JP3048311B2 - Hydroxy radical scavenging activator - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a hydroxy radical scavenging activator containing a lignan glycoside as an active ingredient. More specifically, the present invention relates to a hydroxy radical scavenging activator containing a lignan glycoside having a specific structure obtained from sesame seed as a raw material as an active ingredient. The hydroxy radical scavenging activator of the present invention is widely used in the fields of foods, pharmaceuticals, agricultural chemicals, cosmetics, and the like.

[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., 21st.
9, Vol. 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.
Year).

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
It is an object of the present invention to provide a novel hydroxy radical scavenging activator, especially a hydroxy radical scavenging activator utilizing components in sesame seeds.

[0011]

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

That is, the gist of the present invention is to provide a hydroxyl radical containing one kind of lignan glycoside selected from the group consisting of sesaminol triglycoside, P-1 and sesamolinol (hereinafter referred to as lignan glycoside) as an active ingredient. Erasing activator.

[0013] The lignan glycoside according to the present invention comprises sesaminol triglycoside or P
-1 and one kind of lignan moiety selected from the group consisting of sesamolinol, and glucose at its hydroxyl group,
It is a lignan glycoside composed of a sugar moiety in which 1 to 3 sugar residues of galactose or fructose are bonded. The lignan glycoside targeted in the present invention is preferably a water-soluble glucoside lignan in which the sugar residue is a diglucoside residue and / or a triglucoside residue, and more preferably the following structural formula (I-a) It contains the indicated sesaminol triglucoside.

[0014]

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

The lignan glycoside according to the present invention can be produced by a chemical synthesis method, but it is possible to use sesame seeds or their crushed products or their defatted lees as raw materials.
Alternatively, the inventors of the present application have filed a patent (Japanese Patent Application No. 5-3).
For example, as described in JP-A No. 16079 and JP-A No. 7-145066, it is convenient to prepare the germinated product of sesame seed by the method described below. The extracted component obtained by such a method and the purified product thereof can be used as a hydroxy radical scavenging activator.

First, sesame seeds that have not been subjected to high-temperature treatments such as decoction, such as 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, a large amount of the lignan glycoside according to the present invention can be produced and accumulated in the humidified material or the germinated product of the sesame seed.

After the sesame seeds swollen or germinated with water are separated from the culture 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 comprises the lignan glycoside,
In addition, it is a mixture containing 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 other than the lignan glycoside according to the present invention (substances that are fat-soluble and water-soluble compared to the lignan glycoside according to the present invention) in the aqueous lower alcohol extract, It is desirable to perform the following processing. That is, to remove fat-soluble impurities first,
2 to 10 times (v / w) relative to the aqueous lower alcohol extract
The water-insoluble organic solvent of t), for example, ethyl acetate or n-hexane, and water are added to extract 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. 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.

The crude lignan glycoside thus obtained is composed of sesaminol as a main component and saccharides such as glucose and galactose bound to hydroxyl groups of lignans such as P-1, sesamolinol and pinoresinol. It is a mixture of di- and / or triglucosidic lignans having two or three molecules of glucose bonded thereto. This is a water-soluble substance soluble in n-butanol, ethanol, methanol, water and the like.

The above-mentioned hydrated lower alcohol extract and 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 structure of a lignan glycoside can be obtained by separating each component purified to a single substance with high purity, for example, by acid hydrolysis to separate it into a lignan (aglycone portion) and a saccharide portion, and then trimethylsilylating each of these components to conduct gas chromatography. Offering,
Alternatively, it can be confirmed by analysis using nuclear magnetic resonance spectroscopy and mass spectroscopy.

Next, a method for measuring the erasing activity of active oxygen species will be described below. The hydroxy radical scavenging activity is measured by using an electron spin resonance (ESR) apparatus and a spin trap method (for example, Gow-Chin Ye) using 5,5′-dimethyl-1-pyrroline-N-oxide (hereinafter abbreviated as DMPO).
n and Pin-Der Cuh, J. Agric. Food Chem.,
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 has an ESR
Characteristic quadruplets are shown in the spectrum. At this time,
When a substance having an activity to scavenge hydroxyl radicals coexists 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, and formic acid in the above-mentioned literature ("SOD and active oxygen species regulators-pharmacological action and clinical application"). 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 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 lignan glycoside according to the present invention has an extremely high activity as an active ingredient of a hydroxy radical scavenging activator.

The hydroxy radical scavenging activator of the present invention is a glycan of one kind of lignan selected from the group consisting of sesaminol triglycoside or P-1 and sesamolinol, especially diglucoside lignan and / or triglucoside lignan. Any of the lignan glycosides containing at least one of them can be the active ingredient. These may be chemically synthesized, but may be obtained by removing the above-mentioned hydrated lower alcohol extract from humidified or germinated sesame seeds and removing impurities (fat-soluble substances and water-soluble substances) from the extract. It is desirable to use crude lignan glycosides, and also high-purity lignan glycosides obtained by fractionating these by column chromatography or HPLC.

[0027]

The present invention will be specifically described below with reference to examples and reference examples. Reference Example 1 Quartz sand previously sterilized was spread in a stainless steel vat of 300 cm 2, and 10 g of Chinese sesame seeds were spread thereon, and cultured in a constant temperature bath at 40 ° C. for 2 days while sufficiently spraying distilled water. Sprouted. The germination rate was 89%. A certain amount of germinated material having a similar 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. Then, the extract was washed twice with n-hexane and ethyl acetate to obtain a crude lignan glycoside. This crude lignan glycoside was treated with 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-W5μ, 10 μm).
mmφ × 250mm), UV absorption detector (UV-800
0, manufactured by Tosoh Corporation), and elution was started at 90:10 (v: v) with water: methanol at 60 minutes after 10 minutes.
Using a linear gradient of 90 (v: v), the flow rate was 1 mL / min, and the detection wavelength was 288 nm.

As a result of HPLC analysis, the crude lignan glycoside contains sesaminol diglucoside and sesaminol triglucoside as main components, sesaminol monoglucoside, sesaminol monogalactoside, P-1 diglucoside, and sesamolinol monoglucoside. , Sesamolinol diglucoside, pinoresinol diglucoside and pinoresinol triglucoside, each containing 35% of sesaminol triglucoside,
Sesaminol diglucoside accounted for 45%.

Each component of the lignan glycoside was isolated and analyzed by the methods described below. That is, 1 g of crude lignan glycoside was subjected to reverse phase partition column chromatography (YMC-GELODS-A 60200/60).
(Yamamura Chemical Co., Ltd.) 60 g was suspended in 20% (v / v) water-containing methanol and packed in a glass column). (V / v) step by step 40:60
(V: v) using aqueous methanol raised to
It was eluted at 0.8 mL / min. Among the eluted fractions containing 100 mL of aqueous methanol, fractions containing components corresponding to the components detected by the HPLC analysis were concentrated and dried to obtain lignan glycoside fractions. Each of the fractions was repeatedly subjected to preparative HPLC, and purified until each lignan glycoside component became single.

The purified product of each lignan glycoside fraction was treated with 1N
Hydrolysis by heating at 100 ° C. for 30 minutes using hydrochloric acid,
Extracted with ethyl acetate. The ethyl acetate layer obtained here was used as a sample for lignan (aglycone part) analysis, and the aqueous layer was used as a sample for sugar part analysis. The ethyl acetate layer was concentrated to dryness at 40 ° C. or less, then trimethylsilylated by adding TMS-PZ (manufactured by Tokyo Chemical Industry Co., Ltd.), and subjected to gas chromatography (GLC) for quantitative analysis (external standard: sesamin).

GLC conditions are as follows. GLC apparatus: 5890 manufactured by Hewlett-Packard Company, column: DB-1
7HT (15m x 0.319mm, film thickness:
0.15 μm, manufactured by J & W SCIENTIFIC), injection method: split method (split ratio 1/1)
0), column temperature: 270 ° C., carrier gas: He.

Further, the aqueous layer was subjected to a pretreatment filter for HPLC (pore size: 0.2 μm, Meishori disk W-13).
2. Tosoh Corp.), 5 ml of acetone was added to the filtrate, and the mixture was concentrated to dryness under reduced pressure. TMS-PZ (manufactured by Tokyo Kasei Kogyo Co., Ltd.) was added to trimethylsilylate, which was subjected to GLC for quantitative analysis. (External standard: glucose, galactose, fructose).

GLC conditions are as follows: Column: DB-1701
(15m × 0.25mm, film thickness: 1.0μm
, J & WSCIENTIFIC), injection method: split method (split ratio 1/50), column temperature: 18
Except that the temperature is set to 0 ° C., it is the same as in the case of the lignan (aglycone part) analysis.

Example 1 The elimination activity of a lignan glycoside component on hydroxyl radicals 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 an aqueous solution. The purified product (for example, sesaminol triglucoside) obtained in Reference Example 1 was used in a concentration of 0 to 1.0 μmol in the reaction solution.
/ ML, and each DMPO-
The spectrum of the OH adduct was measured with an ESR device. E
The measurement conditions for SR are as follows: ESR device (JEOL, JES
-RE1X), magnetic field: 334.5 ± 5 mT, output:
8 mV, modulation: 100 kHz, measured at room temperature, response time:
0.1 s, and manganese ion (Mn2 +) in magnesium oxide was used as a standard substance. ESR spectra of the elimination activity of hydroxy radicals by sesaminol triglucoside are shown in FIGS. 1 (A) to 1 (D). ESR spectrum without sesaminol triglucoside (Fig. 1
Compared with (A)), the spectrum clearly decreased as the added concentration of sesaminol triglucoside increased (FIG. 1).
(B), FIG. 1 (C) and FIG. 1 (D)). This has revealed that sesaminol triglucoside has an activity to scavenge hydroxyl radicals.

Example 2 In the method described in Example 1, the types of lignan glycoside components to be added to the reaction mixture for measuring hydroxyl radicals (each purification of sesaminol diglucoside and sesamolinol diglycoside obtained in Reference Example 1) ESR spectra were measured at different concentrations and the concentrations, and the relationship between the integrated value and the added concentration was determined for each glycoside component. As a result, as shown in FIG. 2, sesaminol diglucoside, sesamolinol diglucoside and sesaminol triglucoside (Example 1)
Of each lignan glycoside component in the reaction solution was 0.0
Strong activity for scavenging hydroxy radicals was observed at an addition amount in the concentration range of 1 to 1.0 μmol / mL. It was shown that sesaminol triglycoside has the strongest scavenging activity.

Example 3 Using the aqueous methanol extract, crude lignan glycoside and column fraction obtained in Reference Example 1 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: 3
0 μmol / mL, 2 μmol / mL and: 0.08
μmol / mL. From this, it became clear that the lignan glycoside according to the present invention retains sufficient hydroxy radical scavenging ability not only in a purified product of each component but also in a mixture.

[0038]

According to the present invention, it is possible to provide a hydroxy radical scavenging activator comprising, as an active ingredient, sesaminol triglyceride or a lignan glycoside selected from P-1 or sesamolinol. The lignan glycoside is easily obtained from germinated sesame seeds. Further, the hydroxy radical scavenging activator 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 food, 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 a change in hydroxy radical scavenging activity depending on the amount of a lignan glycoside component (sesaminol triglucoside) added. FIG. FIG. 1 (A): no addition concentration of sesaminol triglucoside, FIG. 1 (B): 0.05 μmol
/ ML, FIG. 1 (C): 0.10 μmol / mL, FIG.
(D): 0.25 μmol / mL. Mn2 + indicates 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 (sesaminol diglucoside, sesamolinol diglucoside and sesaminol) FIG. 4 is a graph showing the relationship between the concentration of triglucoside and triglycoside.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI A61P 43/00 111 A61P 43/00 111 C07H 15/26 C07H 15/26 C09K 15/06 C09K 15/06 // C07G 3/00 C07G 3/00 (58) Field surveyed (Int.Cl. ⁷ , DB name) C07H 17/04 C07H 15/26 A23L 3/3562 A61K 7/00 A61K 31/7048 A61K 35/78 C09K 15/06 C07G 3 / 00 CAPLUS (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. A lignan glycoside selected from the group consisting of a sesaminol triglycoside represented by the following structural formula (Ia), a glycoside of P-1 and a sesamolinol glycoside (hereinafter referred to as a glycan of lignan) Lignan glycoside) as an active ingredient. Embedded image (Ia) (In the above formula (Ia), Glc represents a glucose residue.) 2. The hydroxy radical scavenging activator according to claim 1, wherein the lignan glycoside is a component obtained by extracting humidified or germinated sesame seeds or defatted sesame with hydrated lower alcohol.