JP2008000081A - New cinnamic acid derivative, method for producing the same and fermented product of propolis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new cinnamic acid derivative having a good anticancer activity, a method for producing the same and a fermented product of propolis. <P>SOLUTION: This new cinnamic acid derivative is expressed by formula (1). The method for producing the new cinnamic acid derivative is provided by inoculating the mycelium of a Basidiomycota on propolis, fermentation-treating the propolis by the mycelium and then separating the new cinnamic acid derivative expressed by the formula (1). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a novel cinnamic acid derivative contained in a fermented product obtained by fermenting propolis or Artepillin C with basidiomycetous mycelium, for example, and a propolis fermented product.

  Propolis is a glue-like substance formed by mixing sap of trees collected by bees, plant shoots, exudates, etc. with beeswax, etc. Bees repair the nest with propolis. Propolis has been used for a long time as a folk medicine in the form of topical use or internal use in a wide range of applications such as natural antibacterial components, anti-inflammatory agents, and analgesics. In recent years, research to elucidate the various physiological actions and the like has progressed, and it has been found that various effects can also be exhibited by oral ingestion. For this reason, for example, antibacterial, antioxidant, antitumor, carcinogenesis suppression, immunostimulatory effects, etc., as well as prevention, improvement and treatment effects on lifestyle-related diseases and other diseases, raw materials for health foods and functional foods It has been attracting attention as a practical application. However, propolis is a mixture of various substances such as sap and resin of trees, various medicinal plants that are origin plants, pollen, beeswax, etc., and the substances contained in propolis have not been fully elucidated. It is thought that there are many substances that have not been elucidated or identified.

  Even if it is called propolis, the content varies depending on the production area. In recent years, many researchers have been studying the components of propolis by production area, and it has been disclosed that artepilin C, one of cinnamic acid derivatives, is detected as a major component in Brazilian propolis (for example, , See Non-Patent Document 1).

  So far, a propolis composition containing blended propolis of different origins and containing artepilin C as one of the components has been disclosed (for example, see Patent Document 1). For example, a propolis composition containing quercetin, p-coumaric acid and artepiline C and further containing chrysin, galangin or caffeic acid phenethyl ester is mentioned.

Propolis and Agaricus mycelium are both known to have anticancer and antibacterial effects. For example, until now, using a solvent that dissolves Agaricus, propolis and Amazon enzyme, each extract is extracted separately or in combination, and one, two or three of them are mixed and dissolved, A food and drink product that can be stored by adding to the product is disclosed (see, for example, Patent Document 2).
JP 2004-159563 A (first and second pages) Japanese Patent Laid-Open No. 10-215838 (pages 1 and 2) Shigeru Tazawa et al .: Biopharmaceutical magazine, 54 (6), 164-168, 2000

    In recent years, a diet centered on vegetables has become a diet centered on meat, and the dietary habits have become westernized and the number of cancer patients has increased. So far, it has been known that mycelium of basidiomycetes such as propolis and agaricus each have an anticancer effect. However, further improvement of such anticancer activity is demanded, and in order to meet such a demand, fermented foods by combining propolis and agaricus have been developed, and search for new compounds caused by changes in components due to fermentation has been continued. Yes.

  Then, the place made into the objective of this invention is providing the novel cinnamic acid derivative which can exhibit a favorable anticancer action, its manufacturing method, and a propolis fermented material.

  As a result of diligent research, the present inventors have conducted a novel cinnamic acid derivative having a good anticancer effect by subjecting propolis or artepilin C to fermentation treatment using mycelium of a basidiomycete such as Agaricus and then separating it. It was found that can be obtained.

  In order to achieve the above object, the novel cinnamic acid derivative of the invention described in claim 1 is a compound having a structure of the following formula (1).

請求項２に記載の発明は、請求項１に記載の新規な桂皮酸誘導体を製造する方法であって、担子菌類の菌糸体をプロポリスに接種し、前記菌糸体によりプロポリスを発酵処理した後、式（１）で示される新規な桂皮酸誘導体を分離することを特徴とするものである。

The invention according to claim 2 is a method for producing the novel cinnamic acid derivative according to claim 1, wherein the mycelium of basidiomycetes is inoculated into propolis, and the propolis is fermented with the mycelium, A novel cinnamic acid derivative represented by the formula (1) is isolated.

  The invention according to claim 3 is a method for producing the novel cinnamic acid derivative according to claim 1, wherein the mycelium of basidiomycetes is inoculated into artepillin C, and artepilin C is fermented by the mycelium. Thereafter, the novel cinnamic acid derivative represented by the formula (1) is separated.

  The propolis fermented product of the invention described in claim 4 is obtained by fermenting propolis with a mycelium of a basidiomycete and containing a novel cinnamic acid derivative represented by the formula (1) according to claim 1. It is a feature.

According to the present invention, the following effects can be exhibited.
According to the novel cinnamic acid derivative of the invention described in claim 1, the compound of the formula (1) has the basic skeleton of the cinnamic acid derivative and can exhibit a good anticancer activity.

  According to the method for producing the novel cinnamic acid derivative of the invention according to claims 2 and 3, propolis or artepilin C is fermented using mycelium of basidiomycetes. Thereafter, the novel cinnamic acid derivative represented by the formula (1) can be easily provided by a simple operation of separating the novel cinnamic acid derivative.

  According to the propolis fermented product of the invention described in claim 4, in addition to the anticancer component originally possessed by the mycelium of propolis and basidiomycetes, a novel cinnamic acid having a good anticancer activity Contains derivatives. Therefore, when they work synergistically, a dramatic anticancer action can be exerted as compared with propolis, mycelium of basidiomycetes or a mixture of both.

In the following, embodiments that are considered to be the best of the present invention will be described in detail.
The novel cinnamic acid derivative of this embodiment is a compound having a structure represented by the following formula (1).

式（１）の化合物は、ＩＵＰＡＣ命名法では３−[３−ヒドロキシ−２−（１−メチルエチル）−７−（３−メチル−２−ブテニル）−２，３−ジヒドロベンゾフラン−５−イル]アクリル酸と命名でき、抗癌作用のある桂皮酸誘導体の基本骨格を有する構造的特徴がある。

The compound of formula (1) is in the IUPAC nomenclature 3- [3-hydroxy-2- (1-methylethyl) -7- (3-methyl-2-butenyl) -2,3-dihydrobenzofuran-5-yl ] It can be named acrylic acid, and has a structural feature having a basic skeleton of cinnamic acid derivative with anticancer activity.

  This novel cinnamic acid derivative is, for example, a compound obtained by separating propolis or artepilin C from a fermented product fermented with a mycelium of basidiomycetes, and has an anticancer activity. Therefore, the novel cinnamic acid derivative can be used for health foods, functional foods, pharmaceuticals and the like for the purpose of anticancer effect.

  The production method of the novel cinnamic acid derivative is not particularly limited, and may be a known chemical synthesis method or a fermentation method. Among these methods, the fermentation method is preferred because it can be easily obtained by a simple operation of separating a novel cinnamic acid derivative after fermentation treatment of propolis or artepilin C using mycelium of basidiomycetes.

  First, a method for producing a novel cinnamic acid derivative from propolis using mycelium of basidiomycetes will be described. The production method involves inoculating mycelium of basidiomycetes into propolis, fermenting propolis with the mycelium, and then separating a novel cinnamic acid derivative.

  As the basidiomycetes used in the production method of the present embodiment, any kind of agaricus having a laccase activity, cordyceps, birch, and reishi can be used. Among them, it is preferable to use agaricus because a novel cinnamic acid derivative can be obtained most efficiently due to high laccase activity. The mycelium may be collected from the fruiting body of basidiomycetes and used, or may be obtained from a depository or the like and used.

  As said propolis, the propolis extract which extracted the propolis original block by the well-known extraction method is used suitably. Propolis bulk is made by mixing the bee's own secretions and beeswax into an aggregate of gums, sap, plant pigment-based substances and perfume oils, etc. collected by honeybees from specific parts of the tree, mainly shoots and bark. It is a sticky resinous solid natural substance having a dark green or dark brown to dark brown color. The propolis bulk used to obtain the propolis extract can be from any production region such as South American countries including Brazil, Asian countries such as China and Japan, European countries, North American countries, Oceania countries, etc. . Among them, it is preferable to use a Brazilian ingot containing abundant amounts of 2 to 6% by mass of artepillin C which is a cinnamic acid derivative.

  Examples of the propolis extract include a water extract of propolis, an alcohol extract, a hydrous alcohol extract, an organic solvent extract, a supercritical extract, and a micellized extract. Among these, it is preferable to use an alcoholic extract of propolis because a component having a high anticancer activity can be easily extracted in a large amount. The alcohol used for preparing the alcohol extract is preferably a lower alcohol such as methanol, ethanol, butanol, propanol or isopropanol. Moreover, well-known organic solvents, such as acetone and hexane, can be used as an organic solvent used in order to prepare an organic solvent extract.

  In the propolis fermentation treatment of the present embodiment, it is preferable to activate the mycelium of the basidiomycete in advance by pre-culturing the mycelium of the basidiomycete before the propolis fermentation treatment. For example, activated mycelium can be prepared by pre-culturing spores of basidiomycetes before fermentation treatment.

  Further, in the propolis fermentation treatment, treatment conditions such as a medium suitable for culture of mycelium of basidiomycetes, a culture temperature, a culture period, and the like may be appropriately selected. In the fermentation treatment and pre-culture, it is preferable to select a medium capable of growing basidiomycetous mycelium more quickly. Such a medium usually contains sugar, yeast extract, peptone, salts and the like. It should be noted that the medium may contain alcohol, organic solvent, or the like used in preparing the propolis extract at a low concentration that does not inhibit mycelial culture.

  Although the time which implements a fermentation process, ie, fermentation time, is not specifically limited, Preferably it is 0.5 to 300 hours, More preferably, it is 0.5 to 150 hours, More preferably, it is 3.5 to 72 hours. When the fermentation time is less than 0.5 hour, a sufficient amount of novel cinnamic acid derivative cannot be obtained, which is not preferable. Conversely, when the fermentation time exceeds 300 hours, a sufficient amount of novel cinnamic acid derivative cannot be obtained in place of the fermentation time, resulting in poor efficiency and consequently increased production costs.

  Although the temperature which implements a fermentation process, ie, a fermentation process temperature, is not specifically limited, Preferably it is 25-60 degreeC, More preferably, it is 30-50 degreeC, More preferably, it is 35-45 degreeC. When the fermentation treatment temperature is less than 25 ° C., the enzyme activity is low and the reaction is slow, whereas when it exceeds 60 ° C., the enzyme is deactivated and the reaction is not efficient, which is not preferable.

  For example, when using an alcoholic extract having a propolis solid content of 20% by mass as propolis during the fermentation process, the concentration of the propolis solids in the fermentation process liquid is not particularly limited, but preferably 0.05 to 3% by mass, More preferably, it is 0.05-1.5 mass%, More preferably, it is 0.1-1 mass%. When the concentration of propolis solids is less than 0.05% by mass, all the ingredients in propolis may be used as a nutrient source for mycelium, and when this fermented product is used as a product, propolis in the product Since the content is lowered, it is not preferable. Conversely, if it exceeds 3% by mass, the solubility of propolis deteriorates and the alcohol concentration increases, so that the mycelium growth is poor and the laccase activity is also reduced, which is not preferable.

  The separation method is not particularly limited, and for example, after extraction with an organic solvent such as ethyl acetate, it is performed by appropriately combining various column chromatography such as gel filtration chromatography, high performance liquid chromatography, ion exchange chromatography, In that case, it is carried out according to a conventional method.

  Next, a method for producing a novel cinnamic acid derivative from artepilin C using mycelium of basidiomycetes will be described. In this production method, mycelium of basidiomycetes is inoculated into artepillin C, and after propolis is fermented with the mycelium, a novel cinnamic acid derivative is separated.

  Artepillin C used in the production method of the present embodiment is not particularly limited, and may be derived from, for example, propolis, from the asteraceae plant that is the origin of propolis, or by chemical synthesis. It may be. Examples of the Asteraceae plant include Artemisia genus (for example, Artemisia capillaris), Baccharis genus (for example, Baccharis conferia).

  As the propolis, a propolis extract is preferably used as described above, and the production area and extraction method of the propolis bulk are not particularly limited. A propolis extract obtained by alcohol-extracting a Brazilian bulk is particularly preferable because it has a high cinnamic acid derivative content and can be efficiently extracted. Further, as the basidiomycete, as described above, any kind of agaricus having a laccase activity, cordyceps, birch moth, reishi, etc. can be used.

  In this fermentation treatment, as described above, the culture conditions suitable for the culture of mycelium of basidiomycetes, culture temperature, culture period, etc. may be selected as appropriate, and mycelia of basidiomycetes can be grown faster. It is preferable to select a medium.

  Although the time which implements a fermentation process, ie, fermentation time, is not specifically limited, Preferably it is 0.1 to 300 hours, More preferably, it is 0.1 to 150 hours, More preferably, it is 6 to 48 hours. When the fermentation time is less than 0.1 hour, a sufficient amount of a novel cinnamic acid derivative cannot be obtained, which is not preferable. Conversely, when the fermentation time exceeds 300 hours, a sufficient amount of novel cinnamic acid derivative cannot be obtained in place of the fermentation time, resulting in poor efficiency and consequently increased production costs.

  Although the temperature which implements a fermentation process, ie, a fermentation process temperature, is not specifically limited, Preferably it is 25-60 degreeC, More preferably, it is 30-50 degreeC, More preferably, it is 35-45 degreeC. When the fermentation treatment temperature is less than 25 ° C., the enzyme activity is low and the reaction is slow, whereas when it exceeds 60 ° C., the enzyme is deactivated and the reaction is not efficient, which is not preferable.

  For example, when using an alcoholic solution of 20% by mass of artepilin C solid content as artepilin C at the time of fermentation treatment, the concentration of artepilin C solid content in the fermentation treatment solution is not particularly limited, but is preferably 0.01 to 5 masses. %, More preferably, it is 0.05-3 mass%, More preferably, it is 0.1-1 mass%. When the concentration of artepilin C solid content is less than 0.01% by mass, there is a possibility that all artepilin C may be used as a nutrient source for mycelium, and when this fermented product is used as a product, artepilin C in the product Since the content is lowered, it is not preferable. Conversely, if it exceeds 5% by mass, the solubility of artepilin C becomes poor and the alcohol concentration becomes high, so that mycelium growth is poor and laccase activity is also reduced, which is not preferable.

  The separation method is not particularly limited as described above. For example, after extraction with an organic solvent such as ethyl acetate, various column chromatography such as gel filtration chromatography, high performance liquid chromatography, and ion exchange chromatography are appropriately combined. Done.

Next, the propolis fermented product will be described.
The propolis fermented product of this embodiment is obtained by subjecting propolis to fermentation treatment with mycelium of basidiomycetes, and contains the novel cinnamic acid derivative. The form of the propolis fermented product is not particularly limited, and may be in the form of a liquid that has been subjected to fermentation treatment, or may be a form in which a known drying treatment such as spray drying (spray drying) or freeze drying is performed. .

  The propolis fermented product exhibits a significantly higher anticancer effect than that exhibited by propolis alone, basidiomycete mycelium alone, or both. The anticancer effect exerted by the propolis fermented product is effective for both cancer cells and abnormal cells that are becoming cancerous. For this reason, propolis fermented products can be used for health foods, functional foods, pharmaceuticals and the like as anticancer agents or as cancer preventive agents.

  Now, the action in the present embodiment will be described. A novel cinnamic acid derivative is obtained by inoculating mycelium of basidiomycetes on propolis or artepilin C, followed by fermentation, and then separating. The obtained novel cinnamic acid derivative has the basic skeleton of cinnamic acid derivative having anticancer activity and has two hydroxyl groups and one carboxyl group. By having such a structural characteristic, it is estimated that a favorable anticancer action can be exhibited.

  Moreover, fermented propolis can be obtained by subjecting propolis to fermentation with mycelium of basidiomycetes. The propolis fermented product contains a novel cinnamic acid derivative having a good anticancer activity, in addition to the anticancer component inherent to the mycelium of propolis and basidiomycetes. It is considered that a dramatic anticancer action can be exerted by the synergistic action of these contained components.

The effects exhibited by the embodiment will be described collectively below.
-The novel cinnamic acid derivative of this embodiment has the basic skeleton of cinnamic acid derivative, and can exhibit a good anticancer effect.

  -According to the method for producing a novel cinnamic acid derivative of the present embodiment, propolis is an alcohol-extracted propolis bulk produced in Brazil and contains abundant cinnamic acid derivatives. A novel cinnamic acid derivative can be provided. Moreover, according to the production method of the present embodiment, the basidiomycete is Agaricus and has high laccase activity, so that a novel cinnamic acid derivative can be efficiently provided in a short time.

  -The propolis fermented product of the present embodiment is obtained by fermenting Brazilian propolis rich in cinnamic acid derivatives having anti-cancer activity with agaric mycelium having strong anti-cancer activity. Therefore, since a component having a strong anticancer activity is contained in a large amount, a dramatic anticancer activity can be exhibited more than those using other basidiomycetes and propolis.

Examples are given below to describe the embodiment more specifically, but the present invention is not limited to these examples.
<Production of ethanol extract of propolis>
An ethanol extract of propolis was obtained by adding 4 L of a 95 mass% ethanol solution to 1.2 kg of Brazilian propolis bulk, performing ethanol extraction at room temperature for 12 hours, and dewaxing after diatomaceous earth filtration. After concentrating the ethanol extract, 10% by mass of an emulsifier (manufactured by Kirayanin S-100 Maruzen Kasei Co., Ltd., containing 25% by mass of Kiraya extract) was added to prepare an ethanol extract having a propolis solid content of 20% by mass.

<Manufacture of fermented propolis>
Example 1 Production 1 of Propolis Fermented Product Using Agaricus Mycelium
Agaricus mycelium (obtained from New Piers) obtained in a polydextrose broth (PDB) medium (Difco) at a concentration of 2.4 g / L was collected and dried. Next, an agaricus culture solution was prepared by suspending the dried mycelium in a PDB medium having a concentration of 0.24 g / L so as to have a concentration of 0.0035 g per ml. Subsequently, the Agaricus culture solution was precultured by shaking at 25 ° C. and 100 rpm for 6 days using a mini shaker (manufactured by IWAKI). Subsequently, the ethanol extract having a solid content of 20% by mass of propolis is added so that the concentration of the solid content of propolis in the enzyme-treated solution is 0.5% by mass, and the mixture is stirred at 40 ° C. for 3.5 hours, thereby fermenting. Processed. The agaricus suspension after the fermentation treatment was freeze-dried to obtain a propolis fermented product.

Example 2 Production 2 of Propolis Fermented Product Using Agaricus Mycelium
Agaricus mycelium was cultured at 25 ° C. in the dark for 2 to 3 hours using an agar medium made of potato dextrose agar (PDA) (manufactured by Nissui Pharmaceutical Co., Ltd.). Thereafter, Agaricus mycelium grown on the agar medium was scraped off, added to a flask containing 50 ml of PDB medium at a concentration of 2.4 g / L, and left to stand for 5 to 10 days for preculture. Next, the medium in this flask (corresponding to a dry mycelium mass of 113 mg) was changed to a PYS medium (0.3 mass% polypeptone, 0.3 mass% yeast extract, 1 mass% sucrose; all manufactured by Nakarai-Tex Corporation. The sample was added to 5 L, and aerated (precultured) for 10 to 15 days with a mini-jar fermenter (manufactured by Takasaki Scientific Instruments Co., Ltd.) under culture conditions of an aeration rate of 0.5 L / min and a stirring speed of 100 rpm. The mycelium after aeration culture was collected and suspended in a PDB medium having a concentration of 0.24 g / L to prepare an agaricus suspension. Further, the ethanol extract having a propolis solid content of 20% by mass is added to the Agaricus suspension so that the concentration of the propolis solid content in the enzyme-treated solution is 0.5% by mass, followed by stirring at 40 ° C. for 72 hours. The fermentation process was performed. The agaricus suspension after the fermentation treatment was freeze-dried to obtain a propolis fermented product.

<Production of novel cinnamic acid derivatives>
(Example 3, Production of a novel cinnamic acid derivative from ethanol extract of propolis)
By the method of Example 2 above, a propolis fermentation product was produced from an ethanol extract of propolis. The propolis fermentation product was extracted with ethyl acetate and then concentrated to obtain 5.6 g of an extract. The extract was first purified by ODS column chromatography under the following conditions (a), and a 50% by volume methanol-eluted fraction was collected and concentrated to obtain 800 mg of the eluate. Subsequently, the eluate was purified by preparative high performance liquid chromatography (abbreviated preparative HPLC) under the following conditions (b), and after fractionating a peak around a retention time of 20 minutes, concentration was performed to obtain 153 mg of eluate. It was. Finally, the eluate was purified by preparative HPLC under the following conditions (c), and after fractionating a peak at a retention time of about 35 minutes, concentration was performed to obtain 5.2 mg of a novel cinnamic acid derivative. The structure of the novel cinnamic acid derivative was determined by ¹ H-NMR, ¹³ C-NMR, C—H COSY which is a two-dimensional heteronuclear NMR, H—H COSY which is a two-dimensional NMR of the same nucleus, and a remote C—H correlation. Two-dimensional NMR was performed by measuring HMBC, methyl, methylene, methine, DEPT which measures the distinction between quaternary carbon, and MS spectrum.

The measurement conditions are shown below, and the measurement results are shown in Table 1, Table 2, Chemical Formula 3, and FIG. Table 1 shows the ¹ H-NMR chemical shift results of the novel cinnamic acid derivative obtained from the propolis fermented product, and Table 2 shows the ¹³ C-NMR of the novel cinnamic acid derivative obtained from the propolis fermented product. The result of chemical shift is shown. Furthermore, the relationship between the structural formula of the novel cinnamic acid derivative represented by the formula (2) in [Chemical Formula 3] and the assignment numbers of ¹ H-NMR and ¹³ C-NMR was obtained from the propolis fermentation product in FIG. The result of the MS spectrum of a novel cinnamic acid derivative is shown.

Condition (a) ODS column chromatography Column: Chromatorex ODS-DM1020T, inner diameter 60 mm, length 300 mm (manufactured by Fuji Silysia Chemical Ltd.)
Elution solvent: water → water: methanol (v / v) = 70: 30 → 50: 50 → 20: 80 → methanol Elution method: 500 ml of each solvent is eluted stepwise Condition (b) Preparative HPLC
Column: Shiseido CAPCELLPAK ACR, inner diameter 10 mm, length 250 mm (manufactured by Shiseido Co., Ltd.)
Elution solvent: water: methanol: acetic acid (v / v / v) = 24: 75: 1
Flow rate: 6 ml / min
Detection: 330nm
Condition (c) Preparative HPLC
Column: Shiseido CAPCELLPAK ACR, inner diameter 4.6 mm, length 250 mm (manufactured by Shiseido Co., Ltd.)
Elution solvent: water: methanol: acetic acid (v / v / v) = 44: 55: 1
Flow rate: 1 ml / min
Detection: 330nm
Various NMR spectral conditions Apparatus: MERCURY 300 VX 300 MHz (Varian)
Solvent: Methanol-d ₄
MS spectral conditions Apparatus: Applied Biosystems API 365 system (Applied Biosystems Japan Co., Ltd.)
Ionization method: Turbo ion spray positive

次に、プロポリス中の主成分の一つであるアルテピリンＣのみを発酵させたところ、実施例３に示したのと同様の新規な桂皮酸誘導体を得た。以下にその製造方法を示す。

Next, when only arteline C, one of the main components in propolis, was fermented, a novel cinnamic acid derivative similar to that shown in Example 3 was obtained. The manufacturing method is shown below.

<Separation of Artepillin C from Propolis>
51.5 g of Brazilian propolis ethanol extract having a solid content of 20% by mass was purified by silica gel column chromatography under the following conditions (d), and fractions 5 to 10 were collected and concentrated to obtain 13.4 g of eluate. Obtained. Next, the eluate was purified by ODS column chromatography under the following conditions (e), and fractions 20 to 26 were collected and then concentrated to obtain 5.66 g of Artepillin C concentrate. Finally, in order to improve the purity, Artepilin C concentrate was purified again by silica gel column chromatography under the following conditions (f), and fractions 1 to 4 were collected and concentrated to obtain Arteline C 3.8 g.

Condition (d) Silica gel column chromatography Column: PSQ60B, inner diameter 60 mm, length 100 cm (Fuji Silysia Co., Ltd.)
Elution solvent: chloroform: methanol (v / v) = 95: 5
Elution: Elution of 200 ml per fraction Elution confirmation: Thin layer chromatography plate (abbreviated as TLC plate) (Merck)
Condition (e) ODS column chromatography Column: Chromatorex ODS-DM1020T, inner diameter 40 mm, length 500 mm (manufactured by Fuji Silysia Chemical Ltd.)
Elution solvent: water: methanol (v / v) = 20: 80
Elution: Elution of 100 ml per fraction Elution confirmation: Thin layer chromatography plate (abbreviated as TLC plate) (Merck)
Condition (f) Silica gel column chromatography Column: PSQ60B (Fuji Silysia Co., Ltd.)
Elution solvent: chloroform → chloroform: methanol (v / v) = 99: 1 → 98: 2 → 95: 5
Elution: Elution of 100 ml per fraction Elution confirmation: Thin layer chromatography plate (abbreviated as TLC plate) (Merck)
(Example 4, Production of a novel cinnamic acid derivative from Artepilin C)
<Manufacture of fermented artepilin C with Agaricus mycelium>
Agaricus mycelium was cultured at 25 ° C. in the dark for 2 to 3 weeks using an agar medium made of potato dextrose agar (PDA) (manufactured by Nissui Pharmaceutical Co., Ltd.). Thereafter, Agaricus mycelium grown on the agar medium was scraped off, added to a flask containing 50 mL of PDB medium at a concentration of 2.4 g / L, and left to stand for 25 days. Laccase activity is 200 U / ml (in a reaction solution containing syringaldazin as a substrate (pH 6.5, 30 ° C.), the amount of enzyme activity that increases the absorbance at 530 nm by 0.001 per minute is 1 U (unit)) It was prepared so that. Thereafter, an artepilin C ethanol solution having a solid content of 20% by mass in which the above artepilin C is dissolved in ethanol is added so that the concentration of artepilin C solid content in the enzyme treatment reaction system is 0.075% by mass, and 48 at 40 ° C. Fermentation treatment was performed by stirring for a period of time to obtain a fermented artepilin C. The progress of the fermentation process was confirmed by measuring the HPLC chromatogram of the sample by sampling at 4 points at the start, 6 hours, 24 hours, and 48 hours, and confirming the change in the peak area. . The results are shown in Table 3.

その結果、アルテピリンＣ発酵処理開始後、数時間で急激に減り（発酵処理開始後６時間でアルテピリンＣ残り１９質量％）、それに対応して新規桂皮酸誘導体が増えることがわかった。

As a result, it was found that the number decreased drastically within several hours after the start of the artepilin C fermentation process (the remaining 19% by mass of artepilin C within 6 hours after the start of the fermentation process), and the amount of novel cinnamic acid derivatives increased correspondingly.

<Separation of a novel cinnamic acid derivative from Artepilin C fermented product>
The fermented artepilin C was purified by the same conditions and method as in Example 3 to obtain 3.2 mg of a novel cinnamic acid derivative. The same compound as in Example 3 was judged by coincidence of ¹ H-NMR, ¹³ C-NMR, HPLC retention time and UV spectrum analysis results. Table 4 shows the results of ¹ H-NMR chemical shifts of novel cinnamic acid derivatives obtained from Artepilin C fermented products, and Table 5 shows ¹³ C-NMR results of novel cinnamic acid derivatives obtained from Artepilin C fermented products. The result of chemical shift is shown. Further, FIG. 2 (a) was obtained from the HPLC chromatogram chart of the propolis fermentation product, (b) was obtained from the HPLC chromatogram chart of the artepillin C fermentation product, and FIG. 3 (a) was obtained from the propolis fermentation product. The UV spectrum of the novel cinnamic acid derivative is shown in (b), and the UV spectrum of the novel cinnamic acid derivative obtained from the artepiline C fermented product is shown in (b). 2 represents a peak of a novel cinnamic acid derivative.

次に、新規な桂皮酸誘導体の効果を調べるために、癌細胞増殖抑制試験を行った。以下にその試験方法とその結果を示す。

Next, in order to examine the effect of the novel cinnamic acid derivative, a cancer cell growth inhibition test was performed. The test methods and the results are shown below.

Example 5 Cancer Cell Growth Inhibitory Action of Novel Cinnamic Acid Derivative
A leukemia cell line (HL60 cell) (manufactured by Sigma) was cultured at 37 ° C. in the presence of 5% CO _{2 in an} RPI 1640 medium (manufactured by Sigma) containing 10% FCS (Fetal Bovine Serum). After culturing at 1 × 10 ⁵ cells / ml, each sample was added to a concentration of 50 μg / ml within 12 to 24 hours. After further incubation for 24 hours, the number of viable cells was counted by trypan blue staining (manufactured by Sigma). The survival rate of each sample was evaluated when the survival rate of cells without a sample was defined as 100%. The sample was dissolved in DMSO at 10 mg / ml and then diluted to the use concentration with water. The measurement was conducted for a total of four types including a novel cinnamic acid derivative and three types of cinnamic acid derivatives known as artepilin C, durupanin and baccaline as comparative examples. The results are shown in Table 6.

その結果、新規な桂皮酸誘導体は、公知の桂皮酸誘導体であるアルテピリンＣ、ドゥルパニン及びバッカリンと同程度の癌細胞増殖抑制作用を示した。また、その作用は、アルテピリンＣに次いで強いものであった。

As a result, the novel cinnamic acid derivative showed a cancer cell growth inhibitory effect comparable to that of the known cinnamic acid derivatives artepilin C, durupanin and baccaline. The action was strong next to Artepilin C.

Next, the anticancer activity of the propolis fermented product containing the novel cinnamic acid derivative was examined. The test methods and the results are shown below.
(Example 6, anticancer effect of propolis fermentation product)
Sarcoma 180 cells subcultured to ddY mice were collected from mouse ascites and adjusted to the desired cell concentration with physiological saline. Next, Sarcoma 180 cells were subcutaneously transplanted to the back of a ddY male mouse (6 weeks old) purchased from Chubu Scientific Materials Co., Ltd. so as to have a transplant amount of 1 × 10 ⁷ cells / mouse. Mice transplanted with Sarcoma 180 cells were divided into the following 5 groups and administered respectively (10 mice in each group).

First group: physiological saline Second group: dried mycelium of Agaricus mycelium collected in Example 1 suspended in physiological saline Third group: ethanol extract of propolis suspended in physiological saline Group 4: A mixture of the dried mycelium and ethanol extract suspended in physiological saline Group 5: A suspension of the propolis fermented product obtained in Example 1 in physiological saline These were administered intraperitoneally for 28 days at the doses shown in Table 7. On day 28 after transplantation, tumors were removed from each group of mice and their masses were measured. The results are shown in Table 7. In Table 7, # represents p <0.1, * represents p <0.05, and ** represents p <0.01. Here, p represents a statistically meaningful difference by a numerical method called Student's paired t-test. Normally, it can be said that there is a significant difference at p <0.05, and a smaller number means a greater degree of significant difference. The subject of the significant difference here is the physiological saline administration group.

その結果、アガリクス菌糸体投与群及びプロポリス発酵投与群では生理食塩水投与群と比較して、それぞれｐ＜０．０５及びｐ＜０．０１で有意に腫瘍質量が低下した。また、エタノール抽出物投与群及び混合物投与群では、生理食塩水投与群と比較して、いずれも腫瘍質量が低下する傾向が認められた（ｐ＜０．１）。これらの結果より、プロポリス発酵物は、エタノール抽出物、アガリクス菌糸体、及び両者の混合物よりも飛躍的に高い抗癌作用を有していることが確認された。

As a result, in the Agaricus mycelium administration group and the propolis fermentation administration group, the tumor mass was significantly reduced at p <0.05 and p <0.01, respectively, as compared with the physiological saline administration group. In addition, in the ethanol extract administration group and the mixture administration group, the tumor mass tended to decrease compared to the physiological saline administration group (p <0.1). From these results, it was confirmed that the propolis fermented product has a significantly higher anticancer effect than the ethanol extract, agaricus mycelium, and a mixture of both.

It should be noted that the present embodiment can be implemented with the following modifications.
-Fermentation can also be carried out by directly inoculating mycelium of basidiomycetes on the propolis bulk.

  ・ Propolis fermented products containing novel cinnamic acid derivatives can exhibit physiological functions such as antibacterial, antioxidative, and immunostimulatory effects other than anticancer effects, as well as preventive, ameliorating, and therapeutic effects on lifestyle-related diseases and other diseases. It can be used for health foods and pharmaceuticals having effects other than anticancer agents and cancer preventive agents.

  -The carboxyl group or hydroxyl group of the compound of Formula (1) has high reactivity. Therefore, it is used as a derivative of the compound of the formula (1) in which the hydrogen of the carboxyl group or hydroxyl group in the formula (1) is substituted with an alkali metal such as sodium or potassium, or an alkaline earth metal such as magnesium or calcium. be able to.

-A propolis fermented product and an artepilin C fermented product can also be mixed and used.
Further, the technical idea that can be grasped from the embodiment will be described below.
The method for producing a novel cinnamic acid derivative according to claim 2 or 3, wherein the basidiomycete is Agaricus. When comprised in this way, a novel cinnamic acid derivative can be efficiently obtained in a shorter time than the case of other basidiomycetes due to laccase activity with high agaricus.

  The method for producing a novel cinnamic acid derivative according to claim 2, wherein the propolis is an alcohol-extracted Brazilian propolis bulk containing 2-6% by mass of artepilin C. When configured in this way, the cinnamic acid content is high and can be extracted efficiently, so that a large amount of novel cinnamic acid derivatives can be efficiently produced compared to the propolis bulk of other production areas, when using another extraction method. Can be obtained.

  The method for producing a novel cinnamic acid derivative according to claim 2, wherein the fermentation treatment has a fermentation time of 3.5 to 72 hours and a fermentation treatment temperature of 35 to 45 ° C. When constituted in this way, in addition to easily obtaining a novel cinnamic acid derivative, since the fermentation treatment conditions are appropriate, a novel cinnamic acid derivative can be obtained efficiently.

  The method for producing a novel cinnamic acid derivative according to claim 3, wherein in the fermentation treatment, the fermentation time is 6 to 48 hours, and the fermentation treatment temperature is 35 to 45 ° C. When comprised in this way, since a fermentation treatment condition is suitable, a novel cinnamic acid derivative can be obtained efficiently.

  5. The propolis fermented product according to claim 4, wherein the basidiomycete is Agaricus, and the propolis is a product obtained by alcohol-extracting a Brazilian propolis bulk containing 2 to 6% by mass of Artepillin C. . When comprised in this way, since the component with a strong anticancer effect | action is contained in large quantities, a drastic anticancer effect | action can be exhibited rather than what uses other basidiomycetes and propolis.

  A fermented artepilin C obtained by fermenting artepilin C with a mycelium of basidiomycetes and containing a novel cinnamic acid derivative represented by formula (1) according to claim 1. When comprised in this way, the novel cinnamic acid derivative with a favorable anticancer effect | action is contained in addition to the component with the anticancer effect | action which the mycelium of artepilin C and basidiomycetes originally has. Therefore, when they work synergistically, a dramatic anticancer action can be exerted as compared with propolis, mycelium of basidiomycetes or a mixture of both.

The chart which shows the MS spectrum of the novel cinnamic acid derivative obtained from the propolis fermented material of Example 3. (A) is a chart showing the HPLC chromatogram of the propolis fermentation product of Example 2, and (b) is a chart showing the HPLC chromatogram of the artepillin C fermentation product of Example 4. (A) is a chart showing the UV spectrum of the novel cinnamic acid derivative obtained from the propolis fermented product of Example 3, and (b) is the UV of the novel cinnamic acid derivative obtained from the Artepiline C fermented product of Example 4. The chart which shows a spectrum.

Claims (4)

A novel cinnamic acid derivative represented by the following formula (1).

A method for producing the novel cinnamic acid derivative according to claim 1, wherein the mycelium of basidiomycetous fungi is inoculated into propolis, and after the propolis is fermented with the mycelium, the novel cinnamate derivative is expressed by the formula (1). A method for producing a novel cinnamic acid derivative, characterized by separating the cinnamic acid derivative. A method for producing the novel cinnamic acid derivative according to claim 1, wherein a mycelium of basidiomycetes is inoculated into Artepillin C, and Artepilin C is fermented with the mycelium, and then the formula (1 A novel cinnamic acid derivative represented by (2) is isolated. A propolis fermented product obtained by fermenting propolis with a mycelium of a basidiomycete and containing a novel cinnamic acid derivative represented by the formula (1) according to claim 1.

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