JP5221155B2 - Organic compounds and antitumor agents, antioxidants and antibacterial agents - Google Patents

Description

  The present invention relates to organic compounds belonging to flavanones, megastigman glycosides and lignan glycosides, and uses thereof.

As the flavanones (2,3-dihydroflavones), for example, nymphaeol such as nymphaeol-A, nymphaeol-B, nymphaeol-C, isonimpheol-B and the like are known (see Patent Documents 1 to 3). It is known that Nimpheol is contained in the extract of a giant grass (major tree) belonging to the genus Euphorbiaceae (refer to Patent Document 4). The present inventors have reported novel flavanones contained in the grasshopper extract (see Non-Patent Document 1). In addition, megastigman glycosides have been reported (see Non-Patent Document 2). Furthermore, it has been reported that pinoresinol glycoside as a lignan glycoside can be obtained from an extract of sesame (see Patent Document 5).
JP 2005-29560 A Japanese Patent Laying-Open No. 2005-29778 JP 2005-272374 A JP 2007-45754 A JP-A-6-116282 Kawakami Shiori, Liva Harinantenaina, Katsuyoshi Matsunami, Hideaki Otsuka et al., Research on Ingredients of Macaranga tanarius (2), Proceedings of the 54th Annual Meeting of the Japanese Biopharmaceutical Society, p. 209, 2007 issue Radical-scavenging activities of new megastigmane glucosides from Macaranga tanarius (L.) Mull.-Arg., Katsuyoshi Matsunami, Ichiko Takamori, Hideaki Otsuka, Takakazu Shinzato, Mitsunori Aramoto, Kazunari Kondo and YoshioTakeda, Chemical & Pharmaceutical Bulletin, 54 , 1403-1407, (2006)

  The present invention has been made by the isolation of organic compounds and the discovery of useful physiological activities as a result of intensive studies by the present inventors. An object of the present invention is to provide an organic compound that exhibits useful physiological activity and use thereof.

  In order to achieve the above object, the organic compound of the invention described in claim 1 is represented by the following chemical formula (1).

請求項２に記載の発明の抗腫瘍剤は、請求項１に記載の有機化合物を有効成分として含有することを特徴とする。

The antitumor agent of the invention described in claim 2 contains the organic compound described in claim 1 as an active ingredient.

The antioxidant of the invention described in claim 3 contains the organic compound described in claim 1 as an active ingredient.
The antibacterial agent of the invention described in claim 4 contains the organic compound described in claim 1 as an active ingredient.

  The antitumor agent of the invention described in claim 5 is characterized by containing an organic compound represented by the following chemical formula (2) as an active ingredient.

請求項６に記載の発明の抗酸化剤は、下記化学式（２）に示される有機化合物を有効成分として含有することを特徴とする。

The antioxidant of invention of Claim 6 contains the organic compound shown by following Chemical formula (2) as an active ingredient, It is characterized by the above-mentioned.

請求項７に記載の発明の抗菌剤は、下記化学式（２）に示される有機化合物を有効成分として含有することを特徴とする。

The antibacterial agent of the invention described in claim 7 contains an organic compound represented by the following chemical formula (2) as an active ingredient.

請求項８に記載の発明の有機化合物は、下記化学式（３）に示されることを特徴とする。

The organic compound according to an eighth aspect of the present invention is represented by the following chemical formula (3).

請求項９に記載の発明の抗腫瘍剤は、請求項８に記載の有機化合物を有効成分として含有することを特徴とする。

The antitumor agent of the invention described in claim 9 contains the organic compound described in claim 8 as an active ingredient.

The antioxidant of invention of Claim 10 contains the organic compound of Claim 8 as an active ingredient, It is characterized by the above-mentioned.
An organic compound according to an eleventh aspect of the present invention is represented by the following chemical formula (4).

但し、化学式（４）に示されるＭｅはメチル基を示す。

However, Me shown by Chemical formula (4) shows a methyl group.

The antitumor agent of the invention described in claim 12 contains the organic compound described in claim 11 as an active ingredient.
The antioxidant of the invention described in claim 13 contains the organic compound described in claim 11 as an active ingredient.

  ADVANTAGE OF THE INVENTION According to this invention, the organic compound which exhibits useful physiological activity, and its use are provided.

(First embodiment)
A first embodiment embodying the present invention will be described in detail.
The organic compound of the first embodiment is represented by the following chemical formula (1). The organic compound is a novel flavanone compound having a flavanone skeleton.

この有機化合物は、上記化学式に“＊”で示されるように３´´の炭素は不斉炭素であり、その３´´位について互いにエピマーの関係にある３´´Ｒと３´´Ｓとが存在する。すなわち、上記有機化合物は、下記化学式（１Ａ）に示される有機化合物と下記化学式（１Ｂ）に示される有機化合物とを示している。

In this organic compound, as indicated by “*” in the above chemical formula, the 3 ″ carbon is an asymmetric carbon, and 3 ″ R and 3 ″ S, which are in an epimer relationship with each other at the 3 ″ position, Exists. That is, the organic compound is an organic compound represented by the following chemical formula (1A) and an organic compound represented by the following chemical formula (1B).

上記有機化合物は、トウダイグサ科オオバギ属に属するオオバギ（大葉木）の抽出物から単離される。オオバギは、マカランガ・タナリウス（Ｍａｃａｒａｎｇａ ｔａｎａｒｉｕｓ）とも呼ばれる植物であって、常緑広葉樹（雌雄異株）である。オオバギは、沖縄、台湾、中国南部、マレー半島、フィリピン、マレーシア、インドネシア、タイなどの東南アジア、オーストラリア北部などに生育している。また、オオバギは、樹木の中でも成長が極めて早く、荒廃地における成長も可能である。

The said organic compound is isolated from the extract of the bigeye (large leaf tree) which belongs to Euphorbiaceae. The grasshopper is a plant called Macaranga tanarius, and is an evergreen broad-leaved tree (sexed). Weow grows in Okinawa, Taiwan, southern China, the Malay Peninsula, Southeast Asia such as the Philippines, Malaysia, Indonesia, and Thailand, and northern Australia. In addition, weow grows very fast among trees and can grow in degraded areas.

  As a raw material for the plant extract, various organs and components of the plant can be used. As a raw material, a single organ or component may be used, or two or more organs or components may be mixed and used. From the viewpoint of enhancing the antitumor and antioxidant effects of the grasshopper extract, the raw material preferably contains fruits, seeds, flowers, roots, trunks, stem tips, leaf blades, and secretions (wax etc.). Since the tip of the stem includes the growth point of the stem and the leaf bud and is more flexible than the leaf blade, it is easy to perform the extraction operation efficiently. In addition, when the proportions of the respective organs with respect to the whole of the grasshopper are compared, the proportions occupied by the trunk, roots, and leaves are high. For this reason, it is industrially suitable from the viewpoint that it is easy to secure the raw material to use easy-to-handle green grass as a raw material for the extract. These raw materials can be extracted as they are collected, crushed, crushed or ground after collection, crushed, crushed or ground after collection or drying, or crushed, crushed or ground after collection and dried. The operation can be performed. For such crushing, for example, a cutter, a cutter, a crusher, or the like can be used. Moreover, when preparing the grind | pulverized raw material, a mill, a crusher, a grinder etc. can be used, for example. In preparing the ground material, a kneader, a mortar, or the like can be used.

  As an extraction solvent for extracting the wean extract from the above-mentioned raw materials, a mixed solvent of water and an organic solvent, an organic solvent such as a lower alcohol, dimethyl sulfoxide, acetonitrile, acetone, ethyl acetate, hexane, glycerin, propylene glycol and the like. Can be mentioned. Examples of the lower alcohol include methanol, ethanol, propanol, isopropanol, butanol and the like. As an organic solvent, a single species may be used, or a mixed solvent obtained by mixing a plurality of species may be used.

  When a mixed solvent of water and an organic solvent is used as the extraction solvent, the content of the organic solvent in the mixed solvent is preferably 50% by volume or more, more preferably 80% by volume or more. When the content of the organic solvent in the mixed solvent is less than 50% by volume, there is a possibility that the active ingredient contained in the grass can not be efficiently extracted. The organic solvent is preferably a lower alcohol.

  As the extraction operation, the raw material is immersed in the extraction solvent for a predetermined time. In such an extraction operation, a stirring operation, heating, or the like may be performed as necessary to increase the extraction efficiency. Further, in order to reduce impurities extracted from the raw material, a separate water extraction operation or hot water extraction operation may be performed prior to the extraction operation.

  After the extraction operation, a solid-liquid separation operation is performed to separate the grass extract and the raw material residue. As a separation method for such a solid-liquid separation operation, a known separation method such as filtration or centrifugation can be used. The obtained grass extract may be concentrated as necessary.

  In addition, by removing the extraction solvent contained in the grass extract as necessary, a solid grass extract can be obtained. Such removal of the solvent may be performed, for example, by heating under reduced pressure or by freeze-drying.

  In addition, the plantain extract contains nymphaeol and the like in addition to the above organic compounds. Examples of nymphaeols include nymphaeol-A, nymphaeol-B, and nymphaeol-C.

  The organic compound of the first embodiment is isolated by chromatographic purification of the weevil extract. Examples of chromatography include partition chromatography, adsorption chromatography, molecular exclusion chromatography and the like. Here, in the silica gel column chromatography, for example, when a mixed solvent of hexane and ethyl acetate or a mixed solvent of toluene and acetone is used as a developing solvent, the organic compound represented by the chemical formula (1) includes nimpheol-C. Obtained as a crude product. For this reason, the organic compound of 1st Embodiment can also be obtained in the process which refine | purifies Nimpheol C from such a crudely purified product.

  The antitumor agent of 1st Embodiment contains the organic compound shown by the said Chemical formula (1) as an active ingredient. That is, the antitumor agent contains at least one of the organic compound represented by the chemical formula (1A) and the organic compound represented by the chemical formula (1B) as an active ingredient. The antitumor agent exhibits an antitumor action that suppresses the growth of cancer cells. Examples of the cancer cells include lung cancer cells, nasopharyngeal cancer cells, leukemia cells, breast cancer cells and the like. Such antitumor agents are mainly useful as pharmaceuticals.

  The antioxidant of 1st Embodiment contains the organic compound shown by the said Chemical formula (1) as an active ingredient. That is, the antioxidant contains as an active ingredient at least one of the organic compound represented by the chemical formula (1A) and the organic compound represented by the chemical formula (1B). This antioxidant suppresses oxidation of chemical substances such as fats and oils, fragrances, and pigments. In addition, antioxidants eliminate the active oxygen in the ingested living body, thereby enhancing the liver function, reducing the toxicity of acetaldehyde, the antioxidant effect of low density cholesterol (LDL), improving the immune function, etc. Demonstrate the promotion effect. Moreover, an antioxidant exhibits effects, such as skin whitening and anti-aging, about skin, an oral cavity, etc. Such antioxidants are useful as additives for foods and drinks such as health foods, foods, cosmetics, quasi drugs, and the like.

  The antibacterial agent of 1st Embodiment contains the organic compound shown by the said Chemical formula (1) as an active ingredient. That is, the antibacterial agent contains at least one of the organic compound represented by the chemical formula (1A) and the organic compound represented by the chemical formula (1B) as an active ingredient. This antibacterial agent has antibacterial properties against mold, gram negative bacteria, and gram positive bacteria. Such antibacterial agents are useful as additives such as health foods, foods and drinks such as foods, cosmetics, pharmaceuticals, quasi drugs, and detergents.

  The antitumor agent, antioxidant and antibacterial agent may contain, for example, excipients, bases, emulsifiers, stabilizers, solvents, fragrances, sweeteners and the like. The antitumor agent, antioxidant and antibacterial agent may be liquid or solid. These dosage forms are not particularly limited, and examples thereof include powders, powders, granules, tablets, capsules, pills, liquids, injections and the like.

(Second Embodiment)
The second embodiment embodying the present invention will be described focusing on differences from the first embodiment. The second embodiment is different from the first embodiment in that it has a double bond at the 5 ″ position of the organic compound.

  The antitumor agent of 2nd Embodiment contains the organic compound shown by following Chemical formula (2) as an active ingredient. The organic compound is a flavanone compound having a flavanone skeleton.

この有機化合物は、上記化学式に“＊”で示されるように３´´の炭素は不斉炭素であり、その３´´位について互いにエピマーの関係にある３´´Ｒと３´´Ｓとが存在する。すなわち、上記有機化合物は、下記化学式（２Ａ）に示される有機化合物と下記化学式（２Ｂ）に示される有機化合物とを示している。

In this organic compound, as indicated by “*” in the above chemical formula, the 3 ″ carbon is an asymmetric carbon, and 3 ″ R and 3 ″ S, which are in an epimer relationship with each other at the 3 ″ position, Exists. That is, the organic compound is an organic compound represented by the following chemical formula (2A) and an organic compound represented by the following chemical formula (2B).

すなわち、抗腫瘍剤は、化学式（２Ａ）に示される有機化合物、及び、化学式（２Ｂ）に示される有機化合物の少なくとも一方を有効成分として含有する。

That is, the antitumor agent contains at least one of the organic compound represented by the chemical formula (2A) and the organic compound represented by the chemical formula (2B) as an active ingredient.

  The organic compound of 2nd Embodiment is isolated from the extract of a grasshopper like the said 1st Embodiment. Here, in silica gel column chromatography, for example, when a mixed solvent of hexane and ethyl acetate or a mixed solvent of toluene and acetone is used as a developing solvent, the organic compound represented by the chemical formula (2) is a crude compound containing nimpheol-C. Obtained as a purified product. For this reason, it is possible to obtain the organic compound of the second embodiment in the process of purifying nymphaeol C from such a crude product.

  The antioxidant of 2nd Embodiment contains the organic compound shown by Chemical formula (2) as an active ingredient. That is, the antioxidant contains at least one of the organic compound represented by the chemical formula (2A) and the organic compound represented by the chemical formula (2B) as an active ingredient.

  The antibacterial agent of 2nd Embodiment contains the organic compound shown by Chemical formula (2) as an active ingredient. That is, the antibacterial agent contains at least one of an organic compound represented by the chemical formula (2A) and an organic compound represented by the chemical formula (2B) as an active ingredient.

(Third embodiment)
A third embodiment embodying the present invention will be described focusing on differences from the first embodiment. The third embodiment is different from the first embodiment regarding the structure of the organic compound.

  The organic compound of the third embodiment is an organic compound represented by the following chemical formula (3). This organic compound is a novel megastigman glycoside.

第３の実施形態の有機化合物は、上記第１の実施形態と同様にオオバギの抽出物から単離される。抽出操作においては、抽出効率を高めるという観点から、水と有機溶媒の混合溶媒を用いて抽出するのが好ましい。その混合溶媒中における水の比率は、好ましくは１０容量％以上、より好ましくは５０容量％以上である。また、抽出温度を高めることで、抽出効率を高めることが容易である。

The organic compound of 3rd Embodiment is isolated from the extract of a grasshopper like the said 1st Embodiment. In extraction operation, it is preferable to extract using the mixed solvent of water and an organic solvent from a viewpoint of improving extraction efficiency. The ratio of water in the mixed solvent is preferably 10% by volume or more, more preferably 50% by volume or more. Moreover, it is easy to raise extraction efficiency by raising extraction temperature.

  The antitumor agent of 3rd Embodiment contains the organic compound shown by Chemical formula (3) as an active ingredient. The antioxidant of 3rd Embodiment contains the organic compound shown by Chemical formula (3) as an active ingredient. The antitumor agent and antioxidant can be used in the same manner as in the first embodiment, and the dosage form is not particularly limited.

(Fourth embodiment)
A fourth embodiment embodying the present invention will be described focusing on differences from the first embodiment. The fourth embodiment differs from the first embodiment regarding the structure of the organic compound.

  The organic compound of the fourth embodiment is an organic compound represented by the following chemical formula (4). This organic compound is a novel lignan glycoside.

但し、化学式（４）に示されるＭｅはメチル基を示す。

However, Me shown by Chemical formula (4) shows a methyl group.

  The organic compound of 4th Embodiment is isolated from the extract of a grasshopper like the said 1st Embodiment. In extraction operation, it is preferable to extract using the mixed solvent of water and an organic solvent from a viewpoint of improving extraction efficiency. The ratio of water in the mixed solvent is preferably 10% by volume or more, more preferably 50% by volume or more. Moreover, it is easy to raise extraction efficiency by raising extraction temperature.

  The antitumor agent of 4th Embodiment contains the organic compound shown by Chemical formula (4) as an active ingredient. The antioxidant of 4th Embodiment contains the organic compound shown by Chemical formula (4) as an active ingredient. The antitumor agent and antioxidant can be used in the same manner as in the first embodiment, and the dosage form is not particularly limited.

(Effect of embodiment)
The effects exhibited by the first to fourth embodiments will be described below.
(1) Since the organic compound according to the first to fourth embodiments exhibits an antitumor action, an antioxidant action or an antibacterial action, an antitumor agent, an antioxidant and an antibacterial agent are provided.

(2) Among the organic compounds according to the first and second embodiments, the antitumor agent containing the organic compound represented by the chemical formula (1A) as an active ingredient has excellent antitumor activity.
(3) The organic compounds according to the first to fourth embodiments are all obtained from a grasshopper extract. Weow grows very fast among trees and can grow in degraded areas. In this way, the grasshopper does not take time and effort for its cultivation management. In addition, the plantain extract is a plant-derived raw material, and thus has high safety. Therefore, antitumor agents, antioxidants and antibacterial agents are excellent in raw material supply, productivity, safety and the like.

  (4) The organic compounds according to the first and second embodiments can be roughly purified by silica gel column chromatography using a grasshopper extract as a raw material. In the silica gel column chromatography, for example, when a mixed solvent of hexane and ethyl acetate or a mixed solvent of toluene and acetone is used as a developing solvent, the organic compound according to the first and second embodiments includes nimpheol-C. Obtained as a crude product. For this reason, it is possible to obtain the organic compounds of the first and second embodiments in the process of purifying nymphaeol C from such a crude product.

Next, the embodiment will be described more specifically with reference to examples.
<Rough purification of the extract>
A methanol extract was obtained by extracting three times (total amount of methanol 75 L) with methanol using 12.1 kg of dried leaves of grasshopper as a raw material. The methanol extract was distributed using 3 L of hexane, and the methanol layer was concentrated under reduced pressure. The residue was suspended in water and extracted with ethyl acetate to obtain 802 g of an ethyl acetate layer. The ethyl acetate layer thus obtained was purified by silica gel column chromatography (column dimensions: inner diameter 10 cm × height 50 cm). In this purification, a fraction was obtained under the conditions of 1 L of 1 fraction using the developing solvent shown below.

Fraction (1-1): hexane: ethyl acetate = 20 parts by volume: 1 part by volume, 6 L
Fraction (1-2): hexane: ethyl acetate = 5 parts by volume: 1 part by volume, 6 L
Fraction (1-3): hexane: ethyl acetate = 4 parts by volume: 1 part by volume, 6 L
145 g of the above fraction (1-3) was further purified by silica gel column chromatography (inner diameter 10 cm × height 50 cm). In this purification, a fraction was obtained under the conditions of 1 L of 1 fraction using the developing solvent shown below.

Fraction (2-1): Toluene Fraction (2-2): Toluene: Acetone = 100 parts by volume: 1 part by volume, 6 L
Fraction (2-3): Toluene: acetone = 70 parts by volume: 1 part by volume, 3 L
Fraction (2-4): Toluene: acetone = 50 parts by volume: 1 part by volume, 3 L
Fraction (2-5): Toluene: acetone = 40 parts by volume: 1 part by volume, 3 L
Fraction (2-6): Toluene: acetone = 30 parts by volume: 1 part by volume, 3 L
Fraction (2-7): Toluene: acetone = 10 parts by volume: 1 part by volume, 3 L
Fraction (2-8): Toluene: acetone = 5 parts by volume: 1 part by volume, 3 L
102 g of the above fraction (2-5) to fraction (2-8) was used as a crude product.

<Isolation of the organic compound represented by the chemical formula (1)>
The roughly purified product 102 g was purified by column chromatography (column size: inner diameter 11 cm × height 50 cm) using a reverse phase porous resin (trade name: Diaion HP-20, manufactured by Mitsubishi Chemical Corporation) as a filler. did. In the same purification, fractions were obtained under the conditions of 1 fraction and 1 L using the developing solvent shown below.

Fraction (1): Water: methanol = 1 volume part: 3 volume part, 9 L
Fraction (2): Methanol, 9L
Fraction (3): methanol: chloroform = 1 volume part: 1 volume part, 9 L
Fraction (4): Chloroform, 9L
21.8 g of the above fractions (3) and (4) were further purified using reverse phase silica gel column chromatography (column dimensions: inner diameter 5 cm × height 20 cm). In this purification, fractions (1) to (120) were obtained under the condition of 13 g per fraction using a linear gradient solvent shown below as a developing solvent.

Water: methanol = 50 parts by volume: 50 parts by volume (1 L) → methanol (1 L)
Methanol (1 L) → Methanol: Chloroform = 1 volume part: 1 volume part (1 L)
Methanol: chloroform = 1 volume part: 1 volume part (1 L) → chloroform (1 L)
Subsequently, 1.29 g of the obtained fraction was divided into two portions and purified by size exclusion column chromatography using a porous gel (manufactured by GE Healthcare Biosciences, trade name: Sephadex LH-20) as a filler. did. In the same purification, using a mixed solvent of chloroform and methanol (chloroform: methanol = 1 volume part: 1 volume part) as a moving bed, fraction (42) to fraction (55) were obtained under the condition of 1 fraction 2 g. Obtained.

  Subsequently, 390 mg of the obtained fraction was purified by high performance liquid chromatography. In this purification, 21.0 mg of the organic compound represented by the chemical formula (1A) was obtained from the fraction confirmed by the peak of the relative refractive index (RI) appearing at the retention time of 52 minutes.

The details of the high performance liquid chromatography are as follows.
Preparative column: Inertsil ODS (trade name, 6.0 × 250 mm, manufactured by Gaskuro Kogyo Co., Ltd.), Cosmosil 5C18-AR-II (trade name, 20 × 250 mm, manufactured by Nacalai Tesque)
Detector: UV8000 (manufactured by Tosoh Corporation), RI8000 (manufactured by Tosoh Corporation)
Flow rate: 1.6 ml / min or 5.0 ml / min
Moving bed: Mixed solvent of methanol and water (methanol: water = 85 parts by volume: 15 parts by volume)
Detection wavelength of ultraviolet absorption: 254 nm
On the other hand, in the high performance liquid chromatography, a fraction obtained from a non-refractive index (RI) peak having a retention time of 44 to 47 minutes was further purified by high performance liquid column chromatography. In this purification, a mixed solvent of methanol, 2-propanol and water (methanol: 2-propanol: water = 3 volume parts: 3 volume parts: 4 volume parts) is used as the moving bed. In this purification, 18.7 mg of an organic compound represented by the chemical formula (1B) was obtained from the fraction confirmed by the peak of relative refractive index (RI) appearing at a retention time of 199 minutes.

<Isolation of organic compound represented by chemical formula (2)>
The organic compound represented by the chemical formula (2) can be isolated from the fraction obtained by the above <Isolation of the organic compound represented by the chemical formula (1)> with respect to 102 g of the crude product. That is, 102 g of the roughly purified product can be isolated from the fraction (2) obtained using methanol as a developing solvent when it is purified by column chromatography using the above reverse-phase porous resin as a filler. 68.7 g of this fraction (2) was further purified by reverse phase silica gel column chromatography (column size: inner diameter 5 cm × height 20 cm). In this purification, fractions (247) to (276) were obtained under the condition of 1 fraction of 10 g using a linear gradient solvent as shown below as a developing solvent.

Methanol: water = 50 parts by volume: 50 parts by volume (2 L)
→ Methanol (2L)
Subsequently, 2.45 g of the obtained fraction was purified by high performance liquid chromatography. In the same purification, 366 mg of a fraction confirmed at the peak of relative refractive index (RI) appearing at a retention time of 31 to 37 minutes was obtained. In this purification, a mixed solvent of methanol, dichloromethane, and water (methanol: dichloromethane: water = 90 parts by volume: 0.2 parts by volume: 9.8 parts by volume) is used as the moving layer. Subsequently, 366 mg of the obtained fraction was further purified by high performance liquid chromatography. In the same purification, an organic compound represented by the chemical formula (2A) was obtained from a fraction confirmed by a peak of relative refractive index (RI) appearing at a retention time of 48 minutes. Further, an organic compound represented by the chemical formula (2B) was obtained from a fraction confirmed by a peak of specific refractive index (RI) appearing at a retention time of 55 minutes.

<Isolation of organic compound represented by chemical formula (3) and chemical formula (4)>
12.1 kg of dried leaves of grass were extracted three times with methanol (total 45 L), concentrated to about 3 L, and then extracted with 3 L of hexane. The remaining methanol layer was further concentrated to distill off the methanol, then suspended in 3 L of water, extracted and concentrated successively with 3 L of ethyl acetate and 1-butanol, respectively, and hexane layer 70.0 g, ethyl acetate layer 801 g, 1-butanol layer 374 g, and water layer 499 g were obtained.

  Of these, 181 g of the 1-butanol soluble fraction suspended in 2 L of 20 volume% methanol was used with a reverse phase porous resin (trade name: Diaion HP-20, manufactured by Mitsubishi Chemical Corporation) as a filler. Purification was performed by column chromatography (column dimensions: inner diameter 8 cm × height 40 cm). In the same purification, fractions were obtained under the conditions of 1 fraction and 1 L using the developing solvent shown below.

Water: methanol = 4 parts by volume: 1 part by volume, 6L
Water: methanol = 2 parts by volume: 3 parts by volume, 6L
Water: methanol = 3 parts by volume: 2 parts by volume, 6 L
Water: methanol = 1 volume part: 4 volume part, 6 L
Methanol, 6L
Among these, fraction MTB-4 (23.1 g) of methanol: water = 2: 3 → 3: 2 elution fraction was separated from MTB-4-1 by silica gel column chromatography (column size: inner diameter 5 cm × length 50 cm). MTB-4-6 was obtained. In the same purification, a fraction was obtained under the condition of 0.5 L per fraction using the developing solvent shown below.

Chloroform: methanol = 100 parts by volume: 1 part by volume, 6 L
Chloroform: methanol = 25 parts by volume: 1 part by volume, 3 L
Chloroform: methanol = 10 parts by volume: 1 part by volume, 3 L
Chloroform: methanol = 2 volume parts: 1 volume part, 3 L
Chloroform: methanol = 1 volume part: 1 volume part, 3 L
7.66 g of chloroform: methanol = 2: 1 elution fraction MTB-4-5 obtained from silica gel column chromatography was divided into 4 portions and subjected to reverse phase silica gel column chromatography (column size: inner diameter 5 cm × length). 20 cm). In the same purification, MTB4-5-3 which is fraction (126) to fraction (144) under the condition of 10 g of one fraction using a linear gradient solvent of the solvent shown below as a developing solvent. 2.47 g was obtained.

Water: methanol = 90 parts by volume: 10 parts by volume (1 L)
→ Water: Methanol = 50 parts by volume: 50 parts by volume (1 L)
→ water: methanol = 50 parts by volume: 50 parts by volume (0.6 L)
→ water: methanol = 20 parts by volume: 80 parts by volume (0.6 L)
Next, 2.47 g of MTB4-5-3 was purified by droplet countercurrent distribution chromatography (trade name: EYELA DCC-3000, Tokyo Rika Kikai Co., Ltd.). In the same purification, the solvent was eluted by the droplet ascending method using the lower layer of chloroform: methanol: water: 1-propanol = 9: 12: 8: 2 as the stationary phase and the upper layer as the mobile phase. The eluate obtained 154 g of MTB4-5-3-5 as fraction (86) to fraction (15) with 5 g as one fraction. Subsequently, 154 g of MTB4-5-3-5 was purified by high performance liquid column chromatography. In the same purification, a mixed solvent of acetonitrile and water (acetonitrile: water = 20 volume parts: 80 volume parts) was used as the moving bed, and the specific refractive index (RI) peak appearing at a retention time of 17 minutes was confirmed. From this fraction, 2.9 mg of an organic compound represented by the chemical formula (3) was obtained.

  On the other hand, 3.54 g of chloroform: methanol = 1: 1 elution fraction MTB-4-6 obtained from silica gel column chromatography was divided into two portions and subjected to reverse phase silica gel column chromatography (column size: inner diameter 5 cm × long). 20 cm). In the same purification, MTB4-6-2 which is fraction (51) to fraction (105) under the condition of 10 g of one fraction using a linear gradient solvent shown below as a developing solvent. 306 mg was obtained.

Water: methanol = 90 parts by volume: 10 parts by volume (1 L)
→ Water: Methanol = 50 parts by volume: 50 parts by volume (1 L)
→ Water: Methanol = 50 parts by volume: 50 parts by volume (1 L)
→ Water: Methanol = 30 parts by volume: 70 parts by volume (1 L)
Next, 306 mg of MTB4-6-2 was purified by droplet countercurrent distribution chromatography. In the same purification, the solvent was eluted by the droplet ascending method using the lower layer of chloroform: methanol: water: 1-propanol = 9: 12: 8: 2 as the stationary phase and the upper layer as the mobile phase. The eluate obtained 97.6 mg of MTB4-6-2-2, which was fraction (43) to fraction (60), with 5 g as one fraction. Subsequently, 97.6 mg of MTB4-6-2-2 was purified by high performance liquid column chromatography. In the same purification, a mixed solvent of acetonitrile and water (acetonitrile: water = 20 volume parts: 80 volume parts) was used as the moving bed, and the specific refractive index (RI) peak appearing at a retention time of 24 minutes was confirmed. From this fraction, 6.6 mg of an organic compound represented by the chemical formula (4) was obtained.

<Identification of organic compound represented by chemical formula (1A)>
The organic compound represented by the formula (1A), from the results of high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), was determined with the molecular formula _C 30 _H 36 _{O 6.}

In the data of ¹ HNMR spectrum shown in Table 1, δ1.34, 1.60, 1.69, 1.75, and 1.81 each have 5 methyl hydrogen signals of 5H, δ6.82 (d, J = 8Hz), 6.94 (d, J = 8Hz), two hydrogen signals on the aromatic ring ortho-coupled, and δ12.4, the hydrogen of the hydroxyl group shifted greatly to a low magnetic field due to intramolecular hydrogen bonding. One signal was observed.

In the ¹³ C NMR spectrum data shown in Table 1, out of the total number of 30 carbons, there are 2 signals of sp ³ carbon in which δ75.9 and 76.9 are presumed to be bonded with oxygen atoms, and δ196.6 is carbonyl. By observing one carbon signal, it was speculated that this organic compound had a flavanone skeleton and had one prenyl group and one geranyl group.

更に、ＨＭＢＣスペクトルにより、５位の水酸基（δ１２．４（ｓ））と６位の炭素（δ１０７．０）とに相関が見られ、１´´´位の水素（δ３．３５（ｔ，Ｊ＝７Ｈｚ））は、５位の炭素（δ１６１．３）及び７位の炭素（δ１６３．７）に相関が見られたことから、フラバノン骨格のＡ環の６位にプレニル基が結合していることが明らかとなった。また、１´´位の水素（δ２．８０（ｍ），δ２．７０（ｍ））は、１´位の炭素（δ１２７．２）及び３´位の炭素（δ１４１．０）に相関が見られた。このことから、Ｂ環の２´位にゲラニル基が結合しており、分子式から得られた不飽和度を換算してその３´´´位の炭素と７位の炭素がエーテル結合により閉環していることが明らかとなった。

Further, from the HMBC spectrum, there is a correlation between the hydroxyl group at the 5-position (δ12.4 (s)) and the carbon at the 6-position (δ107.0), and hydrogen at the 1 ′ ″ position (δ3.35 (t, J = 7 Hz)), a correlation was observed between the carbon at the 5-position (δ161.3) and the carbon at the 7-position (δ163.7), so the prenyl group was bonded to the 6-position of the A ring of the flavanone skeleton. It became clear. In addition, the 1′-position hydrogen (δ 2.80 (m), δ 2.70 (m)) is correlated with the 1′-position carbon (δ127.2) and the 3′-position carbon (δ141.0). It was. From this, a geranyl group is bonded to the 2 ′ position of the B ring, and the 3 ′ ″ carbon and the 7th carbon are closed by an ether bond in terms of the degree of unsaturation obtained from the molecular formula. It became clear that.

As described above, the planar structure of the organic compound was identified.
The determination method of stereochemistry is, for example, literature: Cichewicz, RH, Kenyon, VA and Whitman, S., J. Am. Chem. Soc., 126, 14910-14920, (2004), literature: Kikuchi, T., Mori. , Y., Yokoi, T., Nakazawa, S., Kuroda, H., Masada, Y., Kitamura, K. and Kuriyama, K., Chem. Pharm. Bull., 31, 106-113, (1983) Etc. were referenced. As a result, the absolute configuration at the 3 ″ position was determined as 3 ″ R.

Next, physical properties of the organic compound represented by the chemical formula (1A) will be described below.
Property: Yellow amorphous powder Optical rotation: [α] D ³⁰ + 75.9 ° (c = 1.40, CHCl ₃ )
Infrared absorption spectrum: ν max (film) cm ⁻¹ : 3367, 1634, 1601, 1449, 1152
Circular dichroism spectrum: Δε (nm): +25.2 (218), -2.3 (293), +2.0 (330)
(C = 2.13 × 10 ⁻⁵ M, MeOH)
Ultraviolet absorption spectrum: λ max (MeOH) nm (log ε): 220 (4.11), 234 (4.15), 289 (4.11), 335 sh (3.63)
HR-ESI-MS (positive) m / z: 515.2416 [M + Na] ⁺
_{(Calcd for C 30 H 34 O} 6 Na: 515.2424)
<Identification of organic compound represented by chemical formula (1B)>
The identification of the organic compound represented by the chemical formula (1B) was performed in the same manner as the method described in the section <Identification of the organic compound represented by the chemical formula (1A)>.

Table 2 shows ¹ HNMR spectrum and ¹³ CNMR spectrum data of the organic compound represented by the chemical formula (1B).

３´´位の絶対立体配置については、上述した文献を参考にして、３´´Ｓと決定された。

The absolute configuration at the 3 ″ position was determined as 3 ″ S with reference to the above-mentioned literature.

Next, physical properties of the organic compound represented by the chemical formula (1B) will be described below.
Property: Yellow amorphous powder Optical rotation: [α] D ²⁵ + 25.0 ° (c = 1.25, CHCl ₃ )
Infrared absorption spectrum: ν max (film) cm ⁻¹ : 3367, 1637, 1603, 1451, 1155
Circular dichroism spectrum: Δε (nm): +9.2 (226), -3.1 (292), +1.3 (335)
(C = 3.74 × 10 ⁻⁵ M, MeOH)
Ultraviolet absorption spectrum: λ max (MeOH) nm (log ε): 212 (4.46), 230 sh (4.32), 290 (4.18), 335 sh (3.54)
HR-ESI-MS (positive) m / z: 515.2424 [M + Na] ⁺
_{(Calcd for C 30 H 34 O} 6 Na: 515.2404)
<Identification of organic compound represented by chemical formula (2A)>
The organic compound represented by the formula (2A), from the results of HR-ESI-MS, was determined with the molecular formula _C 30 _H 34 _{O 6.}

In the data of ¹ HNMR spectrum shown in Table 3, ^one methyl hydrogen signal for 6H at δ1.75, and three methylhydrogen signals for 3H at δ1.48, 1.69, and 1.81, respectively. , Δ6.84 (d, J = 8 Hz), 6.94 (d, J = 8 Hz), two hydrogen signals on the aromatic ring ortho-coupled, δ5.59 (d, J = 15 Hz), 6 .34 (dd, J = 15, 11 Hz), the trans double bond signal is δ 5.78 (d, J = 11 Hz), 6.34 (dd, J = 15, 11 Hz), C = CH-CH = There was one C hydrogen signal and a hydroxyl group hydrogen signal that was greatly shifted to a low magnetic field due to intramolecular hydrogen bonding at δ12.4.

In the data of ¹³ CNMR spectrum shown in Table 3, two signals of sp ³ carbon in which δ76.1 and 77.2 are assumed to be bonded with oxygen atoms out of 30 carbon atoms and carbonyl carbon at δ196.5 It was speculated that this organic compound had a flavanone skeleton and one prenyl group and one geranyl group.

更に、ＨＭＢＣスペクトルにより、５位の水酸基（δ１２．４（ｓ））と６位の炭素（δ１０７．０）に相関が見られ、１´´´位の水素［δ３．３５（ｔ，Ｊ＝７Ｈｚ））と５位の炭素（δ１６１．３）及び７位の炭素（δ１６３．７）に相関が見られた。このことから、フラバノン骨格のＡ環の６位にプレニル基が結合していることが明らかとなり、また１´´位の水素（δ２．７７（ｍ），δ２．７３（ｍ））と１´位の炭素（δ１２７．３）及び３´位の炭素（δ１４１．１）に相関が見られた。このことから、Ｂ環の２´位にゲラニル基が結合しており、分子式から得られた不飽和度を勘案してその３´´´位の炭素と７位の炭素がエーテル結合により閉環していることが明らかとなった。

Further, the HMBC spectrum shows a correlation between the hydroxyl group at the 5-position (δ12.4 (s)) and the carbon at the 6-position (δ107.0), and hydrogen at the 1 ′ ″ position [δ3.35 (t, J = 7 Hz)) and carbon at the 5th position (δ161.3) and carbon at the 7th position (δ163.7). This reveals that a prenyl group is bonded to the 6-position of the A ring of the flavanone skeleton, and hydrogen (δ 2.77 (m), δ 2.73 (m)) and 1 ′ in the 1 ″ position. Correlation was found between the carbon at the position (δ127.3) and the carbon at the 3 ′ position (δ141.1). From this, a geranyl group is bonded to the 2 ′ position of the B ring, and the carbon at the 3 ′ ″ position and the 7th position are closed by an ether bond in consideration of the degree of unsaturation obtained from the molecular formula It became clear that.

  With respect to the absolute configuration at the 2-position in the organic compound, a CD spectrum was measured and determined to be 2S in the same manner as described in the section <Organic compound represented by chemical formula (1A)>.

The absolute configuration at the 3 ″ position in the organic compound was determined as 3 ″ R with reference to the above-mentioned literature.
Next, physical properties of the organic compound represented by the chemical formula (2A) are shown below.

Property: Yellow amorphous powder Optical rotation: [α] D ³⁰ -9.53 ° (c = 0.485, CHCl ₃ )
Infrared absorption spectrum: ν max (film) cm ⁻¹ : 3434, 1634, 1451, 1153
Circular dichroism spectrum: Δε (nm): −6.8 (239), +0.39 (262), −5.4 (293), +0.86 (323)
(C = 2.86 × 10 ⁻⁵ M, MeOH)
Ultraviolet absorption spectrum: λ max (MeOH) nm (log ε): 237 (4.38), 275 sh (4.02), 289 (4.16), 334 sh (3.54)
HR-ESI-MS (positive) m / z: 491.2424 [M + H] ⁺
_{(Calcd for C 30 H 35 O} 6: 491.2428), 513.2235 [M + Na] + (calcd for C 30 H 34 O 6Na: 513.2247)
<Identification of organic compound represented by chemical formula (2B)>
The identification of the organic compound represented by the chemical formula (2B) was performed in the same manner as the method described in the section <Identification of the organic compound represented by the chemical formula (2A)>.

Table 4 shows ¹ HNMR spectrum and ¹³ CNMR spectrum data of the organic compound represented by the chemical formula (2B).

３´´位の絶対立体配置については、上述した文献を参考にして、３´´Ｓと決定された。

The absolute configuration at the 3 ″ position was determined as 3 ″ S with reference to the above-mentioned literature.

Next, physical properties of the organic compound represented by the chemical formula (2B) will be described below.
Property: Yellow amorphous powder Optical rotation: [α] D ³⁰ + 75.6 ° (c = 0.418, CHCl ₃ )
Infrared absorption spectrum: ν max (film) cm ⁻¹ : 3396, 1632, 1602, 1452, 1153
Circular dichroism spectrum: Δε (nm): +19.1 (232), +1.5 (268), -2.3 (295), +1.3 (331)
(C = 2.47 × 10 ⁻⁵ M, MeOH)
Ultraviolet absorption spectrum: λ max (MeOH) nm (log ε): 237 (4.38), 275 sh (4.01), 292 (4.15), 338 sh (3.51)
HR-ESI-MS (positive) m / z: 491.2423 [M + H] ⁺
_{(Calcd for C 30 H 35 O} 6: 491.2428), 513.2241 [M + Na] + (calcd for C 30 H 34 O 6 Na: 513.2247)
<Identification of organic compound represented by chemical formula (3)>
The organic compound represented by the formula (3), from the results of HR-ESI-MS, was determined with the molecular formula _C 26 _H 34 _{O 11.}

In the data of ¹ HNMR and ¹³ CNMR spectra shown in Table 5, aromatic ring hydrogen of δH 7.10 (2H, s), ester carbonyl carbon of δC168.3, and two sp ² carbons in δC110.4 (d) Was observed, suggesting the existence of a galloyl group. In addition to anomeric hydrogen of δH 4.37 (1H, d, J = 8 Hz) and anomeric carbon of δC 103.3 (d), δC 75.3 (d), δC 78.1 (d), δC 72.1 (d) , ΔC75.6 (d), and δC65.2 (t) signals suggested the presence of glucose esterified at the 6-position. For the remaining 13 carbon signals, δC202.2 (s) suggests the presence of a ketone group, and four sp ² carbons (δC166.0 (s), δC138.0 (d), δC129.5 ( d), δC126.1 (d)) suggests that there are two double bonds. In addition, two double bonds are disubstituted from δH5.69 (1H, ddd, J = 15, 7, ¹ Hz) and δH5.53 (1H, ddd, J = 15, 9, ¹ Hz) in ¹ HNMR. From the trans double bond and δH 5.79 (1H, brs), it was deduced to be a trisubstituted double bond. In addition, there are four methyl carbons (δC27.9 (q), δC27.4 (q), δC23.7 (q), δC21.6 (q)), and δH1. 82 (3H, d, J = 1 Hz), doublet methyl δH1.28 (1H, d, J = 6 Hz), and two singlet methyls (δH0.87 (3H, s), δH0.86 (3H, s )) Observed. The remaining carbon is one quaternary carbon (δC37.0 (s), two methine carbons (δC56.3 (d), δC78.6 (d)), and one methylene carbon (δC49.1 ( t)), the signal of δC78.6 (d) was considered to be oxygen bound, and by taking into account the above-mentioned NMR data and the deficient hydrogen index (unsaturation degree) derived from the molecular formula, Presumed to have a Stigman skeleton.

更に、ＨＭＱＣ、及びＨＭＢＣスペクトルを測定したところ、グルコースの６位の水素（δＨ４．５７（１Ｈ，ｄｄ，Ｊ＝１２，２Ｈｚ），δＨ４．３０（１Ｈ，ｄｄ，Ｊ＝１２，７Ｈｚ））とガロイル基のカルボニル炭素（δＣ１６８．３（ｓ））との間に相関ピークが観測されたことからガロイル基がグルコースの６位にエステル結合していることが明らかになった。またグルコースの１位の水素（δＨ４．３７（１Ｈ，ｄ，Ｊ＝８Ｈｚ））とアグリコン部の９位の炭素δＣ７８．６（ｄ）に相関が見られたことから、グルコースの結合位置を９位と決定した。

Furthermore, when HMQC and HMBC spectra were measured, hydrogen at the 6-position of glucose (δH4.57 (1H, dd, J = 12, 2 Hz), δH 4.30 (1H, dd, J = 12, 7 Hz)) and A correlation peak was observed between the carbonyl carbon of the galloyl group (δC168.3 (s)), and it was revealed that the galloyl group was ester-bonded to the 6-position of glucose. Further, since a correlation was found between hydrogen at position 1 of glucose (δH4.37 (1H, d, J = 8 Hz)) and carbon at position 9 δC78.6 (d) of the aglycone part, the binding position of glucose was determined to be 9 It was decided to rank.

  When the DQF-COSY spectrum was measured, the correlation between the 3 ′ and 4 ′ positions was unclear due to the diagonal peak, but the 1 ′ to 3 ′ and 4 ′ to 6 ′ positions of glucose, and the aglycone Spin coupling from the 6th to 10th positions was observed. In addition, observation of HMBC revealed that the planar structure of the portion excluding the galloyl group was 3-oxo-α-ionollucosides. The 9-dimensional structure was determined to be 9R by δC78.6. Further, when the CD spectrum of the tertiary structure at the 6-position was measured, it was found to be 6R because it showed a value of +32.3 (245).

  As a result, this organic compound was determined to be (6R, 9R) -3-oxo-α-ionol 9-O- (6-O-galloyl) -β-glucopyranoside.

Next, physical properties of the organic compound represented by the chemical formula (3) are shown below.
Property: Amorphous powder Optical rotation: [α] D ²⁵ + 78.4 ° (c = 0.193, CH ₃ OH)
Infrared absorption spectrum: ν max (film) cm ⁻¹ : 3303, 1706, 1648, 1449, 1259, 1031
Ultraviolet absorption spectrum: λ max (MeOH) nm (log ε): 219 (4.8), 275 (4.4)
Circular dichroism spectrum: Δε (nm): +32.3 (245), −1.56 (317)
(C = 1.06 × 10 ⁻⁵ M, MeOH)
HR-ESI-MS (positive-ion mode) m / z: 545.1990 [M + Na] ⁺ (calcd for C ₂₆ H ₃₄ O ₁₁ Na: 545.1993)
<Identification of organic compound represented by chemical formula (4)>
The organic compound represented by the formula (4), from the results of HR-ESI-MS, was determined with the molecular formula _C 33 _H 36 _{O 15.}

In the data of ¹ HNMR and ¹³ CNMR spectra shown in Table 6, aromatic ring hydrogen of δH 7.10 (2H, s), ester carbonyl carbon of δC168.2, and two sp ² carbons in δC110.5 (d) Was observed, suggesting the presence of a galloyl group. In addition to anomeric hydrogen of δH4.83 (1H, d, J = 8 Hz) and anomeric carbon of δC102.7 (d), δC75.0 (d), δC78.0 (d), δC72.2 (d) , ΔC75.8 (d), and δC65.0 (t) signals suggested the presence of glucose esterified at the 6-position.

For the remaining 18 carbon signals, the sp ² carbon (δC150.7 (s), δC149.2 (s), δC147.5 (s), δC147.3 (s), δC137.6 (s), δC133.9 (s), δC120.3 (d), δC119.8 (d), δC118.1 (d), δC116.2 (d), δC111.9 (d), and δC111.3 (d)) Since 12 were observed, there were 2 aromatic rings, of which 4 carbons (δC150.7 (s), δC149.2 (s), δC147.5 (s), δC147.3 (s)) Was thought to be oxygen bound. Also, ¹ HNMR coupling pattern (δH6.96 (1H, d, J = 2 Hz), δH7.02 (1H, d, J = 8 Hz), δH 6.61 (1H, dd, J = 8) in the aromatic ring region. , 2 Hz)), and (δH6.98 (1H, d, J = 2 Hz), δH6.78 (1H, d, J = 8 Hz), δH6.83 (1H, dd, J = 8, 2 Hz)) to 1 , 3,4-substituted benzene was suggested to exist. In addition, oxygen is bonded to these carbons from signals of δC87.7 (d), δC87.1 (d), δC73.0 (t), and δC72.6 (t), and deficient hydrogen derived from the molecular formula By taking into account the exponent (unsaturation) and the remaining methine carbon shifted to a relatively low magnetic field of δC55.5 (d) and δC55.4 (d), the aglycon moiety has a sesamin-type lignan skeleton. I guessed that.

更に、ＨＭＱＣおよびＨＭＢＣスペクトルを測定したところ、グルコースの６位の水素（δＨ４．５６（１Ｈ，ｄｄ，Ｊ＝１２，２Ｈｚ），δＨ４．４４（１Ｈ，ｄｄ，Ｊ＝１２，８Ｈｚ））とガロイル基のカルボニル炭素（δＣ１６８．２（ｓ））との間に相関ピークが観測されたことからガロイル基がグルコースの６位にエステル結合していることが明らかになった。またグルコースの１位の水素（δＨ４．８３（１Ｈ，ｄ，Ｊ＝８Ｈｚ））とアグリコン部の４位の炭素（δＣ１４７．５（ｓ）］に相関が見られたこと、及びＰＮＯＥＳＹスペクトルでグルコースの１位の水素（δＨ４．８３（１Ｈ，ｄ，Ｊ＝８Ｈｚ））とアグリコン部の５位水素（δＨ７．０２（１Ｈ，ｄ，Ｊ＝８Ｈｚ））との間にＮＯＥ相関が観測されたことから、グルコースの結合位置を４位と決定した。更に、ＯＭｅの結合位置については、δＨ３．８４（３Ｈ，ｓ）とδＣ１５０．７（ｓ）に相関が見られるとともに、δＨ３．８７（３Ｈ，ｓ）とδＣ１４９．２（ｓ）に相関が見られたことから、それぞれアグリコン部の３位、３´位に結合していることが明らかになった。その他の部分についてもＨＭＢＣ相関の観測により、この有機化合物の平面構造を決定した。この有機化合物のアグリコン部はｐｉｎｏｒｅｓｉｎｏｌである。

Further, when HMQC and HMBC spectra were measured, hydrogen at the 6-position of glucose (δH4.56 (1H, dd, J = 12, 2 Hz), δH 4.44 (1H, dd, J = 12, 8 Hz)) and galloyl A correlation peak was observed with the carbonyl carbon of the group (δC168.2 (s)), which revealed that the galloyl group was ester-bonded to the 6-position of glucose. In addition, a correlation was found between hydrogen at position 1 of glucose (δH4.83 (1H, d, J = 8 Hz)) and carbon at position 4 of the aglycone (δC147.5 (s)), and glucose in the PNOESY spectrum. NOE correlation was observed between the 1-position hydrogen (δH 4.83 (1H, d, J = 8 Hz)) and 5-position hydrogen (δH 7.02 (1H, d, J = 8 Hz)) Therefore, the binding position of glucose was determined to be position 4. Furthermore, regarding the binding position of OMe, δH3.84 (3H, s) and δC150.7 (s) were correlated, and δH3.87 (3H , S) and δC149.2 (s) were found to be linked to the 3rd and 3 ′ positions of the aglycon part, respectively. Due to this existence The planar structure of the compound was determined. Aglycone portion of the organic compound is Pinoresinol.

  When the CD spectrum was measured to determine the absolute configuration, the values of −0.23 (235) and +1.7 (280) were observed, and it was clear that (+)-pinoresinol was the aglycon. became. As a result, this organic compound was determined to be (+)-pinoresinol 4-O- (6-O-galloyl) -β-glucopyranoside.

Next, physical properties of the organic compound represented by the chemical formula (4) are shown below.
Property: Amorphous powder Optical rotation: [α] D ²⁵ + 18.1 ° (c = 0.440, CH ₃ OH)
Infrared absorption spectrum: ν max (film) cm ⁻¹ : 3367, 1702, 1606, 1514, 1266, 1226
Ultraviolet absorption spectrum: λ max (MeOH) nm (log ε): 276 (4.1), 218 (4.4), 207 (4.6)
Circular dichroism spectrum: Δε (nm): +1.7 (280), +1.8 (256), −0.23 (235), +1.28 (223)
(C = 1.18 × 10 ⁻⁵ M, MeOH)
HR-ESI-MS (positive-ion mode) m / z: 695.1949 [M + Na] ⁺ (calcd for C ₃₃ H ₃₆ O ₁₅ Na: 695.1946)
<Activity test of organic compounds represented by chemical formulas (1) and (2)>
(I) Antitumor activity MTT analysis was performed using KB cells (human nasopharyngeal carcinoma cells) and A549 cells (human lung cancer cells) to examine the growth inhibitory activity against cancer cells. A 96-well plate was used for culture and measurement, and was performed in triplicate.

The medium is DMEM (Dulbecco's Modified Eagle's Medium) 10% FCS (Fetal Calf Serum) containing fetal bovine serum, and kanmycin sulfate, amphotericin B (sigma A9528) 2000-fold solution containing 2000 ml of antibiotics. It was used. Antibiotics were 100 mg γ-irradiated amphotericin B aseptically dissolved in 20 ml sterile MilliQ water, dispensed 1 ml at a time into sterile 1.5 ml tubes and stored frozen at -20 ° C. Aseptic mixture of 1 g (potency) of 4 kanamycin sulfate with ampule for injection and 1 ml of the above-mentioned amphotericin B soln to make a 2000 times solution of antibiotics. FCS was deactivated by treating at 56 ° C. for 30 minutes, dispensed into a 50 ml tube and stored frozen at −20 ° C. The cells were cultured using an incubator at 37 ° C. and 5% CO ₂ .

The MTT reagent was dissolved in PBS and prepared to 5 mg / ml. At the time of use, a 10-fold diluted solution (0.5 mg / ml) was used.
After the organic compound was dried, it was weighed into a 1.5 ml tube, dissolved in DMSO and adjusted to 5 mM, and a further serially diluted sample.

MTT analysis was performed by triplicate. KB cells were seeded on a 96-well plate at 3 × 10 ³ cells / well (90 μl medium) and cultured at 37 ° C. for 24 hours. After culturing, 10 μl of each sample and control diluted 10-fold with the medium was added to each well and cultured at 37 ° C. for 72 hours. Thereafter, the medium was sucked out with an aspirator, 100 μl of MTT solution diluted 10-fold with the medium was added to each well, and cultured at 37 ° C. for 1.5 hours. The medium was discarded with the plate inverted, and 100 μl of DMSO was added to lyse the cells, followed by colorimetric quantification with a spectroscopic microplate reader (λ1 = 540 nm, λ2 = 620 nm, λ1-λ2).

The results of the antitumor activity test are shown in Table 7.
Table 7 shows values obtained by subtracting the survival rate (%), assuming that the control is 100%. Viability (%) is the percentage of each sample relative to the control.

表７に示される結果から明らかなように、ＫＢ細胞及びＡ５４９細胞のいずれについても、化学式（１Ａ）に示される有機化合物が最も強い活性を有することがわかった。

As is clear from the results shown in Table 7, it was found that the organic compound represented by the chemical formula (1A) had the strongest activity for both KB cells and A549 cells.

(Ii) Antioxidant activity DPPH (α, α-diphenyl-β-picrylhydradil) is a purple stable radical having a maximum absorption at 517 nm, and turns into colorless hydrazine by obtaining hydrogen. Radical scavenging activity was measured using this color reaction. First, the organic compound was dissolved in ethanol to prepare 0.5 ml of a sample solution. Subsequently, 0.5 ml of a 0.15 mM DPPH solution (the solvent was ethanol) was added to each sample solution, stirred, and reacted for 1 hour in the dark, and then the absorbance at 517 nm was measured. On the other hand, α-tocopherol was evaluated as a comparative control. Table 8 shows “±” when almost no activity was confirmed, “+” when activity was weak but weak, and “++” when activity was sufficiently confirmed, When strong activity is confirmed, it is indicated by “++++”.

（ｉｉｉ）抗菌活性
グラム陰性菌の指標菌として下記表９に示される菌株について抗菌活性を評価した。上記有機化合物を７０％エタノールに溶解させた後、該試料濃度が０ｐｐｍ、５ｐｐｍ、１０ｐｐｍ、１５ｐｐｍ、２０ｐｐｍ又は５０ｐｐｍになるように調製した標準寒天培地を作製した。続いて、これらの標準寒天培地をオートクレーブ殺菌することにより評価培地を作製し、これらの評価培地に上記各菌を接種してその生育を確認した。表９には、活性がほとんど確認されなかった場合は“±”で示し、活性が確認されたが弱い場合は“＋”で示し、活性が十分に確認された場合は“＋＋”で示し、強い活性が確認された場合は“＋＋＋”で示している。

(Iii) Antibacterial activity Antibacterial activity was evaluated for the strains shown in Table 9 below as indicator bacteria for Gram-negative bacteria. After the organic compound was dissolved in 70% ethanol, a standard agar medium prepared so that the sample concentration was 0 ppm, 5 ppm, 10 ppm, 15 ppm, 20 ppm or 50 ppm was prepared. Subsequently, these standard agar media were sterilized by autoclaving to prepare evaluation media, and each of the above-described bacteria was inoculated into these evaluation media to confirm their growth. Table 9 shows “±” when almost no activity was confirmed, “+” when activity was confirmed but weak, and “++” when activity was sufficiently confirmed, When strong activity is confirmed, it is indicated by “++++”.

＜化学式（３）及び（４）に示される有機化合物の活性試験＞
（ｉｖ）抗腫瘍活性
上記（ｉ）抗腫瘍活性に示される方法と同様にして抗腫瘍活性を評価した場合において、得られる結果を推定した。表１０には、活性がほとんど確認されない場合は“±”で示し、活性が確認されるが弱い場合は“＋”で示し、活性が十分に確認される場合は“＋＋”で示し、強い活性が確認される場合は“＋＋＋”で示している。

<Activity test of organic compounds represented by chemical formulas (3) and (4)>
(Iv) Antitumor activity When the antitumor activity was evaluated in the same manner as in the above (i) antitumor activity, the results obtained were estimated. Table 10 shows “±” when almost no activity is confirmed, “+” when activity is weak but weak, and “++” when activity is sufficiently confirmed, indicating strong activity Is confirmed by “++++”.

（ｖ）抗酸化活性
上記（ｉｉ）抗酸化活性に示される方法と同様にして抗酸化活性を評価した場合において、得られる結果を推定した。表１１には、活性がほとんど確認されない場合は“±”で示し、活性が確認されるが弱い場合は“＋”で示し、活性が十分に確認される場合は“＋＋”で示し、強い活性が確認される場合は“＋＋＋”で示している。

(V) Antioxidant activity When the antioxidant activity was evaluated in the same manner as in the method shown in (ii) Antioxidant activity, the results obtained were estimated. Table 11 shows “±” when almost no activity is confirmed, “+” when activity is weak but weak, and “++” when activity is sufficiently confirmed, indicating strong activity Is confirmed by “++++”.

Claims (13)

An organic compound represented by the following chemical formula (1):

An antitumor agent comprising the organic compound according to claim 1 as an active ingredient. An antioxidant containing the organic compound according to claim 1 as an active ingredient. An antibacterial agent comprising the organic compound according to claim 1 as an active ingredient. An antitumor agent comprising an organic compound represented by the following chemical formula (2) as an active ingredient.

An antioxidant comprising an organic compound represented by the following chemical formula (2) as an active ingredient.

An antibacterial agent comprising an organic compound represented by the following chemical formula (2) as an active ingredient.

An organic compound represented by the following chemical formula (3):

An antitumor agent comprising the organic compound according to claim 8 as an active ingredient. An antioxidant containing the organic compound according to claim 8 as an active ingredient. An organic compound represented by the following chemical formula (4):

However, Me shown by Chemical formula (4) shows a methyl group. An antitumor agent comprising the organic compound according to claim 11 as an active ingredient. The antioxidant containing the organic compound of Claim 11 as an active ingredient.