1 mM hydrogen peroxide solution, 20 μl of 8.8 mM DMPO solution and the following lignan glycoside A reaction solution was prepared by mixing 50 μl of the aqueous solution. The purified product (SG-3) obtained in Reference Example 3 was added so that the concentration in the reaction solution became a predetermined concentration of 0 to 1.0 μmol / ml, and the spectrum of each DMPO-OH adduct was analyzed by ES.
It was measured with an R device. The ESR measurement conditions were determined by using an ESR apparatus (JES-RE1X, manufactured by JEOL Ltd.), and applying a magnetic field of 3
34.5 ± 5 mT, output: 8 mV, modulation: 100 kHz, measured at room temperature, response time: 0.1 s, and manganese ion (Mn ²⁺ ) in magnesium oxide was used as a standard substance. SG
The ESR spectra of the hydroxyl radical scavenging activity of -3 are shown in FIGS. 1 (A) to 1 (D). Compared to the ESR spectrum without SG-3 (FIG. 1 (A)),
The spectrum clearly decreased as the concentration of -3 increased (FIGS. 1 (B), 1 (C) and 1 (D)).
This has revealed that SG-3 has an activity to scavenge hydroxy radicals.

Example 2 In the method described in Example 1, the types of lignan glycoside components (purified products of SG-1 and SG-5 obtained in Reference Example 3) to be added to the reaction solution for measuring hydroxyl radicals and The ESR spectrum was measured while changing the concentration, and the relationship between the integrated value and the added concentration was determined for each glycoside component. As a result, as shown in FIG.
For each of the lignan glycoside components of G-5, a strong activity of scavenging hydroxy radicals was observed at an amount of 0.01 to 1.0 μmol / ml added to the reaction solution.

Example 3 The aqueous methanol extract and crude lignan glycoside obtained in Reference Example 1, the crude lignan glycoside obtained in Reference Example 2, and the column fraction obtained in Reference Example 3 were used as samples. The hydroxy radical scavenging activity was measured in the same manner as in Example 1.
As a result, the addition concentration of each sample showing the activity of eliminating 50% of the hydroxyl radical intensity when no sample was added was:
30 μmol / ml, 2 μmol / ml, 1 μmol / ml
And: 0.1 μmol / ml. From this,
New lignan glycosides (SG-1, SG-3 and SG-
It became clear that 5) retained sufficient hydroxy radical scavenging ability not only in the purified product of each component but also in a mixture.

[0053]

According to the present invention, there can be provided a hydroxy radical scavenger containing the novel lignan glycoside represented by the structural formula (I) as an active ingredient. The lignan glycoside is easily obtained from germinated sesame seeds. In addition, the hydroxy radical scavenger of the present invention has a small amount of strong hydroxyl radical scavenging ability and is expected to be applicable to products in the fields of foods, cosmetics, pharmaceuticals, agricultural chemicals, and the like.

[Brief description of the drawings]

FIG. 1 is an electron spin resonance spectrum of a 5,5′-dimethyl-1-pyrroline-N-oxide-OH adduct, showing changes in hydroxy radical scavenging activity depending on the amount of lignan glycoside component (SG-3) added. FIG. SG
FIG. 1 (A): No additive concentration, FIG. 1 (B):
0.052 μmol / ml, FIG. 1 (C): 0.13 μmol / m
l, FIG. 1 (D): 0.26 μmol / ml. Mn ²⁺ represents a manganese ion (standard substance) in magnesium oxide.

FIG. 2 shows the integrated value of the electron spin resonance spectrum intensity of the 5,5′-dimethyl-1-pyrroline-N-oxide-OH adduct and the lignan glycoside components (SG-1, SG-3 and SG-5). It is a figure which shows the relationship with addition concentration.

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁷ identification code FI A61P 39/06 A61P 39/06 C09K 3/00 C09K 3/00 S (58) Field surveyed (Int.Cl. ⁷ , DB name ) C09K 15/06 A23L 1/30 A61K 7/00 A61K 31/70 A61K 35/78 CA (STN) REGISTRY (STN)

Claims (6)

(57) [Claims] 1. A compound represented by the following structural formula (I): (In the formula (I), R represents one kind of glucosyl residue selected from the group consisting of glucose, galactose and fructose, m represents any one of integers from 1 to 3, and n represents an integer of 0 or 1) A hydroxy radical scavenger comprising a lignan glycoside represented by the following formula: 2. The hydroxy radical scavenger according to claim 1, wherein the lignan glycoside is a glucoside lignan having a diglucoside residue and / or a triglucoside residue as a sugar residue. 3. A lignan glycoside having the following structural formula (II-
a), (II-b) and (II-c) (In the formula (II-a), Glc represents a glucose residue.) (In the formula (II-b), Glc represents a glucose residue.) (In the formula (II-c), Glc represents a glucose residue.)
The hydroxy radical scavenger according to claim 1, wherein the hydroxy radical scavenger comprises at least one of the following. 4. A lignan glycoside obtained by extracting a humidified product of sesame seeds or a crushed germinated product of sesame seeds or a defatted lees thereof with a lower alcohol containing water, and removing a fat-soluble substance and a water-soluble substance from the extract. Claims 1 to 3
4. The hydroxy radical scavenger according to any one of 3. 5. A lignan glycoside which is obtained by extracting a humidified product of sesame seeds or a crushed germinated product or a defatted lees thereof with a lower alcohol containing water, allowing a sugar chain hydrolase to act on the extract in an aqueous solution. The hydroxy radical scavenger according to any one of claims 1 to 3, which is a component obtained by removing a fat-soluble substance and a water-soluble substance and concentrating a non-hydrolysate. 6. The method according to claim 6, wherein the sugar chain hydrolase is α-glucosidase, β-glucosidase, α-galactosidase, β-glucosidase.
The hydroxyl radical scavenger according to claim 5, which is at least one or more of galactosidase, cellulase and amylase.