WO2013073085A1 - Apoptosis inducer - Google Patents

Apoptosis inducer Download PDF

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WO2013073085A1
WO2013073085A1 PCT/JP2012/005806 JP2012005806W WO2013073085A1 WO 2013073085 A1 WO2013073085 A1 WO 2013073085A1 JP 2012005806 W JP2012005806 W JP 2012005806W WO 2013073085 A1 WO2013073085 A1 WO 2013073085A1
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human
cells
cancer cells
apoptosis
organic solvents
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PCT/JP2012/005806
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French (fr)
Japanese (ja)
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絢矢 川井
崇光 清水
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ホクト株式会社
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Priority to JP2013544092A priority Critical patent/JP5845280B2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to an apoptosis inducer comprising an ergosterol derivative as an active ingredient.
  • Ergosterol is a precursor of vitamin D and is a kind of sterol, and is known to be biosynthesized in fungi such as mushrooms.
  • an angiogenesis inhibitory effect on cancer cells has been reported (Non-patent Document 1).
  • ergosterol does not induce apoptosis on cancer cells (Non-patent Document 2).
  • the ergosterol derivative refers to a compound that is chemically modified with ergosterol as a base.
  • ergosterol derivatives there are some studies on anticancer effects, and the apoptosis-inducing action of ergosterol derivatives has been reported (Non-patent Document 3, Non-patent Document 4, Non-patent Document 5, Non-patent Document). 6).
  • ergosterol derivatives have been reported to suppress melanin production (Patent Document 1, 2003-267873).
  • ergosterol derivatives have different anticancer effects due to differences in chemical structure due to the introduction, oxidation, or reduction of functional groups, and in particular have an apoptotic effect on human lung adenocarcinoma cells. What brings is not yet invented.
  • Himematsutake is a basidiomycete belonging to the genus Agaricaceae, and has the scientific name Agaricusicblazei Murrill.
  • the common name of Himematsutake is often referred to as Agaricus or Agaricus mausoleum. In general, the Agaricus name is more prominent.
  • agaricus is a Japanese reading of the agaric genus Agaricus, which includes so-called mushrooms (Agaricus bisporus (J.Lange) Imbach var.albidus (J.Lange) Sing.). Then, the name of Himematsutake is used to identify the species.
  • Non-patent Document 7 Anti-cancer treatment with immune enhancement is a treatment that eliminates cancer cells by activating immune cells present in the body without administering drugs, so there is no damage to normal cells,
  • the use of ingredients derived from natural products is excellent in that there are almost no side effects.
  • the effects vary widely among individuals, and further resection is necessary for advanced cancer, and it was difficult to achieve complete relief with immunotherapy alone.
  • Patent Document 2 describes an apoptosis-inducing action on human lung large cell carcinoma cells and human gastric cancer cells as a steroid derivative obtainable from the fruit body of Himematsutake or its crushed material. Has been. Furthermore, it was revealed that this steroid derivative has an apoptosis-inducing action on human colon cancer cells and human hepatoma cells (Patent Document 3, 2010-229077).
  • Himematsutake induces apoptosis in lung adenocarcinoma cells with high mortality. Furthermore, it is not known at all that Himematsutake also induces apoptosis in human cervical cancer cells and human tongue cancer cells. Anticancer agents are often specific depending on the type of cancer, and there is a problem that an apoptotic effect must be found for each type of cancer cell.
  • the problem to be solved by the present invention is an apoptosis inducer comprising an ergosterol derivative as an active ingredient, and particularly an apoptosis inducer for human lung adenocarcinoma cells, human cervical cancer cells and human tongue cancer cells. Is to provide.
  • the present inventor has conducted extensive studies to solve the above-mentioned problems.
  • the ergosterol derivative represented by the formula (1) has been found from a fat-soluble extract of Japanese apricot, and further, human lung adenocarcinoma cells, human cervix
  • the present invention has been completed by finding that apoptosis is induced in cancer cells and human tongue cancer cells.
  • the invention according to claim 1 can be arbitrarily selected from human lung adenocarcinoma cells, human tongue cancer cells or human cervical cancer cells containing an ergosterol derivative represented by formula (1) as an active ingredient. It is an apoptosis inducer for one or more selected cells. . . . . (1)
  • the invention according to claim 2 is a fat-soluble extract obtainable from Himematsutake, which contains an ergosterol derivative represented by the formula (1) as an active ingredient. And an apoptosis inducer for one or more cells arbitrarily selected from human tongue cancer cells or human cervical cancer cells.
  • the invention according to claim 3 is one or two of human lung adenocarcinoma cells, human tongue cancer cells or human cervical cancer cells containing an ergosterol derivative represented by formula (1) as an active ingredient.
  • Apoptosis-inducing agent for cells of more than one species as a primary extraction, obtain an extract of dried powder of Himematsutake with one or more organic solvents selected from protic polar organic solvents, An organic solvent layer concentrate obtained by adding distilled water and one or more organic solvents selected from low-polar organic solvents to the extract obtained by the primary extraction and stirring the mixture is obtained.
  • one or more organic solvents selected from a protic polar organic solvent and a low polar organic solvent are used. 1 selected Elution with an organic solvent mixture obtained by mixing two or more organic solvents to obtain a plurality of fractions, from which human lung adenocarcinoma cells, human tongue cancer cells or human cervical cancer cells. It is a method for producing an apoptosis-inducing agent, characterized in that it is produced by selecting a fraction that causes apoptosis in one or two or more cells arbitrarily selected from the above.
  • the effect of the invention according to claim 1 is that the ergosterol derivative represented by formula (1) induces apoptosis in human lung adenocarcinoma cells, human tongue cancer cells or human cervical cancer cells. Cells can be killed.
  • a human lung adenocarcinoma cell, a human tongue cancer cell or a human cervical cancer is obtained by using a fat-soluble extract of Himematsutake containing an ergosterol derivative represented by the formula (1). It can induce apoptosis of cells and kill the cancer cells. Furthermore, since it is derived from a natural product, it can provide a substance having an anticancer effect without side effects.
  • the invention according to claim 3 is one or two of human lung adenocarcinoma cells, human tongue cancer cells or human cervical cancer cells containing an ergosterol derivative represented by formula (1) as an active ingredient. It is possible to provide a method for producing an apoptosis-inducing agent for cells of more than one species from Himematsutake.
  • the substance that induces apoptosis of human lung adenocarcinoma cells, human tongue cancer cells or human cervical cancer cells according to the present invention is an ergosterol derivative represented by the formula (1). Therefore, since the structure is clear, a synthesized product can be used if the ergosterol derivative can be synthesized.
  • the ergosterol derivative according to the present invention is contained in the mycelium or fruit body of Himematsutake, it can be extracted from the mycelium or fruit body of Himematsutake.
  • the structure of the ergosterol derivative according to the present invention is clearly fat-soluble.
  • a mycelium, fruit body or a mixture of mycelium and fruit body of Himematsutake can be used.
  • the fruit body is further more preferably a dry powder of the fruit body. use.
  • the dried fruit body of Himematsutake can be dried by sun drying, hot air drying, hot air drying or freeze drying.
  • One or two or more organic solvents selected from protic polar organic solvents are added to the dried fruit bodies of Japanese apricot peas to obtain a mixed solution, which is allowed to stand for a certain period of time or stirred, and then the mixed solution is suction filtered. Collect the extract. About the residue at this time, the above-mentioned organic solvent is further added, and the extract can be recovered more by repeating the standing and suction filtration for a fixed time. The recovered extract can be concentrated under reduced pressure to evaporate the organic solvent to obtain a primary extract.
  • examples of the organic solvent having a protic polarity include 1-butanol, 2-propanol, 1-propanol, ethanol, and methanol.
  • any one of ethanol, methanol, or 1-propanol is used alone or a mixture of two or more thereof Used as a solvent.
  • the standing or stirring time is preferably 24 hours or more, more preferably 48 hours.
  • the extract obtained in the first extraction is added with distilled water and one or more organic solvents selected from low-polarity organic solvents, mixed and allowed to stand, then the mixture is separated, then distilled water Leave only the organic solvent layer except the layer.
  • the organic solvent layer can be dehydrated with anhydrous sodium sulfate and then concentrated under reduced pressure to obtain a secondary extract.
  • the low-polar organic solvent includes methylene chloride, ethyl acetate, chloroform, diethyl ether, toluene, benzene, hexane, etc., and preferably any one of ethyl acetate, chloroform, hexane, or a mixture of two or more. Used as a solvent.
  • the secondary extract can be fractionated to further increase the purity. That is, a secondary extract can be adsorbed on silica gel, and then the silica gel adsorbate is eluted with an organic solvent, and fractionated into 1 to 10 fractions as appropriate, whereby a fraction can be obtained.
  • the organic solvent for eluting the silica gel adsorbent is one or more organic solvents selected from low polar organic solvents and one or two or more organic solvents selected from protic polar organic solvents.
  • An organic solvent obtained by mixing is preferable, and an organic solvent obtained by appropriately mixing hexane, ethyl acetate, and methanol is more preferable.
  • the fraction of the secondary extract is eluted using a solvent mixed with or a single organic solvent.
  • the eluted liquid is fractionated for each arbitrary elution amount, and arbitrary fractions are collected and concentrated under reduced pressure to obtain a secondary fraction.
  • acetonitrile or acetone can be used, but acetonitrile is preferably used.
  • the low polarity organic solvent include methylene chloride, ethyl acetate, chloroform, diethyl ether, toluene, benzene, hexane and the like, and preferably ethyl acetate is used.
  • the secondary fraction it is confirmed whether or not the extracted component is isolated by thin layer chromatography. If the extracted component is not isolated, further fractionation is performed.
  • the amount of elution to be collected is preferably 5 ml or less, more preferably 1 ml.
  • a low polarity organic solvent methylene chloride, ethyl acetate, chloroform, diethyl ether, toluene, benzene, hexane, etc. can be used, but chloroform is more preferably used.
  • 1-butanol, 2-propanol, 1-propanol, ethanol, methanol and the like can be used as the protic polar organic solvent, but methanol is more preferably used.
  • the extracted components are dissolved in an organic solvent, and the solution is applied with a point at a height of 7 mm from the lower end of the thin layer chromatography plate using a capillary tube.
  • the thin layer chromatography plate coated with the extraction component is stood in a sealed container containing a mixed solvent of a low polarity organic solvent and a protic polarity organic solvent, and the organic solvent is spread over the entire plate. After the development, the organic solvent is sufficiently dried, and then the component is developed with an anisaldehyde coloring reagent to confirm the isolation of the component.
  • the chemical structure represented by the formula (1) can be confirmed by analyzing the infrared absorption spectrum, 1 HNMR spectrum, and 13 CNMR spectrum.
  • the agaricus extract that is, the third fraction, is dissolved in dimethyl sulfoxide and then added. After 24 hours, a cell proliferation measurement reagent is added to each, and after 1 hour, the absorbance is measured to calculate the cell viability.
  • the cell survival rate is calculated by setting the absorbance value of the untreated group to 100% and comparing the absorbance value of the treated group with the absorbance value of the treated group.
  • the third fraction is added to human lung adenocarcinoma cells that have been subcultured in advance. After 24 hours, apoptotic cells are detected using flow cytometry according to the TUNEL (TdT-mediated dUTP nick end labeling) method.
  • the TUNEL method is a method for specifically staining and detecting cells fragmented with DNA by apoptosis. That is, if cells are detected by the TUNEL method, it becomes clear that apoptosis has occurred.
  • the above-mentioned third fraction can be expected to have an anticancer effect by confirming the apoptosis of human lung adenocarcinoma cells, human cervical cancer cells and human tongue cancer cells.
  • the said 3rd fraction is a fat-soluble extract extracted from Himematsutake, and safety is ensured from the eating experience of Himematsutake.
  • the fat-soluble extract obtained from Himematsutake which induces apoptosis of human lung adenocarcinoma cells extracted by the extraction method described above, is mixed with excipients, binders, disintegrants, lubricants, emulsifiers, and stabilization. Add one or more agents, coloring agents, fragrances, etc., mix into foods in the usual way, compress into tablets, granulate or encapsulate into health foods or anticancer agents it can.
  • the fat-soluble extract from Himematsutake that induces apoptosis of human lung adenocarcinoma cells may be an anti-cancer agent as it is or just powdered by drying, such as cooking rice or tea at home.
  • a small-pack may be packed with a fat-soluble extract from Himematsutake that induces apoptosis of human lung adenocarcinoma cells or a dry powdered anticancer agent thereof.
  • the fat-soluble extract from Himematsutake which induces apoptosis of human lung adenocarcinoma cells, is added to various processed foods as it is or dried and powdered. It can be.
  • FIG. 1 shows the production process of the ergosterol derivative represented by the formula (1) from Himematsutake, and an example of extraction of the ergosterol derivative according to the present invention from Himematsutake is described in detail below.
  • the extract was concentrated under reduced pressure, an arbitrary amount of distilled water and an arbitrary amount of ethyl acetate were added thereto, and the mixture was mixed and stirred in a separatory funnel. After removing the distilled water layer and adding only anhydrous ethyl sulfate to the ethyl acetate layer for dehydration treatment, the solution was concentrated under reduced pressure to obtain 20.73 g of an ethyl acetate soluble fraction.
  • A549 cells were used as human lung adenocarcinoma cells.
  • Human lung adenocarcinoma cells subcultured in advance were adjusted to an initial concentration of 1 ⁇ 10 4 cells / 100 ⁇ l and cultured in a flat bottom plate.
  • the ergosterol derivatives obtained in the third fractionation are dissolved in dimethyl sulfoxide, respectively, and diluted with medium to give concentrations of 0, 2.5, 5.0, 7.5, 10.0 ⁇ g / ml. And added to the cultured cells.
  • the concentration of dimethyl sulfoxide at this time was 1% with respect to the medium.
  • a cell proliferation measurement reagent was added to each, and after another 2 hours, the absorbance was measured, and the cell viability was calculated from the measured values.
  • the medium RPMI 1640 containing 10% fetal bovine serum and 1% streptomycin penicillin was used.
  • the calculation method for cell viability is that the absorbance of the control (cell group cultured in a cell culture medium containing 1% dimethyl sulfoxide) is 100%, and the absorbance value of the ergosterol derivative added group is compared with the absorbance value of the control. Calculated.
  • the cell viability is shown in FIG.
  • the ergosterol derivative according to the present invention markedly caused cell death in human lung adenocarcinoma cells.
  • Human lung adenocarcinoma cells previously subcultured are cultured on a flat bottom plate at an initial concentration of 2 ⁇ 10 5 cells / 2 ml, and the concentration of the ergosterol derivative according to the present invention is 0, 1.0, 4.0 ⁇ g / ml. Each was diluted with a medium and added to the cultured human lung adenocarcinoma cells. After 24 hours, apoptotic cells were detected using flow cytometry according to the TUNEL method.
  • the results of verification of the apoptosis-inducing action by the TUNEL method are as shown in FIG. 3, and the cells in which apoptosis was induced were remarkably detected. This revealed that the cell death of human lung adenocarcinoma cells caused by the ergosterol derivative according to the present invention is apoptosis.
  • HeLa cells Human cervical cancer cells
  • HSC-3 cells human tongue cancer cells
  • Human lung adenocarcinoma cells, human cervical cancer cells and human tongue cancer cells subcultured in advance were each cultured in a flat bottom plate at an initial concentration of 2 ⁇ 10 5 cells / 2 ml.
  • the ergosterol derivative was diluted with a medium so as to be 5.0 ⁇ g / ml, and then added to each cell cultured in a flat plate. After 24 hours, apoptotic cells were detected using flow cytometry according to the TUNEL method.
  • the ergosterol derivative according to the present invention exerts an apoptosis-inducing effect on human lung adenocarcinoma cells, human cervical cancer cells and human tongue cancer cells.
  • the ergosterol derivative according to the present invention can be expected to induce apoptosis in lung adenocarcinoma, cervical cancer, and tongue cancer, and can also be extracted from edible matsutake mushrooms. Because it is a natural material, it can be expected to be safe and can be used in the pharmaceutical and health food industries.

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Abstract

The present invention addresses the problem of providing a substance, said substance having an effect of inducing apoptosis of human lung cancer cells, human uterine cervical cancer cells and human tongue cancer cells, and a method for producing the same. An ergosterol derivative according to the present invention can induce apoptosis of human lung adenocarcinoma cells, human uterine cervical cancer cells and human tongue cancer cells. Moreover, the ergosterol derivative according to the present invention can be found in a fat-soluble extract of Agaricus blazei Murrill that is a natural material, thus has been eaten by humans, has a high safety and is usable as an anticancer drug and a health food.

Description

アポトーシス誘導剤Apoptosis inducer
　本発明は、エルゴステロール誘導体を有効成分とするアポトーシス誘導剤に関するものである。 The present invention relates to an apoptosis inducer comprising an ergosterol derivative as an active ingredient.
　エルゴステロールとはビタミンＤの前駆物質でステロールの一種であり、キノコなどの菌類において生合成されることが知られている。エルゴステロールの抗がん作用についての研究として、がん細胞に対する血管新生阻害作用の報告がされている（非特許文献１）。しかし、エルゴステロールはがん細胞に対してアポトーシスを誘導することは無い（非特許文献２）。 Ergosterol is a precursor of vitamin D and is a kind of sterol, and is known to be biosynthesized in fungi such as mushrooms. As a study on the anticancer effect of ergosterol, an angiogenesis inhibitory effect on cancer cells has been reported (Non-patent Document 1). However, ergosterol does not induce apoptosis on cancer cells (Non-patent Document 2).
　また、エルゴステロール誘導体とは、エルゴステロールを母体とし、化学修飾された化合物をいう。エルゴステロール誘導体についても、抗がん作用に関するいくつかの研究があり、エルゴステロール誘導体のアポトーシス誘導作用の報告がされている（非特許文献３、非特許文献４、非特許文献５、非特許文献６）。他にもエルゴステロール誘導体にはメラニン生成抑制の報告もある（特許文献１　２００３－２６７８７３）。 In addition, the ergosterol derivative refers to a compound that is chemically modified with ergosterol as a base. As for ergosterol derivatives, there are some studies on anticancer effects, and the apoptosis-inducing action of ergosterol derivatives has been reported (Non-patent Document 3, Non-patent Document 4, Non-patent Document 5, Non-patent Document). 6). In addition, ergosterol derivatives have been reported to suppress melanin production (Patent Document 1, 2003-267873).
　しかしながら、上記の文献からも明白なところ、エルゴステロール誘導体は官能基の導入，酸化あるいは還元などによる化学構造の違いにより、抗がん効果が異なり、特にヒト肺腺がん細胞にアポトーシスの効果をもたらすものは未だに発明されていない。 However, as is apparent from the above literature, ergosterol derivatives have different anticancer effects due to differences in chemical structure due to the introduction, oxidation, or reduction of functional groups, and in particular have an apoptotic effect on human lung adenocarcinoma cells. What brings is not yet invented.
　一方、ヒメマツタケとは、ハラタケ科ハラタケ属の担子菌であって学名Agaricus blazei Murrill のことをいう。ヒメマツタケの俗称としてアガリクスまたはアガリクス茸などの名前で呼ばれることも多く、一般的にはアガリクスの名称のほうが著名である。しかしながらアガリクスとは、ハラタケ属の学名Agaricusを日本語読みしたものであり、いわゆるマッシュルーム（Agaricus bisporus (J.Lange) Imbach var.albidus (J.Lange) Sing.）なども含まれてしまうため、本願では、種を特定するためヒメマツタケの名称を使用する。 On the other hand, Himematsutake is a basidiomycete belonging to the genus Agaricaceae, and has the scientific name Agaricusicblazei Murrill. The common name of Himematsutake is often referred to as Agaricus or Agaricus mausoleum. In general, the Agaricus name is more prominent. However, agaricus is a Japanese reading of the agaric genus Agaricus, which includes so-called mushrooms (Agaricus bisporus (J.Lange) Imbach var.albidus (J.Lange) Sing.). Then, the name of Himematsutake is used to identify the species.
　ヒメマツタケは、βグルカンを含有することで、免疫増強作用などが知られていた　（非特許文献７）。免疫増強作用による抗がん治療法は、薬剤を投与すること無く、体内に存在する免疫細胞を活性化することでがん細胞を消滅させる治療法なので、正常細胞への傷害性が無いこと、また天然物由来の成分を使用するため副作用もほとんど無い点で優れている。しかしながら、その効果については個人差も大きく、さらに進行がんにいたっては切除が必要であり、免疫療法だけでの全快は困難であった。 Himematsutake has been known to have an immune enhancement effect by containing β-glucan (Non-patent Document 7). Anti-cancer treatment with immune enhancement is a treatment that eliminates cancer cells by activating immune cells present in the body without administering drugs, so there is no damage to normal cells, In addition, the use of ingredients derived from natural products is excellent in that there are almost no side effects. However, the effects vary widely among individuals, and further resection is necessary for advanced cancer, and it was difficult to achieve complete relief with immunotherapy alone.
　ところが、最近はヒメマツタケにも特定のがん細胞に対してアポトーシスを誘導することがわかってきた（特許文献２　２００８－１１５１５８）。特許文献２にはヒメマツタケ子実体またはその破砕物から得ることができるステロイド誘導体にヒト肺大細胞癌細胞およびヒト胃癌細胞に対するアポトーシス誘導作用が記載されており、ヒメマツタケ由来ステロイド誘導体によるアポトーシス誘導作用が明らかにされている。さらにこのステロイド誘導体はヒト大腸癌細胞並びにヒト肝癌細胞に対してもアポトーシス誘導作用を有することが明らかとされた(特許文献３　２０１０－２２９０７７)。 Recently, however, it has been found that Japanese matsutake also induces apoptosis in specific cancer cells (Patent Document 2, 2008-115158). Patent Document 2 describes an apoptosis-inducing action on human lung large cell carcinoma cells and human gastric cancer cells as a steroid derivative obtainable from the fruit body of Himematsutake or its crushed material. Has been. Furthermore, it was revealed that this steroid derivative has an apoptosis-inducing action on human colon cancer cells and human hepatoma cells (Patent Document 3, 2010-229077).
　しかしながら、死亡率の高い肺腺がんの細胞に対してヒメマツタケがアポトーシスを誘導するということは全く知られていない。さらに、他にもヒト子宮頸がん細胞やヒト舌がん細胞に対してもヒメマツタケがアポトーシス誘導を示すことは全く知られていない。抗がん剤は、がんの種類によって特異的な場合が多く、各種がん細胞ごとにアポトーシス効果を見出さなければならないという問題があった。 However, it is not known at all that Himematsutake induces apoptosis in lung adenocarcinoma cells with high mortality. Furthermore, it is not known at all that Himematsutake also induces apoptosis in human cervical cancer cells and human tongue cancer cells. Anticancer agents are often specific depending on the type of cancer, and there is a problem that an apoptotic effect must be found for each type of cancer cell.
特開２００３－２６７８７３号公報JP 2003-267873 A 特開２００８－１１５１５８号公報JP 2008-115158 A 特開２０１０－２２９０７７号公報JP 2010-229077 A
　 本発明が解決しようとする課題は、エルゴステロール誘導体を有効成分とするアポトーシス誘導剤であって、特にヒト肺腺がん細胞、ヒト子宮頸がん細胞およびヒト舌がん細胞に対するアポトーシス誘導剤を提供することである。 The problem to be solved by the present invention is an apoptosis inducer comprising an ergosterol derivative as an active ingredient, and particularly an apoptosis inducer for human lung adenocarcinoma cells, human cervical cancer cells and human tongue cancer cells. Is to provide.
　本発明者は、上記課題を解決すべく鋭意研究を重ねたところ、式（１）で表されるエルゴステロール誘導体をヒメマツタケの脂溶性抽出物から見いだし、さらにヒト肺腺がん細胞、ヒト子宮頸がん細胞およびヒト舌がん細胞に対してアポトーシスを誘導させることを見いだして発明を完成させた。 The present inventor has conducted extensive studies to solve the above-mentioned problems. As a result, the ergosterol derivative represented by the formula (1) has been found from a fat-soluble extract of Japanese apricot, and further, human lung adenocarcinoma cells, human cervix The present invention has been completed by finding that apoptosis is induced in cancer cells and human tongue cancer cells.
　すなわち、請求項１にかかる発明は、式（１）で表されるエルゴステロール誘導体を有効成分とするヒト肺腺がん細胞、ヒト舌がん細胞又はヒト子宮頸がん細胞の中から任意に選ばれる１種又は２種以上の細胞に対するアポトーシス誘導剤である。
Figure JPOXMLDOC01-appb-C000001
 ．．．．（１） That is, the invention according to claim 1 can be arbitrarily selected from human lung adenocarcinoma cells, human tongue cancer cells or human cervical cancer cells containing an ergosterol derivative represented by formula (1) as an active ingredient. It is an apoptosis inducer for one or more selected cells.
Figure JPOXMLDOC01-appb-C000001
 . . . . (1)
　請求項２にかかる発明は、ヒメマツタケから得ることができる脂溶性抽出物であって、式（１）で表されるエルゴステロール誘導体を有効成分として含有することを特徴とするヒト肺腺がん細胞、ヒト舌がん細胞又はヒト子宮頸がん細胞の中から任意に選ばれる１種又は２種以上の細胞に対するアポトーシス誘導剤である。 The invention according to claim 2 is a fat-soluble extract obtainable from Himematsutake, which contains an ergosterol derivative represented by the formula (1) as an active ingredient. And an apoptosis inducer for one or more cells arbitrarily selected from human tongue cancer cells or human cervical cancer cells.
　請求項３にかかる発明は、式（１）で表されるエルゴステロール誘導体を有効成分とするヒト肺腺がん細胞、ヒト舌がん細胞又はヒト子宮頸がん細胞の中から１種又は２種以上の細胞に対するアポトーシス誘導剤が、第一次抽出として、ヒメマツタケの乾燥粉末をプロトン性極性の有機溶媒から選ばれる１または２以上の有機溶媒で抽出物を得て、第二次抽出として、当該第一次抽出で得られた抽出物に蒸留水と低極性の有機溶媒から選ばれる１または２以上の有機溶媒とを加えて撹拌した後に分離して得られる有機溶媒層の濃縮物を得て、さらに、第二次抽出物の分画として、当該濃縮物をシリカゲルカラムに吸着させた後に、プロトン性極性の有機溶媒から選択される１ないし２以上の有機溶媒と低極性の有機溶媒から選択される１ないし２以上の有機溶媒を混合してなる有機溶媒混合液で溶出して複数の分画を得て、当該分画からヒト肺腺がん細胞、ヒト舌がん細胞又はヒト子宮頸がん細胞の中から任意に選ばれる１種又は２種以上の細胞にアポトーシスを起こさせる分画を選択して製造されることを特徴とするアポトーシス誘導剤の製造方法である。 The invention according to claim 3 is one or two of human lung adenocarcinoma cells, human tongue cancer cells or human cervical cancer cells containing an ergosterol derivative represented by formula (1) as an active ingredient. Apoptosis-inducing agent for cells of more than one species, as a primary extraction, obtain an extract of dried powder of Himematsutake with one or more organic solvents selected from protic polar organic solvents, An organic solvent layer concentrate obtained by adding distilled water and one or more organic solvents selected from low-polar organic solvents to the extract obtained by the primary extraction and stirring the mixture is obtained. Further, as a fraction of the second extract, after the concentrate is adsorbed on a silica gel column, one or more organic solvents selected from a protic polar organic solvent and a low polar organic solvent are used. 1 selected Elution with an organic solvent mixture obtained by mixing two or more organic solvents to obtain a plurality of fractions, from which human lung adenocarcinoma cells, human tongue cancer cells or human cervical cancer cells It is a method for producing an apoptosis-inducing agent, characterized in that it is produced by selecting a fraction that causes apoptosis in one or two or more cells arbitrarily selected from the above.
　請求項１にかかる発明の効果は、式（１）で表されるエルゴステロール誘導体によりヒト肺腺がん細胞、ヒト舌がん細胞又はヒト子宮頸がん細胞に対してアポトーシスを誘導し当該がん細胞を死滅させることができる。 The effect of the invention according to claim 1 is that the ergosterol derivative represented by formula (1) induces apoptosis in human lung adenocarcinoma cells, human tongue cancer cells or human cervical cancer cells. Cells can be killed.
　請求項２にかかる発明は、式（１）で表されるエルゴステロール誘導体が含有されているヒメマツタケの脂溶性抽出物により、ヒト肺腺がん細胞、ヒト舌がん細胞又はヒト子宮頸がん細胞に対してアポトーシスを誘導し当該がん細胞を死滅させることができる。さらに天然物由来のため副作用の無い抗がん作用をもつ物質を提供することができる。 According to a second aspect of the present invention, a human lung adenocarcinoma cell, a human tongue cancer cell or a human cervical cancer is obtained by using a fat-soluble extract of Himematsutake containing an ergosterol derivative represented by the formula (1). It can induce apoptosis of cells and kill the cancer cells. Furthermore, since it is derived from a natural product, it can provide a substance having an anticancer effect without side effects.
　請求項３にかかる発明は、式（１）で表されるエルゴステロール誘導体を有効成分とするヒト肺腺がん細胞、ヒト舌がん細胞又はヒト子宮頸がん細胞の中から１種又は２種以上の細胞に対するアポトーシス誘導剤をヒメマツタケから製造する方法を提供することができる。 The invention according to claim 3 is one or two of human lung adenocarcinoma cells, human tongue cancer cells or human cervical cancer cells containing an ergosterol derivative represented by formula (1) as an active ingredient. It is possible to provide a method for producing an apoptosis-inducing agent for cells of more than one species from Himematsutake.
本発明にかかるエルゴステロール誘導体の製造についての概略工程図である。It is a schematic process drawing about manufacture of an ergosterol derivative concerning the present invention. ヒメマツタケ由来エルゴステロール誘導体がヒト肺腺がん細胞に対して濃度依存的に細胞死を誘導したことを示す図である。It is a figure which shows that the himesterol derivative derived from a Japanese matsutake induced cell death with respect to a human lung adenocarcinoma cell concentration-dependently. ヒメマツタケ由来エルゴステロール誘導体がヒト肺腺がん細胞に対して濃度依存的にアポトーシスを誘導したことを示す図である。It is a figure which shows that an ergosterol derivative derived from Japanese matsutake induced apoptosis in human lung adenocarcinoma cells in a concentration-dependent manner. ヒメマツタケ由来エルゴステロール誘導体がヒト肺腺がん細胞（Ａ５４９）、ヒト舌がん細胞（ＨＳＣ－３)及びヒト子宮頸がん細胞（ＨｅＬａ）に対してアポトーシスを誘導したことを示す図である。It is a figure which shows that an ergosterol derivative | guide_body derived from a Japanese matsutake induced apoptosis with respect to a human lung adenocarcinoma cell (A549), a human tongue cancer cell (HSC-3), and a human cervical cancer cell (HeLa).
　 本発明にかかるヒト肺腺がん細胞、ヒト舌がん細胞又はヒト子宮頸がん細胞に対してアポトーシスを誘導する物質は、式（１）で表されるエルゴステロール誘導体である。
従って、構造が明確なため、当該エルゴステロール誘導体を合成することができれば、合成物を使用することができる。 The substance that induces apoptosis of human lung adenocarcinoma cells, human tongue cancer cells or human cervical cancer cells according to the present invention is an ergosterol derivative represented by the formula (1).
Therefore, since the structure is clear, a synthesized product can be used if the ergosterol derivative can be synthesized.
　また、本発明にかかるエルゴステロール誘導体はヒメマツタケの菌糸体又は子実体に含有されるため、ヒメマツタケの菌糸体又は子実体から抽出することもできる。 Further, since the ergosterol derivative according to the present invention is contained in the mycelium or fruit body of Himematsutake, it can be extracted from the mycelium or fruit body of Himematsutake.
　本発明にかかるエルゴステロール誘導体は脂溶性であるのはその構造上明確である。 The structure of the ergosterol derivative according to the present invention is clearly fat-soluble.
　ヒメマツタケから脂溶性抽出物を得るためには、ヒメマツタケの菌糸体、子実体または菌糸体と子実体との混合物を用いることができるが、好ましくは子実体をさらにより好ましくは子実体の乾燥粉末を使用する。 In order to obtain a fat-soluble extract from Himematsutake, a mycelium, fruit body or a mixture of mycelium and fruit body of Himematsutake can be used. Preferably, the fruit body is further more preferably a dry powder of the fruit body. use.
　ヒメマツタケの乾燥子実体は、天日干し、温風乾燥、熱風乾燥または凍結乾燥などで乾燥することができる。 The dried fruit body of Himematsutake can be dried by sun drying, hot air drying, hot air drying or freeze drying.
　次に第一次抽出について説明する。 Next, the first extraction will be described.
　ヒメマツタケの乾燥子実体あるいはその粉末にプロトン性極性の有機溶媒から選択される１または２以上の有機溶媒を加えて混合液とし、一定時間静置しまたは撹拌してその後、混合液を吸引濾過し、抽出液を回収する。このときの残渣については、さらに上記有機溶媒を添加して一定時間の静置および吸引濾過を繰り返すことにより、抽出物をより多く回収することができる。回収した抽出液を減圧濃縮して有機溶媒を蒸発させ第一次抽出物を得ることができる。 One or two or more organic solvents selected from protic polar organic solvents are added to the dried fruit bodies of Japanese apricot peas to obtain a mixed solution, which is allowed to stand for a certain period of time or stirred, and then the mixed solution is suction filtered. Collect the extract. About the residue at this time, the above-mentioned organic solvent is further added, and the extract can be recovered more by repeating the standing and suction filtration for a fixed time. The recovered extract can be concentrated under reduced pressure to evaporate the organic solvent to obtain a primary extract.
　ここで、プロトン性極性の有機溶媒としては、１－ブタノール、２－プロパノール、１－プロパノール、エタノール又はメタノール等があり、好ましくはエタノール、メタノール又は１－プロパノールの何れかを単独または２以上の混合溶媒として用いる。 Here, examples of the organic solvent having a protic polarity include 1-butanol, 2-propanol, 1-propanol, ethanol, and methanol. Preferably, any one of ethanol, methanol, or 1-propanol is used alone or a mixture of two or more thereof Used as a solvent.
　また、静置又は撹拌時間は２４時間以上が好ましく、より好ましくは４８時間静置である。 Further, the standing or stirring time is preferably 24 hours or more, more preferably 48 hours.
　次に第二次抽出について説明する。 Next, the second extraction will be described.
　第一次抽出で得られた抽出物に蒸留水と低極性の有機溶媒から選択される１または２以上の有機溶媒とを添加して混合し、静置した後混合液が分離したら、蒸留水層を除いて有機溶媒層のみを残す。次に当該有機溶媒層を無水硫酸ナトリウムで脱水した後、減圧濃縮して第二次抽出物を得ることができる。 If the extract obtained in the first extraction is added with distilled water and one or more organic solvents selected from low-polarity organic solvents, mixed and allowed to stand, then the mixture is separated, then distilled water Leave only the organic solvent layer except the layer. Next, the organic solvent layer can be dehydrated with anhydrous sodium sulfate and then concentrated under reduced pressure to obtain a secondary extract.
　ここで、上記低極性の有機溶媒には、塩化メチレン、酢酸エチル、クロロホルム、ジエチルエーテル、トルエン、ベンゼン、ヘキサン等があり、好ましくは酢酸エチル、クロロホルム、ヘキサンの何れかを単独または２以上の混合溶媒として用いる。 Here, the low-polar organic solvent includes methylene chloride, ethyl acetate, chloroform, diethyl ether, toluene, benzene, hexane, etc., and preferably any one of ethyl acetate, chloroform, hexane, or a mixture of two or more. Used as a solvent.
　第二次抽出物は、さらに純度を上げるために分画することができる。すなわち、第二次抽出物をシリカゲルに吸着させ、その後当該シリカゲル吸着物を有機溶媒で溶出させて、１乃至１０の画分に適宜に分画することで分画物を得ることができる。 The secondary extract can be fractionated to further increase the purity. That is, a secondary extract can be adsorbed on silica gel, and then the silica gel adsorbate is eluted with an organic solvent, and fractionated into 1 to 10 fractions as appropriate, whereby a fraction can be obtained.
　ここで、上記シリカゲル吸着物を溶出させる有機溶媒は、低極性の有機溶媒から選択される１または２以上の有機溶媒とプロトン性極性の有機溶媒から選択される１または２以上の有機溶媒とを混合して得られる有機溶媒が好ましく、より好ましくはヘキサンと酢酸エチルとメタノールとを適宜に混合して得られる有機溶媒である。 Here, the organic solvent for eluting the silica gel adsorbent is one or more organic solvents selected from low polar organic solvents and one or two or more organic solvents selected from protic polar organic solvents. An organic solvent obtained by mixing is preferable, and an organic solvent obtained by appropriately mixing hexane, ethyl acetate, and methanol is more preferable.
　次に第二次抽出物の分画物についてさらに純度を高めて単離する方法について説明する。 Next, a method for isolating the fraction of the secondary extract with higher purity will be described.
第二次抽出物の分画物を珪藻土に吸着させたのち、オクタデシルシリル化シリカゲル（ＯＤＳ）を充填したオープンカラムを使用して、蒸留水と非プロトン性極性の有機溶媒と低極性の有機溶媒とを混合した溶媒、あるいはそれぞれ単独の有機溶媒を用いて、第二次抽出物の分画物を溶出させる。溶出させた液は、任意の溶出量ごとに分取して、任意の分画を集めて減圧濃縮し、第二次分画物を得る。 After adsorbing the fraction of the secondary extract onto diatomaceous earth, using an open column filled with octadecylsilylated silica gel (ODS), distilled water, aprotic polar organic solvent and low polar organic solvent The fraction of the secondary extract is eluted using a solvent mixed with or a single organic solvent. The eluted liquid is fractionated for each arbitrary elution amount, and arbitrary fractions are collected and concentrated under reduced pressure to obtain a secondary fraction.
　上記第二次分画物を得る場合の非プロトン性極性の有機溶媒には、アセトニトリルやアセトンなどを使用することができるが、好ましくはアセトニトリルを用いる。また、低極性の有機溶媒については、塩化メチレン、酢酸エチル、クロロホルム、ジエチルエーテル、トルエン、ベンゼン、ヘキサン等があり、好ましくは酢酸エチルを用いる。 As the aprotic polar organic solvent for obtaining the second fraction, acetonitrile or acetone can be used, but acetonitrile is preferably used. Examples of the low polarity organic solvent include methylene chloride, ethyl acetate, chloroform, diethyl ether, toluene, benzene, hexane and the like, and preferably ethyl acetate is used.
　第二次分画物について、薄層クロマトグラフィ―法により抽出成分が単離されているか否かを確認し、抽出成分が単離されていない場合には、さらに分画を行う。 For the secondary fraction, it is confirmed whether or not the extracted component is isolated by thin layer chromatography. If the extracted component is not isolated, further fractionation is performed.
　第二次分画物を珪藻土に吸着させたのち、シリカゲルを充填したオープンカラムを使用して、低極性の有機溶媒とプロトン性極性の有機溶媒とを混合した溶媒、あるいはそれぞれ単独の有機溶媒を用いて、第二次分画物を溶出させる。溶出させた液は、任意の溶出量ごとに分取して、任意の分画を集めて減圧濃縮し、第三次分画物を得る。 After adsorbing the secondary fraction on diatomaceous earth, using an open column packed with silica gel, a solvent that is a mixture of a low-polar organic solvent and a protic-polar organic solvent, or a single organic solvent for each. To elute the secondary fraction. The eluted liquid is collected for each arbitrary elution amount, and arbitrary fractions are collected and concentrated under reduced pressure to obtain a third fraction.
　ここで、上記第三次分画物を得る場合に分取する溶出量は５ｍｌ以下が好ましくより好ましくは１ｍｌである。また、低極性の有機溶媒としては、塩化メチレン、酢酸エチル、クロロホルム、ジエチルエーテル、トルエン、ベンゼン、ヘキサン等を用いることができるがより好ましくはクロロホルムを用いる。さらに、プロトン性極性の有機溶媒としては、１－ブタノール、２－プロパノール、１－プロパノール、エタノール、メタノール等を用いることができるが、より好ましくは、メタノールを用いる。 Here, when the third fraction is obtained, the amount of elution to be collected is preferably 5 ml or less, more preferably 1 ml. Moreover, as a low polarity organic solvent, methylene chloride, ethyl acetate, chloroform, diethyl ether, toluene, benzene, hexane, etc. can be used, but chloroform is more preferably used. Further, 1-butanol, 2-propanol, 1-propanol, ethanol, methanol and the like can be used as the protic polar organic solvent, but methanol is more preferably used.
　第三次分画物についても、薄層クロマトグラフィ―法により抽出成分が単離されているか否かを確認する。 薄層クロマトグラフィー法での抽出成分の単離の確認は次の方法ですることができる。 For the third fraction, confirm whether or not the extracted component is isolated by thin layer chromatography. Confirmation of isolation of the extracted component by the thin-layer chromatography method can be performed by the following method.
抽出成分を有機溶媒で溶解し、その溶液を毛細管を用いて薄層クロマトグラフィープレートの下端から７mmの高さに点で塗布する。当該抽出成分の塗布された薄層クロマトグラフィープレートを低極性の有機溶媒とプロトン性極性の有機溶媒とを混合した溶媒の入った密閉容器内に立てかけ、有機溶媒をプレート全体に展開させる。展開後は有機溶媒を十分に乾燥させた後、アニスアルデヒド発色試薬で成分を発色させ、成分の単離を確認する。 The extracted components are dissolved in an organic solvent, and the solution is applied with a point at a height of 7 mm from the lower end of the thin layer chromatography plate using a capillary tube. The thin layer chromatography plate coated with the extraction component is stood in a sealed container containing a mixed solvent of a low polarity organic solvent and a protic polarity organic solvent, and the organic solvent is spread over the entire plate. After the development, the organic solvent is sufficiently dried, and then the component is developed with an anisaldehyde coloring reagent to confirm the isolation of the component.
　抽出成分の単離を確認したのち、赤外吸収スペクトル、1ＨＮＭＲスペクトル、13ＣＮＭＲスペクトルを解析することで、式（１）で表される化学構造を確認することができる。 After confirming the isolation of the extracted component, the chemical structure represented by the formula (1) can be confirmed by analyzing the infrared absorption spectrum, 1 HNMR spectrum, and 13 CNMR spectrum.
　次にがん細胞に対するアポトーシス誘導について説明する。 Next, apoptosis induction for cancer cells will be described.
　あらかじめ継代培養したヒト肺腺がん細胞（Ａ５４９細胞、東北大学加齢医学研究所、ＴＫＧ ０１８４）、ヒト子宮頸がん細胞（ＨｅＬａ細胞、東北大学加齢医学研究所、ＴＫＧ ０３３１）およびヒト舌がん細胞（ＨＳＣ－３細胞、東北大学加齢医学研究所、ＴＫＧ ０４８４）に、上記アガリクス抽出物すなわち第三次分画物をジメチルスルホキシドに溶解させてから添加する。２４時間経過後、それぞれに細胞増殖測定試薬を添加して、１時間経過後、吸光度を測定することで細胞生存率を算出する。細胞生存率の算出方法は未処理群の吸光度の値を細胞生存率１００％として、処理群の吸光度の値と比較して細胞生存率を求める。 Pre-cultured human lung adenocarcinoma cells (A549 cells, Tohoku University Institute for Aging Medicine, TKG 184), human cervical cancer cells (HeLa cells, Tohoku University Institute for Aging Medicine, TKG 0331) and humans To the tongue cancer cells (HSC-3 cells, Tohoku University Institute of Aging Medicine, TKG-0484), the agaricus extract, that is, the third fraction, is dissolved in dimethyl sulfoxide and then added. After 24 hours, a cell proliferation measurement reagent is added to each, and after 1 hour, the absorbance is measured to calculate the cell viability. The cell survival rate is calculated by setting the absorbance value of the untreated group to 100% and comparing the absorbance value of the treated group with the absorbance value of the treated group.
あらかじめ継代培養したヒト肺腺がん細胞に第三次分画物を添加する。２４時間経過後、TUNEL（TdT-mediated dUTP nick end labeling）法に従い、フローサイトメトリーを用いてアポトーシス細胞を検出する。TUNEL法とはアポトーシスによってDNAが断片化した細胞を特異的に染色し検出する方法をいう。すなわち、TUNEL法で細胞が検出されればアポトーシスを起こしていることが明白となる。 The third fraction is added to human lung adenocarcinoma cells that have been subcultured in advance. After 24 hours, apoptotic cells are detected using flow cytometry according to the TUNEL (TdT-mediated dUTP nick end labeling) method. The TUNEL method is a method for specifically staining and detecting cells fragmented with DNA by apoptosis. That is, if cells are detected by the TUNEL method, it becomes clear that apoptosis has occurred.
　また、上記第三次分画物は、ヒト肺腺がん細胞、ヒト子宮頸がん細胞およびヒト舌がん細胞のアポトーシスを起こすことが確認されることで、抗がん作用が期待できる。そして、上記第三次分画物は、ヒメマツタケから抽出された脂溶性抽出物であり、ヒメマツタケの食経験からすれば安全性も確保される。 In addition, the above-mentioned third fraction can be expected to have an anticancer effect by confirming the apoptosis of human lung adenocarcinoma cells, human cervical cancer cells and human tongue cancer cells. And the said 3rd fraction is a fat-soluble extract extracted from Himematsutake, and safety is ensured from the eating experience of Himematsutake.
　以上に説明した抽出方法で抽出されたヒト肺腺がん細胞のアポトーシスを誘導するヒメマツタケから得ることができる脂溶性抽出物に、賦形剤、結合剤、崩壊剤、潤滑剤、乳化剤、安定化剤、着色剤、香料などを１または複数加えて、通常の方法で食品に配合し、錠剤に打錠し、顆粒化しまたはカプセルに内包した状態にして健康食品または抗がん剤とすることができる。 The fat-soluble extract obtained from Himematsutake, which induces apoptosis of human lung adenocarcinoma cells extracted by the extraction method described above, is mixed with excipients, binders, disintegrants, lubricants, emulsifiers, and stabilization. Add one or more agents, coloring agents, fragrances, etc., mix into foods in the usual way, compress into tablets, granulate or encapsulate into health foods or anticancer agents it can.
　また、上記ヒト肺腺がん細胞のアポトーシスを誘導するヒメマツタケからの脂溶性抽出物は、そのままあるいは乾燥して粉末化しただけの抗がん剤であっても良く、例えば、家庭で炊飯やお茶に簡単に加えられるように、ヒト肺腺がん細胞のアポトーシスを誘導するヒメマツタケからの脂溶性抽出物またはその乾燥粉末化した抗がん剤を小パックに詰めておいても良い。 Further, the fat-soluble extract from Himematsutake that induces apoptosis of human lung adenocarcinoma cells may be an anti-cancer agent as it is or just powdered by drying, such as cooking rice or tea at home. In order to be easily added, a small-pack may be packed with a fat-soluble extract from Himematsutake that induces apoptosis of human lung adenocarcinoma cells or a dry powdered anticancer agent thereof.
　さらに、ヒト肺腺がん細胞のアポトーシスを誘導するヒメマツタケからの脂溶性抽出物は、そのままあるいは乾燥して粉末化したものを様々な加工食品に添加することで、抗がん作用のある健康食品とすることができる。 Furthermore, the fat-soluble extract from Himematsutake, which induces apoptosis of human lung adenocarcinoma cells, is added to various processed foods as it is or dried and powdered. It can be.
　以下、実施例を挙げて本発明をさらに詳細に説明する。ただし、本発明の構成は、下記実施例に記載された内容に限られるものではなく、また、実施例の記述については、特許請求の範囲を限定し、あるいは特許請求の範囲を減縮するように解すべきものでもない。そして、本発明の構成は、特許請求の範囲に記載された技術的範囲内において種々の変形が可能である。 Hereinafter, the present invention will be described in more detail with reference to examples. However, the configuration of the present invention is not limited to the contents described in the following embodiments, and the description of the embodiments limits the scope of claims or reduces the scope of claims. It is not something to be understood. The configuration of the present invention can be variously modified within the technical scope described in the claims.
　式（１）で表されるエルゴステロール誘導体のヒメマツタケからの製造工程について図１に示すとともに、以下に本発明にかかるエルゴステロール誘導体のヒメマツタケからの抽出例を詳説する。 FIG. 1 shows the production process of the ergosterol derivative represented by the formula (1) from Himematsutake, and an example of extraction of the ergosterol derivative according to the present invention from Himematsutake is described in detail below.
　（第一次抽出）
　アガリクスの子実体、９７１１．５７ｇをエタノール２０lに浸漬し、室温で２４時間静置した。その後、混合液を吸引濾過し、エタノールと残渣を分離して回収した。次に当該残渣を、再度エタノール２０lに浸漬し、室温で４８時間静置した後、混合液を吸引濾過し、エタノールと残渣を分離して回収した。更に当該残渣をエタノール２０lに浸漬し、室温で２４時間静置した後、混合液を吸引濾過して、エタノールを回収した。回収されたエタノール全てを一つにまとめて抽出液とした。抽出液を減圧濃縮し、そこへ、任意の量の蒸留水と任意の量の酢酸エチルを加え、分液ロート内で混合して攪拌した。蒸留水層を除いて、酢酸エチル層のみに対して無水硫酸ナトリウムを加え脱水処理した後、減圧濃縮して、酢酸エチル可溶画分２０．７３ｇを得た。 (Primary extraction)
Agaricus fruit bodies, 9711.57 g, were immersed in 20 l of ethanol and allowed to stand at room temperature for 24 hours. Thereafter, the mixed solution was subjected to suction filtration, and ethanol and the residue were separated and recovered. Next, the residue was again immersed in 20 l of ethanol and allowed to stand at room temperature for 48 hours, and then the mixed solution was suction filtered to separate and recover the ethanol and the residue. Further, the residue was immersed in 20 l of ethanol and allowed to stand at room temperature for 24 hours, and then the mixed solution was subjected to suction filtration to recover ethanol. All of the collected ethanol was combined into one extract. The extract was concentrated under reduced pressure, an arbitrary amount of distilled water and an arbitrary amount of ethyl acetate were added thereto, and the mixture was mixed and stirred in a separatory funnel. After removing the distilled water layer and adding only anhydrous ethyl sulfate to the ethyl acetate layer for dehydration treatment, the solution was concentrated under reduced pressure to obtain 20.73 g of an ethyl acetate soluble fraction.
　（第二次抽出およびその分画）
酢酸エチル可溶画分のうち２０．２８ｇを珪藻土に吸着させ、シリカゲルを充填したオープンカラムにアプライし、ヘキサンと酢酸エチルとメタノールを任意の混合割合で混合した溶媒、あるいはそれぞれ単独の溶媒を用いて、抽出物を溶出させた。溶出した液は２００ｍｌずつ分取し、ヘキサン：酢酸エチル＝１：１乃至１：３の混合比率で溶出された分画をまとめ、減圧濃縮して、分画物Ｆ－５（２５０ｍｇ）を得た。 (Secondary extraction and its fractionation)
Of the ethyl acetate-soluble fraction, 20.28 g was adsorbed on diatomaceous earth, applied to an open column packed with silica gel, and a solvent in which hexane, ethyl acetate, and methanol were mixed at an arbitrary mixing ratio, or a single solvent was used. The extract was eluted. The eluted solution was collected in 200 ml portions, and the fractions eluted at a mixing ratio of hexane: ethyl acetate = 1: 1 to 1: 3 were combined and concentrated under reduced pressure to obtain a fraction F-5 (250 mg). It was.
　（第二次分画）
　抽出物Ｆ－５（２５０ｍｇ）を珪藻土に吸着させ、ＯＤＳを充填したオープンカラムにアプライし、蒸留水とアセトニトリルと酢酸エチルを任意の混合割合で混合した溶媒、あるいはそれぞれ単独の溶媒を用いて、抽出物を溶出させた。溶出した液は８ｍｌずつ分取し、アセトニトリル：酢酸エチル＝７：３乃至酢酸エチルで溶出された分画をまとめ、減圧濃縮して、分画物Ｇ－１０（２４．４ｍｇ）を得た。 (Secondary fractionation)
Extract F-5 (250 mg) was adsorbed on diatomaceous earth, applied to an open column filled with ODS, and distilled water, acetonitrile, and ethyl acetate mixed at an arbitrary mixing ratio, or each using a single solvent, The extract was eluted. The eluted solution was collected in an amount of 8 ml each, and fractions eluted with acetonitrile: ethyl acetate = 7: 3 to ethyl acetate were combined and concentrated under reduced pressure to obtain a fraction G-10 (24.4 mg).
　（第三次分画）
　分画物Ｇ－１０（２４．４ｍｇ）を珪藻土に吸着させ、シリカゲルを充填したオープンカラムにアプライし，クロロホルムとメタノールを任意の混合割合で混合した溶媒、あるいはそれぞれ単独の溶媒を用いて、抽出物を溶出させた。溶出した液は１ｍｌずつ分取し、クロロホルム：メタノール＝４９：１で溶出された分画をまとめ、減圧濃縮して、単離成分２．６ｍｇを得た。 (Third fractionation)
Fraction G-10 (24.4 mg) was adsorbed on diatomaceous earth, applied to an open column packed with silica gel, and extracted with a solvent in which chloroform and methanol were mixed in any mixing ratio, or each with a single solvent. The product was eluted. The eluted solution was collected in 1 ml portions, and the fractions eluted with chloroform: methanol = 49: 1 were combined and concentrated under reduced pressure to obtain 2.6 mg of an isolated component.
　（構造解析）
　単離した成分は、赤外吸収スペクトル、1ＨＮＭＲスペクトル、13ＣＮＭＲスペクトルを解析した結果、5α,9α-epidioxy-(22E)-ergosta-7,22-diene-3β,6β-diolであることが分かった。 (Structural analysis)
As a result of analyzing the infrared absorption spectrum, 1 HNMR spectrum and 13 CNMR spectrum, the isolated component is 5α, 9α-epidioxy- (22E) -ergosta-7,22-diene-3β, 6β-diol. I understood.
（１）ヒト肺腺がん細胞の細胞死誘導試験 (1) Cell death induction test of human lung adenocarcinoma cells
ヒト肺腺がん細胞はＡ５４９細胞を使用した。 A549 cells were used as human lung adenocarcinoma cells.
　あらかじめ継代培養したヒト肺腺がん細胞を初回濃度１×１０４個/１００μｌの濃度に調整して平底プレート内で培養した。第三次分画で得られたエルゴステロール誘導体をそれぞれジメチルスルホキシドに溶解させ、濃度が０、２．５、５．０、７．５、１０．０ μｇ/ｍｌになるようにそれぞれ培地で希釈し、培養した細胞に添加した。このときのジメチルスルホキシドの濃度は培地に対して１％だった。当該添加後２４時間経過後、それぞれに細胞増殖測定試薬を添加し、さらに２時間経過後、それぞれの吸光度を測定し、その測定値から細胞生存率を算出した。なお、培地としては１０％牛胎児血清、１％ストレプトマイシン・ペニシリンを含むＲＰＭＩ１６４０を使用した。細胞生存率の算出方法はコントロール(１％ ジメチルスルホキシドを含む細胞培地で培養した細胞群)の吸光度を生存率１００％とし、エルゴステロール誘導体添加群の吸光度の値とコントロールの吸光度の値を比較して算出した。上記細胞生存率を図２に示した。本発明にかかるエルゴステロール誘導体は顕著にヒト肺腺がん細胞に対して細胞死を引き起こした。 Human lung adenocarcinoma cells subcultured in advance were adjusted to an initial concentration of 1 × 10 4 cells / 100 μl and cultured in a flat bottom plate. The ergosterol derivatives obtained in the third fractionation are dissolved in dimethyl sulfoxide, respectively, and diluted with medium to give concentrations of 0, 2.5, 5.0, 7.5, 10.0 μg / ml. And added to the cultured cells. The concentration of dimethyl sulfoxide at this time was 1% with respect to the medium. After 24 hours from the addition, a cell proliferation measurement reagent was added to each, and after another 2 hours, the absorbance was measured, and the cell viability was calculated from the measured values. As the medium, RPMI 1640 containing 10% fetal bovine serum and 1% streptomycin penicillin was used. The calculation method for cell viability is that the absorbance of the control (cell group cultured in a cell culture medium containing 1% dimethyl sulfoxide) is 100%, and the absorbance value of the ergosterol derivative added group is compared with the absorbance value of the control. Calculated. The cell viability is shown in FIG. The ergosterol derivative according to the present invention markedly caused cell death in human lung adenocarcinoma cells.
（２）アポトーシス誘導作用の検証 (2) Verification of apoptosis-inducing action
　あらかじめ継代培養したヒト肺腺がん細胞を初回濃度２×１０５個/２ｍｌで平底プレートに培養し、本発明にかかるエルゴステロール誘導体の濃度が０、１．０、４．０ μｇ/ｍｌなるようにそれぞれ培地で希釈し、その培養したヒト肺腺がん細胞に添加した。２４時間経過後に、TUNEL法に従い、フローサイトメトリーを用いてアポトーシス細胞を検出した。 Human lung adenocarcinoma cells previously subcultured are cultured on a flat bottom plate at an initial concentration of 2 × 10 5 cells / 2 ml, and the concentration of the ergosterol derivative according to the present invention is 0, 1.0, 4.0 μg / ml. Each was diluted with a medium and added to the cultured human lung adenocarcinoma cells. After 24 hours, apoptotic cells were detected using flow cytometry according to the TUNEL method.
　TUNEL法によるアポトーシス誘導作用の検証の結果は、図３に示したとおりで、アポトーシスが誘導された細胞を顕著に検出した。このことにより、本発明にかかるエルゴステロール誘導体が引き起こしたヒト肺腺がん細胞の細胞死は、アポトーシスであることが明確となった。 The results of verification of the apoptosis-inducing action by the TUNEL method are as shown in FIG. 3, and the cells in which apoptosis was induced were remarkably detected. This revealed that the cell death of human lung adenocarcinoma cells caused by the ergosterol derivative according to the present invention is apoptosis.
　(３)他のがん細胞へのアポトーシス誘導作用 (3) Induction of apoptosis in other cancer cells
ヒト肺腺がん細胞以外の細胞として、ヒト子宮頸がん細胞（ＨｅＬａ細胞）、ヒト舌がん細胞（ＨＳＣ－３細胞）を使用した。 Human cervical cancer cells (HeLa cells) and human tongue cancer cells (HSC-3 cells) were used as cells other than human lung adenocarcinoma cells.
　あらかじめ継代培養したヒト肺腺がん細胞、ヒト子宮頸がん細胞、ヒト舌がん細胞を初回濃度２×１０５個/２ｍｌで平底プレート内でそれぞれ培養した。エルゴステロール誘導体が５.０　μｇ/ｍｌになるように培地で希釈した後、平板プレート内で培養したそれぞれの細胞に添加した。２４時間経過後に、ＴＵＮＥＬ法に従い、フローサイトメトリーを用いてアポトーシス細胞を検出した。 Human lung adenocarcinoma cells, human cervical cancer cells and human tongue cancer cells subcultured in advance were each cultured in a flat bottom plate at an initial concentration of 2 × 10 5 cells / 2 ml. The ergosterol derivative was diluted with a medium so as to be 5.0 μg / ml, and then added to each cell cultured in a flat plate. After 24 hours, apoptotic cells were detected using flow cytometry according to the TUNEL method.
本発明にかかるエルゴステロール誘導体は、ヒト肺腺がん細胞、ヒト子宮頸がん細胞およびヒト舌がん細胞にアポトーシス誘導効果を発揮することが明白となった。 It became clear that the ergosterol derivative according to the present invention exerts an apoptosis-inducing effect on human lung adenocarcinoma cells, human cervical cancer cells and human tongue cancer cells.
　 本発明にかかるエルゴステロール誘導体は、肺腺がん、子宮頸がんおよび舌がんに対してアポトーシス誘導を起こさせる効果が期待でき、さらに食用のヒメマツタケから抽出ができるから食経験もあり、また天然素材のため安全性も期待でき、医薬品や健康食品の産業分野で利用することが可能である。 The ergosterol derivative according to the present invention can be expected to induce apoptosis in lung adenocarcinoma, cervical cancer, and tongue cancer, and can also be extracted from edible matsutake mushrooms. Because it is a natural material, it can be expected to be safe and can be used in the pharmaceutical and health food industries.

Claims (3)

  1. 　式（１）で表されるエルゴステロール誘導体を有効成分とするヒト肺腺がん細胞、ヒト舌がん細胞又はヒト子宮頸がん細胞の中から任意に選ばれる１種又は２種以上の細胞に対するアポトーシス誘導剤。　　
    Figure JPOXMLDOC01-appb-C000002
     ．．．．（１）     One or more cells selected arbitrarily from human lung adenocarcinoma cells, human tongue cancer cells or human cervical cancer cells containing an ergosterol derivative represented by formula (1) as an active ingredient Apoptosis-inducing agent.
    Figure JPOXMLDOC01-appb-C000002
     . . . . (1)
  2. 　ヒメマツタケから得ることができる脂溶性抽出物であって、式（１）で表されるエルゴステロール誘導体を有効成分として含有することを特徴とするヒト肺腺がん細胞、ヒト舌がん細胞又はヒト子宮頸がん細胞の中から任意に選ばれる１種又は２種以上の細胞に対するアポトーシス誘導剤。 A human soluble adenocarcinoma cell, human tongue cancer cell, or human, which is a fat-soluble extract obtainable from Himematsutake, which contains an ergosterol derivative represented by the formula (1) as an active ingredient An apoptosis inducer for one or more cells arbitrarily selected from cervical cancer cells.
  3. 　式（１）で表されるエルゴステロール誘導体を有効成分とするヒト肺腺がん細胞、ヒト舌がん細胞又はヒト子宮頸がん細胞の中から１種又は２種以上の細胞に対するアポトーシス誘導剤が、第一次抽出として、ヒメマツタケの乾燥粉末をプロトン性極性の有機溶媒から選ばれる１または２以上の有機溶媒で抽出物を得て、第二次抽出として、当該第一次抽出で得られた抽出物に蒸留水と低極性の有機溶媒から選ばれる１または２以上の有機溶媒とを加えて撹拌した後に分離して得られる有機溶媒層の濃縮物を得て、さらに、第二次抽出物の分画として、当該濃縮物をシリカゲルカラムに吸着させた後に、プロトン性極性の有機溶媒から選択される１ないし２以上の有機溶媒と低極性の有機溶媒から選択される１ないし２以上の有機溶媒を混合してなる有機溶媒混合液で溶出して複数の分画を得て、当該分画からヒト肺腺がん細胞、ヒト舌がん細胞又はヒト子宮頸がん細胞の中から任意に選ばれる１種又は２種以上の細胞にアポトーシスを起こさせる分画を選択して製造されることを特徴とするアポトーシス誘導剤の製造方法。 Apoptosis-inducing agent for one or more cells selected from human lung adenocarcinoma cells, human tongue cancer cells or human cervical cancer cells containing an ergosterol derivative represented by formula (1) as an active ingredient However, as a first extraction, a dried powder of Japanese apricot is obtained with one or more organic solvents selected from protic polar organic solvents, and obtained as a second extraction by the first extraction. To the extract, distilled water and one or more organic solvents selected from low-polar organic solvents are added and stirred and then separated to obtain an organic solvent layer concentrate. As a fraction of the product, after the concentrate is adsorbed on a silica gel column, 1 to 2 or more organic solvents selected from protic polar organic solvents and 1 or 2 or more organic solvents selected from low polar organic solvents are used. Mixed with organic solvent And a plurality of fractions are obtained by elution with an organic solvent mixture, and the fraction is arbitrarily selected from human lung adenocarcinoma cells, human tongue cancer cells or human cervical cancer cells from the fractions 1 A method for producing an apoptosis-inducing agent, characterized by being produced by selecting a fraction that causes apoptosis in a species or two or more cells.
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Publication number Priority date Publication date Assignee Title
JPH01246299A (en) * 1988-03-28 1989-10-02 Nichirei Corp Ergosterol derivative and production thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01246299A (en) * 1988-03-28 1989-10-02 Nichirei Corp Ergosterol derivative and production thereof

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Title
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YAOITA, Y. ET AL.: "New sterols from two edible mushrooms, Pleurotus eryngii and Panellus serotinus", CHEM PHARM BULL, vol. 50, no. 4, 2002, TOKYO, pages 551 - 3 *

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