JP2988656B2 - Production method of new lignan glycoside - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing useful components derived from natural products, and more particularly to an effective method for producing a novel lignan glycoside from humidified or germinated sesame seeds. Things. The lignan glycoside obtained by the present invention is widely used in the fields of foods, pharmaceuticals, cosmetics, agricultural chemicals and the like.

[0002]

2. Description of the Related Art In recent years, there has been increasing interest in the safety of foods and food additives, and the use of physiologically active substances and additives derived from natural products is expected in place of chemically synthesized products which are regarded as a problem in this regard. ing. In particular, plant raw materials that have been used for food for a long time are expected to be excellent in safety, and search for substances that have useful bioactive or medicinal effects on the human body contained in them and search for these substances in foods and pharmaceuticals. Practical application to products in the field of cosmetics and the like is extremely significant.

However, the quality of most of the plants used as raw materials is greatly affected by natural conditions such as harvest time and weather, and it is often difficult to supply them stably. In many cases, the content is minute or the means and method for separating impurities coexisting in large amounts are complicated. For this reason, attempts have been made to prepare a large amount of these physiologically active components derived from natural products.For example, in tissue culture or molecular biological techniques using cultured cells, aseptic manipulation or cell growth is required. A special technique, a medium, a culture device, and the like are required, and furthermore, long-term breeding is required in order to obtain stable cultured cells, and there are few examples that have been commercialized industrially.
In general, as a conventional technique for separating a very small amount of these physiologically active substances, fractionation by column chromatography or fractionation by preparative HPLC is generally used. Consumes organic solvents
Also, because expensive analytical equipment and column carriers are required,
It often became a problem in terms of energy and cost.

[0004] Sesame is an oil seed that has been used for food since ancient times, and has been widely used as a food material that is not only highly safe for the human body as a food but also excellent in nutrition. Sesame seed is a relatively large and stable available plant material, and currently has 150 to 20
100,000 tons are mainly produced in regions of the world from the tropics to the cold and temperate zones, and 50,000 to 100,000 tons are imported to Japan every year and are eaten in various forms. It is known that lignans are present as characteristic compounds in sesame seeds,
Studies on its antioxidant activity and various physiological functions have been made (for example, edited by Mitsuo Namiki and Sadasaku Kobayashi, "Sesame Science", Asakura Shoten, 1989).

In sesame seeds, sesaminol: tetrahydro-1- [6-hydroxy-3,4- (methylenedioxy) phenyl]-having a superior antioxidant activity as compared with conventional natural antioxidants. 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, 3H-furo [3, 4-C] furan and other phenolic lignans, many of which have been shown to exist as sugar compounds (lignan glycosides) in sesame seeds or their defatted meals (Biosci. B).
iotech. Biochem. , 56, 2087
２０８2088, 1992). Therefore, in order to industrially collect and utilize these lignans having strong antioxidant activity, it was necessary to prepare a larger amount of lignan glycosides and to efficiently hydrolyze them.

On the other hand, it has been reported that sesame seeds produce phenolic antioxidants other than tocopherol and sesamol during the germination process thereof (Journal of the Japan Society of Food Industry, Vol. 32, pp. 407-412; 1985
Year). Also, a method for extracting an antioxidant or an anti-photooxidant from proliferating cells derived from an adult plant of sesame seed has been reported (Abstracts of the 1991 Annual Meeting of the Japanese Society of Agricultural Chemistry, 236 pages, 1991). Tokuhei 4-2147
No. 5, JP-A-5-124949). However, the compounds disclosed therein are all substances different from the above-mentioned phenolic lignans, are novel antioxidants present in a trace amount, and are present in sesame seeds or their defatted lees in relatively large amounts. It is not a knowledge of lignan glycosides present in the lignan glycosides. Therefore, the change in lignan glycoside content during the germination process of sesame seeds has not been clarified so far.

[0007] Lignans are obtained by hydrolyzing lignan glycosides. For example, sesame seeds or defatted meal thereof are extracted with alcohol, and β-glucosidase is allowed to act on the extract to convert sugar chains. A method of cutting and separating using a solvent such as ethyl acetate (Japanese Patent Publication No. 62-4)
No. 4793) is known. However, such a method can hydrolyze only a part of the lignan glycoside in sesame seeds, and its non-hydrolyzed substance was not clear.

[0008]

Accordingly, an object of the present invention is to develop a method for efficiently producing a novel lignan glycoside using sesame seed as a raw material.

[0009]

Means for Solving the Problems In order to achieve the above object, the present inventors proceeded with research to elucidate the relationship between sesame seeds and lignan glycoside components. However, in the process of germinating sesame seeds and the like, a novel lignan glycoside component that has been unknown so far is remarkably increased. Lignan glycosides that can be extracted using the lignan glycosides of the present invention are different from glycosidases of lignans such as sesaminol, P-1, sesamolinol, and pinoresinol, which are conventionally known. The present inventors have found that they are not affected by sugar chain hydrolases such as cellulase and cellulase, and have completed the present invention.

That is, the first of the gist of the present invention is that a sesame seed is subjected to humidification or germination treatment so that the treated product has the following structural formula (I)

Embedded image (Wherein, in the formula (I), R represents a glycosyl residue of glucose, m represents any of integers 1 to 3, and n represents an integer of 0 or 1). Increasing the body, extracting the crushed material or defatted cake of the treated product using a lower alcohol or a hydrated lower alcohol, and then removing a fat-soluble substance and a water-soluble substance from the extract. The second is a method for producing a saccharide, in which a sugar chain hydrolase is further allowed to act in an aqueous solution on a lower alcohol or hydrated lower alcohol extract of a humidified or germinated processed product of sesame seed or defatted cake, Next, the method for producing a novel lignan glycoside according to the present invention comprises removing a fat-soluble substance and a water-soluble substance from the enzyme-treated product and concentrating a non-hydrolysate.

The present inventors have previously extracted the humidified or germinated sesame seeds or defatted sesame seeds with hydrated lower alcohol and desirably further removed oil-soluble and water-soluble impurities. A method for obtaining a lignan glycoside was invented and a patent application was filed (Japanese Patent Application No. 5-316079). The present inventors have further studied and found that the lignan glycoside obtained by the above-described method contains a novel substance which is different from this glycoside and which can be distinguished from undescribed literature. We have filed a patent application for a method for producing a new substance.

Hereinafter, the present invention will be described in detail. First, the novel lignan glycoside referred to in the present invention is a lignan glycoside represented by the above-mentioned structural formula (I), in which two methylenedioxyphenyl groups, which are one of the functional groups characteristic of lignans, are used. It has an aglycone portion and a sugar portion in which one to three sugar residues such as glucose and galactose are bonded to its hydroxyl group. The novel lignan glycosides targeted in the present invention are preferably those of the above-mentioned structural formula (I)
Wherein the saccharide residue is a glucoside lignan in which the disaccharide residue is a diglucoside residue and / or a triglucoside residue, more preferably one represented by the following structural formula (II-a), (II-b) or (II-c) It is.

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

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

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

For example, the above-mentioned structural formula (II-) which is a main component of a novel lignan glycoside isolated from a germinated body of sesame seeds
a) (hereinafter sometimes abbreviated as SG-1), (I
Ib) (hereinafter sometimes abbreviated as SG-3) and (II-c) (hereinafter sometimes abbreviated as SG-5) are physicochemical by instrumental analysis as shown below. Has characteristic values.

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-O
-C) and 1040 (C-O-C). SG-3; UV
λ max (MeOH) nm (log ε) 230 (4.0
0), 280 (3.60) and 310 (3.50),
IRν (cm ⁻¹ ) 3400 (OH), 2900 (CH),
1660, 1610, 1510, 1490, 1450
(Ar), 1260 (Ar-OC) and 1040
(C-O-C). SG-5; UVλmax (MeOH)
nm (log ε) 231 (4.22), 280 (3.81)
And 299 (3.83), IRν (cm ⁻¹ ) 3400
(OH), 2900 (CH), 1670, 1510, 1,
490, 1450 (Ar), 1260 (Ar-OC)
And 1040 (C-O-C).

The components SG-1, SG-3 and SG-5 by mass spectrometry were as follows: SG-1: 856, SG-
3: 694 and SG-5: 710.

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.

Such a novel lignan glycoside has an amphoteric portion of a lipophilic aglycone portion and a hydrophilic sugar portion in its molecule, and has a solubility between water-soluble and fat-soluble. is there. Since the aglycone moiety has a methylenedioxyphenyl group, which is considered to be an active site of various physiological activities including in vivo antioxidant activity, it has a physiologically active function such as a scavenging activity of reactive oxygen species. It is a substance that is expected. As described later, the novel lignan glycoside according to the present invention is a substance which is hardly present in sesame seeds, and is significantly increased by humidifying or germinating sesame seeds, particularly in the initial stage of germination.
Its existence and phenomenon were previously unknown. Furthermore, due to its three-dimensional structure, it has biochemical stability that it is not affected by sugar chain hydrolases such as β-glucosidase and cellulase at all. This means that it has almost the same polarity and molecular weight as the novel lignan glycoside according to the present invention, and conventionally it has been difficult to mutually separate from the glycoside.
This is in contrast to known glycolipids such as sesaminol glycosides and sterol glycosides, oligosaccharides, and the like, which are easily hydrolyzed under the action of the sugar chain hydrolase.

Next, a method for producing the novel lignan glycoside according to the present invention will be described. The first method of the present invention is based on the finding that when the sesame seed is humidified or germinated, the novel lignan glycoside is produced and accumulated in a large amount in the treated product. And the finding that a novel lignan glycoside can be obtained by extracting the crushed or defatted processed product with a lower alcohol or a hydrate thereof by a conventional method. It was made based on. That is, after humidifying or germinating the sesame seeds, the crushed material of the processed material or the defatted lees is extracted with lower alcohol or hydrated lower alcohol, and then the fat-soluble substance and the water-soluble substance are removed from the extract. A process for producing a lignan glycoside as described above.

The novel lignan glycoside of the present invention can be easily produced and accumulated in a humidified or germinated product by humidifying or germinating sesame seeds. If sesame seeds are not subjected to high-temperature treatment such as cultivation, they are used regardless of the type of white sesame or black sesame, domestic, Chinese, Indian, African, etc., cultivation or oil pressing it can. Such sesame seeds can be prepared in a suitable medium that can contain water or water, such as agar, quartz sand, sea sand,
Sprinkle evenly on a preferably sterilized medium such as absorbent cotton, sand, soil, etc., and supplement the water at 10 to 50 ° C., preferably 30 to 40 ° C. in a timely manner for 5 to 100 hours, preferably 2 to 100 hours.
Incubate for 4 to 72 hours. The cultivation may be carried out under illuminated or dark conditions, but it is convenient to cultivate the room day and night using sunlight.

After the sesame seeds swollen or germinated with water are separated from the culture medium, they are put into a pulverizer such as a food mixer, a blender or a homogenizer to be pulverized to obtain pulverized products. The pulverized product may be a defatted cake obtained by extracting and removing an oil component with a fat-soluble organic solvent such as n-hexane. Next, a lower alcohol or a hydrated lower alcohol capable of extracting a novel lignan glycoside is used in an amount of 1 to 10 times the volume of
After adding weight (hereinafter referred to as “fold (v / wt)”) and repeating the pulverization and extraction operations as necessary to remove solids by a conventional method such as decantation, centrifugation, or filtration, water and alcohol are added. The fraction is removed by heating or non-heating at normal pressure or reduced pressure to obtain a lower alcohol extract or a water-containing lower alcohol extract.

Here, the lower alcohol or water-containing lower alcohol is a straight-chain or side-chain lower alcohol having 1 to 4 carbon atoms, for example, methanol, ethanol, n-
Propanol, isopropanol, n-butanol or the like or a mixture thereof with water, and alcohol concentration of 30 to 10
0% capacity / capacity (hereinafter expressed as% (v / v)), preferably 50 to 100% (v / v), more preferably 50 to 100% (v / v)
80% (v / v), most preferably 70-80% (v / v)
The one adjusted to v) is preferable. In the present invention, a water-containing lower alcohol is preferred. However, an alcohol concentration of less than 30% (v / v) is not preferable because a large amount of the water-soluble polysaccharide containing no target substance is extracted. The extract may be appropriately concentrated, but it is necessary to remove at least an alcohol component in order to remove impurities by enzymatic hydrolysis described later. The lower alcohol or water-containing lower alcohol extract thus obtained is a mixture containing the novel lignan glycoside and various other sugar chain compounds.

In the present invention, the following treatment can be performed to remove impurities other than the target substance in the extract. That is, in order to remove fat-soluble impurities, 2 to 10 times (v / wt) of a water-insoluble organic solvent with respect to the extract,
For example, chloroform or n-hexane and water are added for extraction, and the mixture is 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. In order to remove water-soluble impurities, a small amount, preferably 1 to 5 times (v
/ Wt) aqueous alcohol (alcohol concentration 30-100)
% (V / v)), and this is added dropwise to a relatively large amount, preferably 10- to 200-fold (v / wt) alcohol, which is gently stirred. After standing, the precipitate is removed by centrifugation or fractional filtration and the like, 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. 1 to 100 times (v
/ V). These operations are repeated if necessary. The alcohol used for such treatment may be the same as the lower alcohols used for extracting the crushed sesame seed.

The crude lignan glycoside can be further purified to a single component by column chromatography using octadecyl silica (ODS) or silica gel as a carrier, if necessary. That is, for example, OD
S was filled in a glass or stainless steel tube to form a column, and water was flowed through the column to equilibrate the column. Then, the lower alcohol or the aqueous lower alcohol extract or the crude lignan glycoside was loaded at a load ratio of 0.1. ５5% (wt / v), and a hydroalcoholic solvent (alcohol is methanol, ethanol, n-propanol, isopropanol, n-
Normal pressure or pressurization (200 kg / cm
^The hydroalcoholic solvent is supplied under a condition of up to about ² , preferably 50 to 150 kg / cm ² ) until a predetermined fraction elutes. The eluted fraction obtained here can be further subjected to high-performance liquid chromatography (HPLC), preparative liquid chromatography or the like using the above-mentioned carrier, if necessary, to further purify each component.

According to the second method of the present invention, in addition to the findings of the first method, the novel lignan glycoside of the present invention is not affected by sugar chain hydrolases such as glycosidase and cellulase. This is based on knowledge. That is, a glycan hydrolase is further acted on in an aqueous solution by humidifying or germinating the sesame seeds and extracting the ground or defatted lees of the treated product with a lower alcohol or a hydrated lower alcohol. Producing a novel lignan glycoside characterized in that the non-hydrolyzate is concentrated by removing insolubles as necessary and then removing fat-soluble substances and water-soluble substances from solubles. Is the law.

In this method, a lower alcohol or a water-containing lower alcohol extract obtained by the above-mentioned first method is used as a raw material for treatment. Such an extract is dispersed or dissolved in 1 to 100 times (v / wt) water or a buffer solution (pH 2 to 6), and 0.1 to 30% (wt / w) based on the mixture.
t), preferably 1 to 10% (wt / wt) of a sugar chain hydrolase is added, and the sugar chain is added at 10 to 50 ° C for 1 to 50 hours, preferably 5 to 15 hours, preferably with gentle stirring. Is hydrolyzed. By this enzymatic reaction, a sterol glycoside or glycolipid having similar solvent partitioning properties to the novel lignan glycoside according to the present invention, a sesaminol glycoside,
Most of the flavonoid glycosides and carbohydrates are hydrolyzed,
It is further classified into monosaccharides having higher water solubility or relatively low molecular sugar compounds (oligosaccharides and the like) similar thereto and aglycones (lignans). On the other hand, the novel lignan glycoside according to the present invention is not affected by such enzymes and is not hydrolyzed.

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. Furthermore, these enzyme agents may be immobilized on a suitable base material such as activated carbon, celite, synthetic resin, ion exchange resin, gel or the like to enable continuous use and recovery and reuse.

After completion of the enzymatic reaction, a 1 to 100-fold (v / v) low-polarity, water-insoluble or poorly water-soluble organic solvent such as n-hexane, chloroform, diethyl ether, petroleum ether or the like is added to the reaction mixture. Extract. Excluding the organic solvent phase,
Concentrate the aqueous phase. At this time, the novel lignan glycoside according to the present invention is concentrated on the aqueous phase side. Thereby, fat-soluble impurities such as fatty acid glycerides, phospholipids, lignans and sterols, and aglycone components derived from glycosides other than the novel lignan glycoside according to the present invention, which are cleaved by the enzyme, are extracted. , These can be excluded. Repeat if necessary.

The residual liquid (the above-mentioned aqueous phase concentrate) from which the fat-soluble impurities have been removed in this manner is mixed with an organic solvent having an intermediate polarity and being hardly soluble or insoluble in water, such as n-butanol.
Ethyl acetate, methyl ethyl ketone, etc. 1 to 100 times (v
/ V) Add and extract again. Repeat if necessary. As a result, sugars, proteins,
Most of water-soluble impurities such as fibers can be removed as an aqueous phase. The components extracted into this organic solvent phase are:
Since it is only a non-water-soluble component such as a monosaccharide or an oligosaccharide having a higher polarity than a fat-soluble substance such as sterol or lignan, this is separated, and a known concentration method such as drying under reduced pressure is used. By concentrating to dryness, an extract fraction containing a large amount of the target novel lignan glycoside can be obtained.

The novel lignan glycoside thus obtained is
A mixture of the compounds represented by the structural formula (I), and particularly a mixture of the compounds represented by the structural formula (II-a),
It is a novel lignan glycoside represented by (II-b) and (II-c).

The extract obtained by the method of the present invention can be used as it is for products in various fields such as foods, pharmaceuticals, cosmetics, and agricultural chemicals. In particular, each component of the novel lignan glycoside is purified to a single high purity. When necessary, a separation and purification means such as column chromatography which is usually performed may be applied.

The chemical structure of each component of the novel lignan glycoside according to the present invention is such that each of the purified components highly purified by the above-mentioned method is hydrolyzed with hydrochloric acid, for example, to form a lignan portion (aglycone portion) and a sugar portion. These can be subjected to trimethylsilylation and subjected to gas chromatography, or analyzed and confirmed by nuclear magnetic resonance spectroscopy, mass spectroscopy, and the like.

[0032]

The present invention will be specifically described below with reference to examples. Example 1 Quartz sand previously sterilized was spread on a stainless steel vat of 300 cm ² , and 10 g of Chinese sesame seeds were spread thereon, and cultured in a 40 ° C. constant temperature bath while sufficiently spraying distilled water.
Sprouted. The germination rate was 80% or more. A certain amount of germinated bodies having a similar germination state were sampled with time, and each was crushed with a blender together with 100 ml of hydrated methanol (80% (v / v)). After the residue was filtered and the filtrate was concentrated to dryness, the obtained solid was washed with n-hexane to remove fat-soluble substances, then extracted with water-butanol to remove water-soluble substances, and extracted with aqueous methanol extract. (Lignan glycoside extract) was obtained. 100 parts of each aqueous methanol extract
It was redissolved in ml of the same aqueous methanol and subjected to HPLC to analyze the composition.

The HPLC measurement conditions were as follows: pump (CCPM,
Column (SokenPak ODS-W manufactured by Tosoh Corporation)
5μ, 10mmφ × 250mm), UV absorption detector (UV
-8000, manufactured by Tosoh Corporation), and elution was performed using a linear gradient of water: methanol starting at 90:10 (v: v) and 60: 60: 60: 60 (v: v) after 60 minutes. The flow rate was 1 ml / min, and the detection wavelength was 280 nm.

The respective contents were determined from the peak areas of the novel lignan glycoside components SG-1 to SG-5 detected by HPLC analysis, and the changes over time are shown in FIG. Sesame seeds were hardly present in the seeds as they germinated.
G-1, SG-3 and SG-5 were produced, and reached almost a constant value after 48 hours of culture. The content of these total glycoside components on day 7 of culture was 2.5% (wt /
wt) and 5.0% (wt / w) per hydrated methanol extract.
y).

The chemical structure of each lignan glycoside component was confirmed by the following method using each purified product highly 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 is concentrated to dryness at 40 ° C. or less, subjected to trimethylsilylation treatment with TMS-PZ (manufactured by Tokyo Chemical Industry Co., Ltd.), and then subjected to gas chromatography (GLC).
The lignans were subjected to quantitative analysis (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.

Example 2 In the same manner as in Example 1, 10 g of Indian sesame seeds
The cells were cultured at 7 ° C. for 2 days and germinated. The germination rate was 87%. This germinated body and 100 ml of n-hexane were placed in a blender and crushed, and the crushed liquid was refluxed at 65 ° C. for 5 hours. The reflux was then filtered to give a defatted residue, to which was added 100 ml of ethanol (98% (v / v)) to form a dispersion, which was extracted with stirring overnight. The extract was separated by filtration, and the filtrate was concentrated to dryness under reduced pressure. The solid obtained here was washed with n-hexane to remove fat-soluble substances, and then extracted with water-n-butanol to remove water-soluble substances, thereby obtaining an ethanol extract (lignan glycoside extract). Was. When subjected to HPLC and analyzed for composition in the same manner as in Example 1, SG
The content of all the components -1 to SG-5 was 2.7% (wt / wt) per dry matter of the germinated body, and 4.5 per ethanol extract.
% (Wt / wt).

Example 3 In the same manner as in Example 1, 10 g of Chinese sesame seeds
The cells were cultured at 2 ° C. for 2 days and germinated. The germination rate was 90%. The germinated body and 100 ml of aqueous ethanol (50%
(V / v)) to obtain a solid that was concentrated to dryness. On the other hand, the germinated body and 100 ml of a 20 mM acetate buffer (pH 5.0) were placed in a blender and crushed to obtain a crushed liquid. The solid was added to the crushed liquid, shaken at 50 ° C. for 15 hours, and a hydrolysis reaction was performed with a sugar chain hydrolase present in the sesame germ. Then, the same volume of n-hexane was added to the reaction solution and vigorously shaken. This extraction was repeated three times. 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 was repeated twice. washing the n-butanol phase twice with the same volume of distilled water,
It was concentrated to dryness under reduced pressure to obtain an n-butanol extract. When subjected to HPLC analysis in the same manner as in Example 1 to determine the composition and content of the novel lignan glycoside in the n-butanol extract using sesamin as an external standard, SG-3, SG-3 and SG-5 were determined. The seeds are the major components, which are present in the extract at about 110 mg, the composition of which is SG-1 28%,
SG-3 was 39% and SG-5 was 33%.

Example 4 In the same manner as in Example 1, 10 g of Chinese sesame seeds
The cells were cultured at 2 ° C. for 2 days and germinated. The germination rate was 89%. The germinated body and 100 ml of n-hexane were placed in a blender and crushed. The crushed liquid was refluxed at 65 ° C. for 5 hours. The reflux was then filtered to give a defatted residue,
A dispersion was formed by adding water-containing methanol (80% (v / v)), and the mixture was extracted with stirring overnight. The extract was separated by filtration, and the filtrate was concentrated to dryness under reduced pressure. Extract 2
The resultant was dispersed in 100 ml of 0 mM citrate buffer (pH 4.0), 200 mg of β-glucosidase (manufactured by Funakoshi) was added, and the mixture was shaken at 50 ° C. for 15 hours. Add the same volume of n to the reaction solution.
-Hexane was added and shaken vigorously. This extraction was repeated three times. 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 was repeated twice. The n-butanol phase was washed twice with the same volume of distilled water and concentrated to dryness under reduced pressure to obtain n-butanol.
-A butanol extract was obtained. H by the method described in Example 3
As a result of PLC analysis, the novel lignan glycoside in the n-butanol extract was mainly composed of SG-1, SG-3 and SG-5, which were present in the extract in about 150 mg. SG-1 is 25%, SG-3 is 42%,
SG-5 was 33%.

Example 5 In Example 4, 100 mg of β-glucosidase (manufactured by Funakoshi) and 100 mg of α-glucosidase were used instead of 200 mg of β-glucosidase.
A mixed enzyme of 100 mg of glucosidase (manufactured by Wako Pure Chemical Industries, Ltd.), 1 g of cellulase (manufactured by Boehringer Mannheim) and 1 g of amylase (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the other components were treated and analyzed in the same manner. As a result, the novel lignan glycoside in the n-butanol extract is mainly composed of SG-1, SG-3 and SG-5, which are contained in the extract at about 13%.
0 mg was present, and the composition was 30% for SG-1, 40% for SG-3, and 30% for SG-5.

Example 6 The same procedure as in Example 1 was carried out except that sesame seeds were humidified in place of the germinated material of the sesame seeds and a humidified substance kept at 10 ° C. for one day was used for HPLC analysis. , SG-1 to SG-5 were 2.0% (wt / wt) per dry matter of the germinated body and 4.1% (wt / wt) per hydrated methanol extract.

[0044]

According to the production method of the present invention, it is possible to easily and inexpensively and easily obtain a large amount of a novel lignan glycoside without using special equipment, equipment, reagents and the like. Become. That is, humidification or germination of sesame seeds can significantly increase the amount of novel lignan glycosides that hardly exist in sesame seeds. In addition, a novel lignan glycoside can be efficiently produced by solvent extraction and enzymatic treatment of a humidified or germinated body of sesame seeds.

[Brief description of the drawings]

FIG. 1 shows a humid culture of sesame seeds, after crushing, 80% (v /
v) Plotting the content of novel lignan glycoside components (SG-1 to SG-5) and the like present in the extract when extracted with methanol, together with the sesame seed culture time. The horizontal axis shows the cultivation time of sesame seeds, and the vertical axis shows the novel lignan glycoside and other components of the extract and their relative contents.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 31/70 613 A61K 31/70 613 35/78 35/78 CY C09K 15/08 C09K 15/08 C12P 19/58 C12P 19 / 58

Claims (1)

(57) [Claims] The present invention provides a method for treating sesame seeds by humidifying or germinating to increase the amount of a novel lignan glycoside represented by the following structural formula (I). A method for producing the lignan glycoside, comprising extracting with a lower alcohol or a water-containing lower alcohol, and then removing a fat-soluble substance and a water-soluble substance from the extract. Embedded image (In the formula (I), R represents a glycosyl residue of glucose, m represents any of integers 1 to 3, and n represents an integer of 0 or 1.)