JP5403942B2 - Glutathione production promoter and preventive / therapeutic agent for diseases caused by glutathione deficiency - Google Patents
Glutathione production promoter and preventive / therapeutic agent for diseases caused by glutathione deficiency Download PDFInfo
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- JP5403942B2 JP5403942B2 JP2008123940A JP2008123940A JP5403942B2 JP 5403942 B2 JP5403942 B2 JP 5403942B2 JP 2008123940 A JP2008123940 A JP 2008123940A JP 2008123940 A JP2008123940 A JP 2008123940A JP 5403942 B2 JP5403942 B2 JP 5403942B2
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- glutathione
- liquiritigenin
- isoliquiritigenin
- isoliquiritin
- lycochalcone
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Description
本発明は、グルタチオン産生促進剤、グルタチオンの欠乏に起因する疾患の予防・治療剤及び飲食品に関するものである。 The present invention relates to a glutathione production promoter, a preventive / therapeutic agent for diseases caused by deficiency of glutathione, and food and drink.
グルタチオンは、グルタミン酸、システイン、グリシンの３つのアミノ酸からなるトリペプチドであり、細胞内の主要なシステイン残基を有する化合物である。このグルタチオンは、細胞内においてラジカルの捕捉、酸化還元による細胞機能の調節、解毒機構への関与及び各種酵素のＳＨ供与体等としての役割を果たすことが知られている。 Glutathione is a tripeptide composed of three amino acids, glutamic acid, cysteine, and glycine, and is a compound having a major cysteine residue in the cell. This glutathione is known to play a role as a SH donor for various enzymes, such as radical scavenging, regulation of cell function by redox, involvement in detoxification mechanisms, and various enzymes.
このような役割を果たすグルタチオンの細胞内濃度が低下すると、紫外線曝露による細胞傷害、炎症、黒色化、シミ又はソバカスの生成、急性又は慢性アルコール肝障害、肝臓病、慢性腎不全、喫煙等が要因の肺疾患、特発性肺線維症、白内障、虚血性心疾患、パーキンソン病、アルツハイマー病、胃潰瘍、成人呼吸器障害症候群、免疫不全、骨髄形成不全、後天性免疫不全症候群、潜伏性ウイルス感染症、生理学的な加齢に伴う老化現象、及び癌化等の病状が引き起こされることが知られている。このようなグルタチオンの細胞内濃度の低下によって引き起こされる病状の治療には、従来、細胞内グルタチオン濃度の低下した細胞にグルタチオンを取り込ませることを目的として、グルタチオンを含有するグルタチオン製剤が用いられていた（特許文献１参照）。しかしながら、グルタチオン製剤の経口摂取による治療は、治療対象部位によっては思うような効果が期待できず、また、グルタチオン製剤の静脈注射による治療は、経口摂取による治療より効果は期待できるものの、痛みを伴うこと、通院が必要なこと等の問題を抱えていた。 When the intracellular concentration of glutathione that plays such a role decreases, factors such as cytotoxicity due to UV exposure, inflammation, blackening, generation of spots or freckles, acute or chronic alcohol liver injury, liver disease, chronic renal failure, smoking, etc. Lung disease, idiopathic pulmonary fibrosis, cataract, ischemic heart disease, Parkinson's disease, Alzheimer's disease, gastric ulcer, adult respiratory dysfunction syndrome, immunodeficiency, myelogenesis deficiency, acquired immune deficiency syndrome, latent viral infection, It is known that pathological conditions such as aging and canceration accompanying physiological aging are caused. In the treatment of pathological conditions caused by such a decrease in glutathione intracellular concentration, a glutathione-containing preparation containing glutathione has been conventionally used for the purpose of incorporating glutathione into cells having a decreased intracellular glutathione concentration. (See Patent Document 1). However, treatment with oral ingestion of glutathione preparations cannot be expected to be effective depending on the site to be treated, and treatment with intravenous injection of glutathione preparations is expected to be more effective than treatment with oral ingestion, but is painful And had problems such as the need to visit the hospital.
したがって、生体内細胞におけるグルタチオンの産生を促進することにより細胞内グルタチオン濃度を上昇させることができれば、加齢により衰える酸化ストレスに対する防御能を高めるとともに、紫外線の照射による酸化ストレスに対する傷害を抑制することにつながり、皮膚の老化、シミ等の色素沈着症の予防、治療又は改善が期待できるとともに、グルタチオンの欠乏によって起こる各種臓器の機能低下や種々の疾患の予防又は治療が期待できると考えられる。このような考えに基づき、グルタチオン産生促進作用を有するものとして、ビルベリー抽出物及びウォルナット抽出物（特許文献２参照）、クチナシ属植物の抽出物（特許文献３参照）等が知られている。 Therefore, if the intracellular glutathione concentration can be increased by promoting the production of glutathione in cells in vivo, the ability to protect against oxidative stress that declines with aging will be enhanced and the damage to oxidative stress caused by ultraviolet irradiation will be suppressed. Therefore, it is expected that the prevention, treatment or improvement of pigmentation diseases such as aging of skin and spots, and the prevention or treatment of various organ functions and various diseases caused by deficiency of glutathione are expected. Based on this idea, bilberry extract and walnut extract (see Patent Document 2), gardenia plant extract (see Patent Document 3), and the like are known as having glutathione production promoting action.
また、生体内において、グルタチオンは、活性酸素種を除去する作用を有する還元型グルタチオン、及び還元型グルタチオンと活性酸素種との反応により生産される酸化型グルタチオンとして存在する。近年、この酸化型グルタチオンが、神経細胞の興奮を抑制し、睡眠を促進する睡眠促進物質であることが確認されている。そのため、グルタチオンの産生を促進し、還元型グルタチオンのみならず、酸化型グルタチオンの細胞内濃度をも上昇させることができれば、不眠症等の睡眠障害の予防、治療又は改善に有効であると考えられている。このような考えに基づき、従来、酸化型グルタチオンを有効成分とする催眠剤が知られている（特許文献４参照）。 In vivo, glutathione exists as reduced glutathione having an action of removing reactive oxygen species, and oxidized glutathione produced by the reaction of reduced glutathione and reactive oxygen species. In recent years, it has been confirmed that this oxidized glutathione is a sleep promoting substance that suppresses excitation of nerve cells and promotes sleep. Therefore, if it is possible to promote the production of glutathione and increase the intracellular concentration of oxidized glutathione as well as reduced glutathione, it is considered effective for the prevention, treatment or improvement of sleep disorders such as insomnia. ing. Based on such an idea, a hypnotic agent containing oxidized glutathione as an active ingredient has been known (see Patent Document 4).
生体細胞においては、γ−グルタミルシステインシンテターゼの作用により、システインとグルタミン酸とが反応してγ−グルタミルシステインが生合成され、グルタチオンシンテターゼの作用により、γ−グルタミルシステインとグリシンとが反応してグルタチオンが生合成されることが知られている。そのため、グルタチオンの前駆体であるγ−グルタミルシステインを生合成するための触媒的役割を果たすγ−グルタミルシステインシンテターゼの発現を促進することで、生体細胞内グルタチオン濃度を上昇させることができると考えられる。
本発明は、安全性の高い天然物の中からグルタチオン産生促進作用及びγ−グルタミルシステインシンテターゼｍＲＮＡ発現促進作用を有するものを見出し、それを有効成分とするグルタチオン産生促進剤を提供することを目的とする。 It is an object of the present invention to provide a glutathione production promoter having an activity of promoting glutathione production and γ-glutamylcysteine synthetase mRNA expression among highly safe natural products and using it as an active ingredient. To do.
上記課題を解決するために、本発明のグルタチオン産生促進剤及びグルタチオンの欠乏に起因する疾患の予防・治療剤は、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡからなる群より選ばれる１種又は２種以上を有効成分として含有することを特徴とする。 In order to solve the above problems, the glutathione production promoter of the present invention and the prophylactic / therapeutic agent for diseases caused by glutathione deficiency are selected from the group consisting of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A. 1 type or 2 types or more are contained as an active ingredient, It is characterized by the above-mentioned.
また、本発明の飲食品は、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡからなる群より選ばれる１種又は２種以上を配合したことを特徴とする。 Moreover, the food / beverage products of this invention mix | blended 1 type (s) or 2 or more types chosen from the group which consists of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin, and lycochalcone A, It is characterized by the above-mentioned.
本発明によれば、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡからなる群より選ばれる１種又は２種以上を有効成分として含有し、安全性に優れたグルタチオン産生促進剤、グルタチオンの欠乏に起因する疾患の予防・治療剤及び飲食品を提供することができる。 According to the present invention, glutathione production promoter, glutathione excellent in safety, containing as an active ingredient one or more selected from the group consisting of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A It is possible to provide a prophylactic / therapeutic agent for diseases caused by deficiency of food and food and drink.
以下、本発明について説明する。
〔グルタチオン産生促進剤，グルタチオンの欠乏に起因する疾患の予防・治療剤〕
本発明のグルタチオン産生促進剤及びグルタチオンの欠乏に起因する疾患の予防・治療剤は、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡからなる群より選ばれる１種又は２種以上を有効成分として含有する。
The present invention will be described below.
[Glutathione production promoter, preventive and therapeutic agent for diseases caused by glutathione deficiency]
The glutathione production promoter and the prophylactic / therapeutic agent for diseases caused by glutathione deficiency according to the present invention are effective for one or more selected from the group consisting of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A. Contains as a component.
リクイリチン（liquiritin）、リクイリチゲニン（liquiritigenin）、イソリクイリチン（isoliquiritin）、イソリクイリチゲニン（isoliquiritigenin）及びリコカルコンＡ（licochalcone A）は、フラボノイド類の一種であり、これらの化合物を含有する植物抽出物から単離・精製することにより製造することができる。 Liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and licochalcone A are a type of flavonoids and are simply derived from plant extracts containing these compounds. It can be produced by separation and purification.
リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン又はリコカルコンＡを含有する植物抽出物は、植物の抽出に一般に用いられている方法によって得ることができる。リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン又はリコカルコンＡを含有する植物としては、例えば、甘草等が挙げられる。 A plant extract containing liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin or lycochalcone A can be obtained by a method generally used for plant extraction. Examples of plants containing liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin or lycochalcone A include licorice and the like.
甘草には、Glychyrrhiza glabra、Glychyrrhiza inflata、Glychyrrhiza uralensis、Glychyrrhiza aspera、Glychyrrhiza eurycarpa、Glychyrrhiza pallidiflora、Glychyrrhiza yunnanensis、Glychyrrhiza lepidota、Glychyrrhiza echinata、Glychyrrhiza acanthocarpa等、様々な種類のものがあり、これらのうち、いずれの種類の甘草を抽出原料として使用してもよいが、特にGlychyrrhiza glabraを抽出原料として使用することが好ましい。 Licorice includes Glychyrrhiza glabra, Glychyrrhiza inflata, Glychyrrhiza uralensis, Glychyrrhiza aspera, Glychyrrhiza eurycarpa, Glychyrrhiza pallidiflora, Glychyrrhiza yunnanensis, Glychyrrhiza lepidchy, Glychyrrhly Licorice may be used as an extraction raw material, but it is particularly preferable to use Glychyrrhiza glabra as an extraction raw material.
抽出原料として使用し得る甘草の構成部位としては、例えば、葉部、枝部、樹皮部、幹部、茎部、果実部、花部等の地上部、根部又はこれらの部位の混合物等が挙げられるが、好ましくは根部である。 Examples of the constituent parts of licorice that can be used as an extraction raw material include above-ground parts such as leaves, branches, bark parts, trunk parts, stem parts, fruit parts, flower parts, root parts, or a mixture of these parts. Is preferably the root.
リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン又はリコカルコンＡを含有する植物抽出物は、抽出原料を乾燥した後、そのまま又は粗砕機を用いて粉砕し、抽出溶媒による抽出に供することにより得ることができる。乾燥は天日で行ってもよいし、通常使用される乾燥機を用いて行ってもよい。また、ヘキサン等の非極性溶媒によって脱脂等の前処理を施してから抽出原料として使用してもよい。脱脂等の前処理を行うことにより、植物の極性溶媒による抽出処理を効率よく行うことができる。 The plant extract containing liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin or lycochalcone A can be obtained by drying the raw material for extraction, pulverizing it as it is or using a crusher, and subjecting it to extraction with an extraction solvent. it can. Drying may be performed in the sun or using a commonly used dryer. Moreover, after performing pretreatment, such as degreasing, with a nonpolar solvent such as hexane, it may be used as an extraction raw material. By performing pretreatment such as degreasing, extraction treatment with a polar solvent of a plant can be performed efficiently.
抽出溶媒としては、極性溶媒を用いるのが好ましく、例えば、水、親水性有機溶媒等が挙げられ、これらを単独で又は２種以上を組み合わせて、室温又は溶媒の沸点以下の温度で使用することが好ましい。 As the extraction solvent, it is preferable to use a polar solvent, and examples thereof include water and hydrophilic organic solvents. These may be used alone or in combination of two or more at room temperature or a temperature below the boiling point of the solvent. Is preferred.
抽出溶媒として使用し得る水としては、純水、水道水、井戸水、鉱泉水、鉱水、温泉水、湧水、淡水等のほか、これらに各種処理を施したものが含まれる。水に施す処理としては、例えば、精製、加熱、殺菌、濾過、イオン交換、浸透圧調整、酸性化、アルカリ化、緩衝化等が含まれる。したがって、本発明において抽出溶媒として使用し得る水には、精製水、熱水、イオン交換水、生理食塩水、有機酸酸性水、アンモニアアルカリ水、リン酸緩衝液、リン酸緩衝生理食塩水等も含まれる。 Examples of water that can be used as the extraction solvent include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, and those subjected to various treatments. Examples of the treatment applied to water include purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, acidification, alkalization, buffering, and the like. Accordingly, water that can be used as the extraction solvent in the present invention includes purified water, hot water, ion-exchanged water, physiological saline, organic acid acidic water, ammonia alkaline water, phosphate buffer, phosphate buffered saline, and the like. Is also included.
抽出溶媒として使用し得る親水性有機溶媒としては、メタノール、エタノール、プロピルアルコール、イソプロピルアルコール等の炭素数１〜５の低級脂肪族アルコール；アセトン、メチルエチルケトン等の低級脂肪族ケトン；１，３−ブチレングリコール、プロピレングリコール、グリセリン等の炭素数２〜５の多価アルコール等が挙げられる。 Examples of hydrophilic organic solvents that can be used as extraction solvents include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene. Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycol, propylene glycol and glycerin.
２種以上の極性溶媒の混合液を抽出溶媒として使用する場合、その混合比は適宜調整することができる。例えば、水と低級脂肪族アルコールとの混合液を使用する場合には、水１０容量部に対して低級脂肪族アルコール１〜９０容量部を混合するのが好ましく、水と低級脂肪族ケトンとの混合液を使用する場合には、水１０容量部に対して低級脂肪族ケトン１〜４０容量部を混合するのが好ましく、水と多価アルコールとの混合液を使用する場合には、水１０容量部に対して多価アルコール１０〜９０容量部を混合するのが好ましい。 When using the liquid mixture of 2 or more types of polar solvents as an extraction solvent, the mixing ratio can be adjusted suitably. For example, when using a mixed liquid of water and a lower aliphatic alcohol, it is preferable to mix 1 to 90 parts by volume of a lower aliphatic alcohol with respect to 10 parts by volume of water. When using a mixed solution, it is preferable to mix 1 to 40 parts by volume of a lower aliphatic ketone with 10 parts by volume of water, and when using a mixed solution of water and a polyhydric alcohol, water 10 It is preferable to mix 10 to 90 parts by volume of a polyhydric alcohol with respect to the volume part.
抽出処理は、抽出原料に含まれる可溶性成分を抽出溶媒に溶出させ得る限り特に限定はされず、常法に従って行うことができる。例えば、抽出原料の５〜１５倍量（質量比）の抽出溶媒に、抽出原料を浸漬し、常温又は還流加熱下で可溶性成分を抽出させた後、濾過して抽出残渣を除去することにより抽出液を得ることができる。得られた抽出液から溶媒を留去するとペースト状の濃縮物が得られ、この濃縮物をさらに乾燥すると乾燥物が得られる。 The extraction treatment is not particularly limited as long as the soluble component contained in the extraction raw material can be eluted in the extraction solvent, and can be performed according to a conventional method. For example, the extraction raw material is immersed in an extraction solvent 5 to 15 times (mass ratio) of the extraction raw material, the soluble components are extracted at room temperature or under reflux, and then filtered to remove the extraction residue. A liquid can be obtained. When the solvent is distilled off from the obtained extract, a paste-like concentrate is obtained, and when this concentrate is further dried, a dried product is obtained.
以上のようにして得られた抽出液、当該抽出液の濃縮物又は当該抽出液の乾燥物からリクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン又はリコカルコンＡを単離・精製する方法は、特に限定されるものではなく、常法により行うことができる。 The method for isolating and purifying liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin or lycochalcone A from the extract obtained as described above, the concentrate of the extract or the dried product of the extract is particularly limited. It can be carried out by a conventional method.
例えば、植物抽出物を、シリカゲルやアルミナ等の多孔質物質、スチレン−ジビニルベンゼン共重合体やポリメタクリレート等の多孔性樹脂等を用いたカラムクロマトグラフィーに付して、水、アルコールの順で溶出させ、アルコールで溶出される画分として得ることができる。 For example, a plant extract is subjected to column chromatography using a porous material such as silica gel or alumina, a porous resin such as styrene-divinylbenzene copolymer or polymethacrylate, and eluted in the order of water and alcohol. And can be obtained as a fraction eluted with alcohol.
カラムクロマトグラフィーにて溶出液として用いられるアルコールは、特に限定されるものではなく、例えば、メタノール、エタノール、プロピルアルコール、イソプロピルアルコール等の炭素数１〜５の低級脂肪族アルコール又はそれらの水溶液等が挙げられる。 The alcohol used as the eluent in the column chromatography is not particularly limited, and examples thereof include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, isopropyl alcohol, and their aqueous solutions. Can be mentioned.
さらに、カラムクロマトグラフィーにより得られたアルコール画分を、ＯＤＳを用いた逆相シリカゲルクロマトグラフィー、再結晶、液−液向流抽出、イオン交換樹脂を用いたカラムクロマトグラフィー等の任意の有機化合物精製手段を用いて精製してもよい。 Further, the alcohol fraction obtained by column chromatography can be purified by any organic compound such as reverse phase silica gel chromatography using ODS, recrystallization, liquid-liquid countercurrent extraction, column chromatography using ion exchange resin, etc. It may be purified using means.
以上のようにして得られるリクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン又はリコカルコンＡは、グルタチオン産生促進作用を有しているため、その作用を利用してグルタチオン産生促進剤の有効成分として使用することができる。なお、上記化合物は、γ−グルタミルシステインシンテターゼｍＲＮＡ発現促進作用も有しているため、その作用を利用してγ−グルタミルシステインシンテターゼｍＲＮＡ発現促進剤の有効成分としても使用することができる。 Since liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin or lycochalcone A obtained as described above has a glutathione production promoting action, it is used as an active ingredient of a glutathione production promoting agent using this action. be able to. In addition, since the said compound also has a gamma-glutamylcysteine synthetase mRNA expression promotion effect, it can be used also as an active ingredient of a gamma-glutamylcysteine synthetase mRNA expression promoter using the action.
また、上記化合物は、そのグルタチオン産生促進作用を通じて、グルタチオンの産生を促進することができるため、グルタチオンの欠乏に起因する疾患（例えば、活性酸素種等の酸化ストレスによって起こる疾患；肝炎、肝機能障害等の肝臓疾患；慢性腎不全；特発性肺線維症、成人呼吸器障害症候群等の呼吸器系疾患；白内障；虚血性心疾患；パーキンソン病；アルツハイマー病：胃潰瘍等の消化器系疾患；癌；骨髄形成不全；後天性免疫不全症候群；潜伏性ウイルス感染症等）の予防・治療剤の有効成分としても使用することができる。 Further, since the above compound can promote the production of glutathione through its glutathione production promoting action, diseases caused by deficiency of glutathione (for example, diseases caused by oxidative stress such as reactive oxygen species; hepatitis, liver dysfunction Liver diseases such as: chronic renal failure; idiopathic pulmonary fibrosis, respiratory diseases such as adult respiratory syndrome; cataract; ischemic heart disease; Parkinson's disease; Alzheimer's disease: gastrointestinal diseases such as gastric ulcer; It can also be used as an active ingredient of a prophylactic / therapeutic agent for bone marrow dysplasia; acquired immune deficiency syndrome; latent virus infection, etc.
さらに、上記化合物は、グルタチオン産生促進作用を有しており、その作用を利用して酸化型グルタチオンの細胞内濃度を上昇させることができるため、不眠症等の睡眠障害の予防、治療又は改善剤の有効成分としても使用することができる。 Furthermore, since the above compound has a glutathione production promoting action and can increase the intracellular concentration of oxidized glutathione using this action, it prevents, treats or improves sleep disorders such as insomnia It can also be used as an active ingredient.
なお、本発明のグルタチオン産生促進剤又はグルタチオンの欠乏に起因する疾患の予防・治療剤においては、上記化合物のうちのいずれか１種を上記有効成分として使用してもよいし、上記化合物より選ばれる２種以上の化合物を混合して上記有効成分として使用してもよい。上記化合物より選ばれる２種以上の化合物を混合して上記有効成分として使用する場合、それらの配合比は、それらの作用の程度に応じて適宜決定すればよい。 In the present invention, the glutathione production promoter or the prophylactic / therapeutic agent for diseases caused by glutathione deficiency, any one of the above compounds may be used as the active ingredient, or selected from the above compounds. Two or more compounds may be mixed and used as the active ingredient. When two or more compounds selected from the above compounds are mixed and used as the active ingredient, their blending ratio may be appropriately determined according to the degree of their action.
本発明のグルタチオン産生促進剤又はグルタチオンの欠乏に起因する疾患の予防・治療剤は、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡからなる群より選ばれる１種又は２種以上のみからなるものであってもよいし、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡからなる群より選ばれる１種又は２種以上を製剤化したものであってもよい。 The glutathione production promoter of the present invention or the prophylactic / therapeutic agent for diseases caused by glutathione deficiency is selected from only one or more selected from the group consisting of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A. It may be that prepared by formulating one or two or more selected from the group consisting of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A.
リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡからなる群より選ばれる１種又は２種以上の化合物は、デキストリン、シクロデキストリン等の薬学的に許容し得るキャリアーその他任意の助剤を使用して、常法に従い、粉末状、顆粒状、錠剤状、液状等の任意の剤形に製剤化することができる。この際、助剤としては、例えば、賦形剤、結合剤、崩壊剤、滑沢剤、安定剤、矯味・矯臭剤等を使用することができる。また、上記化合物は、他の組成物（例えば、飲食品等）に配合して使用することができるほか、軟膏剤、外用液剤、貼付剤等として使用することができる。 One or more compounds selected from the group consisting of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A use pharmaceutically acceptable carriers such as dextrin and cyclodextrin and other optional auxiliaries. Then, according to a conventional method, it can be formulated into an arbitrary dosage form such as powder, granule, tablet, and liquid. In this case, as an auxiliary agent, for example, an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a flavoring / flavoring agent, and the like can be used. Moreover, the said compound can be mix | blended and used for other compositions (for example, food-drinks etc.), and can be used as an ointment, an external solution, a patch, etc.
なお、本発明のグルタチオン産生促進剤又はグルタチオンの欠乏に起因する疾患の予防・治療剤は、必要に応じて、グルタチオン産生促進作用を有する他の天然抽出物等を配合して有効成分として使用することができる。 The glutathione production promoter of the present invention or the prophylactic / therapeutic agent for diseases caused by glutathione deficiency is used as an active ingredient, if necessary, with other natural extracts having a glutathione production promoting action. be able to.
本発明のグルタチオン産生促進剤又はグルタチオンの欠乏に起因する疾患の予防・治療剤の投与方法としては、一般に経皮投与、経口投与、静脈投与等が挙げられるが、疾患の種類に応じて、その予防・治療等に好適な方法を適宜選択すればよい。 Examples of the administration method of the glutathione production promoter or the prophylactic / therapeutic agent for diseases caused by glutathione deficiency according to the present invention include transdermal administration, oral administration, intravenous administration, etc., depending on the type of the disease, A suitable method for prevention / treatment or the like may be appropriately selected.
また、本発明のグルタチオン産生促進剤又はグルタチオンの欠乏に起因する疾患の予防・治療剤の投与量も、疾患の種類、重症度、患者の個人差、投与方法、投与期間等によって適宜増減すればよい。 Further, the dose of the glutathione production promoter or the prophylactic / therapeutic agent for diseases caused by glutathione deficiency of the present invention can be appropriately increased or decreased depending on the type, severity, individual differences of patients, administration method, administration period, etc. Good.
本発明のグルタチオン産生促進剤は、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡからなる群より選ばれる１種又は２種以上の化合物が有するグルタチオン産生促進作用を通じて、加齢により衰える酸化ストレスに対する防御能を高めるとともに、紫外線の照射による酸化ストレスに対する傷害を抑制することができ、皮膚の老化、シミ等の色素沈着症や、グルタチオンの欠乏によって起こる各種臓器の機能低下等による種々の疾患を予防、治療又は改善することができる。ただし、本発明のグルタチオン産生促進剤は、これらの用途以外にもグルタチオン産生促進作用を発揮することに意義のあるすべての用途に使用することができる。 The glutathione production promoter of the present invention is an oxidation that decays with aging through a glutathione production promoting action of one or more compounds selected from the group consisting of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A. Various diseases caused by increased ability to protect against stress, suppress damage to oxidative stress caused by UV irradiation, pigmentation such as skin aging and spots, and decreased function of various organs caused by glutathione deficiency Can be prevented, treated or ameliorated. However, the glutathione production promoter of the present invention can be used for all purposes other than these uses that are meaningful for exerting the glutathione production promoting action.
後述する実施例において示されるように、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡは、皮膚線維芽細胞及び表皮角化細胞におけるグルタチオンの産生を効果的に促進することができるため、これらの化合物を含有するグルタチオン産生促進剤によれば、皮膚線維芽細胞及び表皮角化細胞内で活性酸素種を消去することができ、皮膚線維芽細胞及び表皮角化細胞を酸化的ストレスによる傷害等から保護することができ、結果として、皮膚の老化等を効果的に防止することができる。 As shown in the examples described later, liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A can effectively promote the production of glutathione in dermal fibroblasts and epidermal keratinocytes, According to the glutathione production promoter containing these compounds, reactive oxygen species can be eliminated in skin fibroblasts and epidermal keratinocytes, and skin fibroblasts and epidermal keratinocytes are damaged by oxidative stress. As a result, it is possible to effectively prevent skin aging and the like.
また、グルタチオンがチロシナーゼの成熟化を抑制する作用を有していることから、グルタチオンの産生を促進することによってチロシナーゼの成熟化をより抑制することができ、メラノサイトにおけるメラニンの生成を抑制することができる。そのため、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡからなる群より選ばれる１種又は２種以上を含有するグルタチオン産生促進剤によれば、メラノサイトにおけるグルタチオンの産生を効果的に促進し、チロシナーゼの成熟化をより抑制することができるため、結果として、皮膚色素沈着症、皮膚の黒化、シミ、ソバカス等を予防することができる。 In addition, since glutathione has an action of suppressing tyrosinase maturation, it is possible to further suppress tyrosinase maturation by promoting the production of glutathione and to suppress the production of melanin in melanocytes. it can. Therefore, according to the glutathione production promoter containing one or more selected from the group consisting of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A, it effectively promotes the production of glutathione in melanocytes. Since tyrosinase maturation can be further suppressed, skin pigmentation, skin darkening, spots, buckwheat, and the like can be prevented as a result.
さらに、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡは、肝細胞におけるグルタチオンの産生を効果的に促進することができるため、これらの化合物を含有するグルタチオン産生促進剤は、グルタチオンの欠乏に起因する肝臓疾患を効果的に予防・治療することができる。 Furthermore, since liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A can effectively promote glutathione production in hepatocytes, glutathione production promoters containing these compounds are deficient in glutathione. It is possible to effectively prevent and treat liver diseases caused by the disease.
なお、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡからなる群より選ばれる１種又は２種以上の化合物をγ−グルタミルシステインシンテターゼｍＲＮＡ発現促進剤に含有せしめることで、これらの化合物が有するγ−グルタミルシステインシンテターゼｍＲＮＡ発現促進作用を通じて、グルタチオンの前駆体であるγ−グルタミルシステインの生合成を促進することができる。 By adding one or more compounds selected from the group consisting of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A to the γ-glutamylcysteine synthetase mRNA expression promoter, these compounds can be used. The biosynthesis of γ-glutamylcysteine, which is a precursor of glutathione, can be promoted through the action of promoting γ-glutamylcysteine synthetase mRNA expression.
本発明のグルタチオンの欠乏に起因する疾患の予防・治療剤は、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡからなる群より選ばれる１種又は２種以上の化合物が有するグルタチオン産生促進作用を通じて、グルタチオンの欠乏に起因する疾患（例えば、活性酸素種等の酸化ストレスによって起こる疾患；肝炎、肝機能障害等の肝臓疾患；慢性腎不全；特発性肺線維症、成人呼吸器障害症候群等の呼吸器系疾患；白内障；虚血性心疾患；パーキンソン病；アルツハイマー病：胃潰瘍等の消化器系疾患；癌；骨髄形成不全；後天性免疫不全症候群；潜伏性ウイルス感染症等）を予防・治療することができるとともに、酸化型グルタチオンの細胞内濃度を上昇させることができるため、不眠症等の睡眠障害を予防、治療又は改善することができる。 The prophylactic / therapeutic agent for diseases caused by glutathione deficiency according to the present invention is glutathione production promotion possessed by one or more compounds selected from the group consisting of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A. Diseases caused by glutathione deficiency through action (for example, diseases caused by oxidative stress such as reactive oxygen species; liver diseases such as hepatitis and liver dysfunction; chronic renal failure; idiopathic pulmonary fibrosis, adult respiratory disorder syndrome, etc. Respiratory system disease; Cataract; Ischemic heart disease; Parkinson's disease; Alzheimer's disease: Gastrointestinal system diseases such as gastric ulcer; Cancer; Myelogenesis deficiency; Acquired immune deficiency syndrome; Insomnia because it can increase the intracellular concentration of oxidized glutathione The sleep disorders prevention, can be treated or improved.
〔飲食品〕
本発明の飲食品は、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡからなる群より選ばれる１種又は２種以上を配合したものである。
[Food and Drink]
The food / beverage products of this invention mix | blend 1 type (s) or 2 or more types chosen from the group which consists of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin, and lycochalcone A.
リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡは、グルタチオン産生促進作用を有しており、消化管で消化されるようなものではないことが確認されており、また安全性に優れているため、一般食品、健康食品、保健機能食品又は栄養補助食品等、任意の飲食品に配合するのに好適である。この場合、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡからなる群より選ばれる１種又は２種以上の化合物をそのまま配合してもよいし、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡからなる群より選ばれる１種又は２種以上の化合物により製剤化したグルタチオン産生促進剤を配合してもよい。 Liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A have a glutathione production promoting action, and it has been confirmed that they are not digested in the digestive tract, and are excellent in safety. Therefore, it is suitable for blending into any food or drink such as general food, health food, health functional food or nutritional supplement. In this case, one or two or more compounds selected from the group consisting of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A may be blended as they are, or liquiritin, liquiritigenin, isoliquiritin, isoliquiritigen it may be blended formulated glutathione production enhancer with one or more compounds selected from the group consisting of threonine and licochalcone a.
上記リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡからなる群より選ばれる１種若しくは２種以上の化合物、又はグルタチオン産生促進剤を飲食品に配合する場合、それらにおける有効成分の配合量は、使用目的、性別、症状等を考慮して適宜調整することができるが、添加対象飲食品の一般的な摂取量を考慮して成人１日あたりの化合物摂取量が約０．１〜１０００ｍｇになるようにするのが好ましい。 In the case where one or more compounds selected from the group consisting of the above-mentioned liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A, or a glutathione production promoter is added to food and drink, the amount of the active ingredient in them Can be adjusted as appropriate in consideration of the purpose of use, gender, symptoms, etc., but taking into account the general intake of foods and drinks to be added, the daily intake of the compound is about 0.1 to 1000 mg It is preferable that
上記リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡからなる群より選ばれる１種若しくは２種以上の化合物、又はグルタチオン産生促進剤を配合し得る飲食品は、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡの有するグルタチオン産生促進作用を妨げない限り、特に限定されるものではない。 The liquiritin, liquiritigenin, isoliquiritin, one or more compounds selected from the group consisting of isoliquiritigenin and licochalcone A, or food or drink can be incorporated glutathione production enhancer is liquiritin, liquiritigenin, isoliquiritin, iso It is not particularly limited as long as it does not interfere with the glutathione production promoting action of liquiritigenin and lycochalcone A.
具体例としては、清涼飲料、炭酸飲料、栄養飲料、果実飲料、乳酸飲料等の飲料（これらの飲料の濃縮原液及び調整用粉末を含む）；アイスクリーム、アイスシャーベット、かき氷等の冷菓；そば、うどん、はるさめ、ぎょうざの皮、しゅうまいの皮、中華麺、即席麺等の麺類；飴、チューインガム、キャンディー、ガム、チョコレート、錠菓、スナック菓子、ビスケット、ゼリー、ジャム、クリーム、焼き菓子等の菓子類；かまぼこ、ハム、ソーセージ等の水産・畜産加工食品；加工乳、発酵乳等の乳製品；サラダ油、てんぷら油、マーガリン、マヨネーズ、ショートニング、ホイップクリーム、ドレッシング等の油脂及び油脂加工食品；ソース、たれ等の調味料；スープ、シチュー、サラダ、惣菜、漬物；その他種々の形態の健康・栄養補助食品；錠剤、カプセル剤、ドリンク剤などに上記リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡからなる群より選ばれる１種若しくは２種以上の化合物、又はグルタチオン産生促進剤を配合することができ、このとき、通常用いられる補助的な原料や添加物を併用することができる。 Specific examples include beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, and lactic acid drinks (including concentrated concentrates and powders for adjustment of these drinks); frozen desserts such as ice cream, ice sherbet and shaved ice; Noodles such as udon, harusame, gyoza skin, sweet husk, Chinese noodles, instant noodles; confectionery such as rice cakes, chewing gum, candy, gum, chocolate, tablet confectionery, snacks, biscuits, jelly, jam, cream, baked goods Fishery and livestock processed foods such as kamaboko, ham and sausage; dairy products such as processed milk and fermented milk; processed oils and fats such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream and dressing; sauces, sauce Seasonings such as soups, stews, salads, prepared dishes, pickles; various other forms of health and nutrition Aid food; tablets, capsules, the liquiritin the like drinks, formulated liquiritigenin, isoliquiritin, one or more compounds selected from the group consisting of isoliquiritigenin and licochalcone A, or a glutathione production enhancer At this time, commonly used auxiliary raw materials and additives can be used in combination.
なお、本発明のグルタチオン産生促進剤、グルタチオンの欠乏に起因する疾患の予防・治療剤又は飲食品は、ヒトに対して好適に適用されるものであるが、それぞれの作用効果が奏される限り、ヒト以外の動物に対して適用することもできる。 The glutathione production promoter of the present invention, the preventive / therapeutic agent for diseases caused by deficiency of glutathione, or foods and drinks are preferably applied to humans, as long as each effect is exhibited. It can also be applied to animals other than humans.
以下、製造例、試験例及び配合例を示し、本発明を具体的に説明するが、本発明は下記の各例に何ら制限されるものではない。 Hereinafter, although a manufacture example, a test example, and a compounding example are shown and this invention is demonstrated concretely, this invention is not restrict | limited to each following example at all.
〔製造例１〕リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡの製造
甘草（Glycyrrhiza inflata）の根部５００ｇに５０容量％エタノール（水とエタノールとの容量比＝１：１）５０００ｍＬを加え、穏やかに撹拌しながら８０℃にて２時間保ち、熱時濾過した。得られた抽出液を４０℃で減圧下にて濃縮し、減圧乾燥機で乾燥して甘草根部５０容量％エタノール抽出物（１２４ｇ）を得た。
[Production Example 1] Production of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A To 500 g of licorice (Glycyrrhiza inflata) roots, 5000 mL of 50 vol% ethanol (volume ratio of water to ethanol = 1: 1) was added. The mixture was kept at 80 ° C. for 2 hours with gentle stirring and filtered while hot. The obtained extract was concentrated under reduced pressure at 40 ° C. and dried with a vacuum dryer to obtain a 50% by volume ethanol extract (124 g) of licorice root.
このようにして得られた甘草根部５０容量％エタノール抽出物１００ｇに水１Ｌを加えて懸濁させ、多孔性樹脂（商品名：ＤＩＡＩＯＮ ＨＰ−２０，和光純薬社製）１Ｌ上に付し、水２０００ｍＬ、３０容量％エタノール２０００ｍＬ、７０容量％エタノール２０００ｍＬ及び９９容量％エタノール２０００ｍＬの順で溶出させた。 1 L of water was added to 100 g of 50% by volume ethanol extract of licorice root thus obtained, suspended, and attached to 1 L of porous resin (trade name: DIAION HP-20, manufactured by Wako Pure Chemical Industries, Ltd.) Elution was performed in the order of 2000 mL of water, 2000 mL of 30% ethanol by volume, 2000 mL of 70% ethanol by volume, and 2000 mL of 99% ethanol by volume.
このようにして得られた９９容量％エタノール画分（固形分：２．８ｇ）を６０容量％メタノール（水とメタノールの容量比＝４：６）に溶解し、ＯＤＳカラム（商品名：クロマトレックスＯＤＳ ＤＭ１０２０Ｔ，富士シリシア化学社製）に付し、移動相として６０容量％メタノール（水とメタノールとの容量比＝４：６）を用いて溶出させ分離し、画分１、画分２及び画分３を得た。得られた画分１、２及び３をそれぞれ減圧下で濃縮し、画分１の濃縮物（２００ｍｇ）、画分２の濃縮物（３００ｍｇ）及び画分３の濃縮物（１００ｍｇ）を得た。 The 99 volume% ethanol fraction (solid content: 2.8 g) thus obtained was dissolved in 60 volume% methanol (volume ratio of water to methanol = 4: 6), and an ODS column (trade name: Chromatrex). ODS DM1020T (manufactured by Fuji Silysia Chemical Co., Ltd.) and eluted and separated using 60% by volume methanol (volume ratio of water to methanol = 4: 6) as the mobile phase, fraction 1, fraction 2 and fraction Minute 3 was obtained. The obtained fractions 1, 2 and 3 were respectively concentrated under reduced pressure to obtain a concentrate of fraction 1 (200 mg), a concentrate of fraction 2 (300 mg) and a concentrate of fraction 3 (100 mg). .
得られた画分１の濃縮物、画分２の濃縮物及び画分３の濃縮物のそれぞれにメタノール１０ｍＬと水１０ｍＬとを加えて一晩放置し、再結晶させることで、それぞれの精製物（１２５ｍｇ（試料２），１８５ｍｇ（試料４），６７ｍｇ（試料５））を得た。 To each of the obtained fraction 1 concentrate, fraction 2 concentrate and fraction 3 concentrate, 10 mL of methanol and 10 mL of water were added and allowed to stand overnight, followed by recrystallization. (125 mg (sample 2), 185 mg (sample 4), 67 mg (sample 5)).
上記７０容量％エタノール画分（固形分：１０．０ｇ）を４０容量％メタノール（水とメタノールの容量比＝６：４）に溶解し、ＯＤＳカラム（商品名：クロマトレックスＯＤＳ ＤＭ１０２０Ｔ，富士シリシア化学社製）に付し、移動相として４０容量％メタノール（水とメタノールとの容量比＝６：４）を用いて溶出させ分離し、画分４及び画分５を得た。得られた画分４及び５をそれぞれ減圧下で濃縮し、画分４の濃縮物（６．７ｇ）及び画分５の濃縮物（３．２ｇ）を得た。得られた画分４の濃縮物及び画分５の濃縮物をそれぞれクロロホルム：メタノール＝５：１（容量比）の混合液に溶解し、ＳｉＯ２カラム（商品名：シリカゲル６０，富士シリシア化学社製）に付し、移動相としてクロロホルム：メタノール＝５：１（容量比）の混合液を用いて溶出させ分離した後、減圧下濃縮した。得られたそれぞれの濃縮物を下記の液体クロマトグラフィーを用いて分画した。 The above 70 volume% ethanol fraction (solid content: 10.0 g) was dissolved in 40 volume% methanol (volume ratio of water to methanol = 6: 4), and ODS column (trade name: Chromatorex ODS DM1020T, Fuji Silysia Chemical Co., Ltd.) And eluted with 40% by volume methanol (volume ratio of water to methanol = 6: 4) as a mobile phase to obtain fraction 4 and fraction 5. The obtained fractions 4 and 5 were respectively concentrated under reduced pressure to obtain a concentrate of fraction 4 (6.7 g) and a concentrate of fraction 5 (3.2 g). The obtained concentrate of fraction 4 and concentrate of fraction 5 were dissolved in a mixed solution of chloroform: methanol = 5: 1 (volume ratio), respectively, and SiO 2 column (trade name: silica gel 60, Fuji Silysia Chemical Ltd.). The product was eluted and separated using a mixed solution of chloroform: methanol = 5: 1 (volume ratio) as a mobile phase, and then concentrated under reduced pressure. Each obtained concentrate was fractionated using the following liquid chromatography.
＜液体クロマトグラフィー条件＞
固定相：ＪＡＩＧＥＬ ＧＳ−３１０（日本分析工業社製）
カラム径：２０ｍｍ
カラム長：５００ｍｍ
移動相流量：５ｍＬ／ｍｉｎ
検出：ＲＩ
<Liquid chromatography conditions>
Stationary phase: JAIGEL GS-310 (manufactured by Nippon Analytical Industrial Co., Ltd.)
Column diameter: 20mm
Column length: 500mm
Mobile phase flow rate: 5 mL / min
Detection: RI
上記液体クロマトグラフィーにて保持時間６０分〜７０分に流出する画分を、リサイクルＨＰＬＣにより精製することで、精製物（３５０ｍｇ，２７０ｍｇ，試料１，３）をそれぞれ得た。上述のようにして得られた各精製物（試料１〜５）を１３Ｃ−ＮＭＲにより分析した結果を下記に示す。 Purified products (350 mg, 270 mg, Samples 1 and 3) were obtained by purifying fractions flowing out at a retention time of 60 minutes to 70 minutes by the above liquid chromatography by recycle HPLC. The result of analyzing each purified product (samples 1 to 5) obtained as described above by 13 C-NMR is shown below.
＜試料１の１３Ｃ−ＮＭＲケミカルシフトδ（帰属炭素，ＤＭＳＯ−ｄ６）：＞
43.1(3-C)，60.7(6''-C)，69.7(4''-C)，73.1(2''-C)，76.5(3''-C)，76.9(5''-C)，78.5(2-C)，100.3(1''-C)，102.4(8-C)，110.4(6-C)，113.4(10-C)，116.1(3',5'-C)，127.7(2',6'-C)，128.1(5-C)，132.2(1'-C)，157.2(4'-C)，162.8(9-C)，164.4(7-C)，189.5(4-C)
< 13 C-NMR chemical shift δ of sample 1 (assigned carbon, DMSO-d6):>
43.1 (3-C), 60.7 (6 ''-C), 69.7 (4 ''-C), 73.1 (2 ''-C), 76.5 (3 ''-C), 76.9 (5 ''-C ), 78.5 (2-C), 100.3 (1 ''-C), 102.4 (8-C), 110.4 (6-C), 113.4 (10-C), 116.1 (3 ', 5'-C), 127.7 (2 ', 6'-C), 128.1 (5-C), 132.2 (1'-C), 157.2 (4'-C), 162.8 (9-C), 164.4 (7-C), 189.5 ( 4-C)
＜試料２の１３Ｃ−ＮＭＲケミカルシフトδ（帰属炭素，ＤＭＳＯ−ｄ６）：＞
43.1(3-C)，78.8(2-C)，102.4(8-C)，110.3(6-C)，113.4(10-C)，114.9(3',5'-C)，127.9(2',6'-C)，127.9(5-C)，129.1(1'-C)，157.3(4'-C)，162.9(9-C)，164.3(7-C)，189.7(4-C)
< 13 C-NMR chemical shift δ of sample 2 (assigned carbon, DMSO-d6):>
43.1 (3-C), 78.8 (2-C), 102.4 (8-C), 110.3 (6-C), 113.4 (10-C), 114.9 (3 ', 5'-C), 127.9 (2' , 6'-C), 127.9 (5-C), 129.1 (1'-C), 157.3 (4'-C), 162.9 (9-C), 164.3 (7-C), 189.7 (4-C)
＜試料３の１３Ｃ−ＮＭＲケミカルシフトδ（帰属炭素，ＤＭＳＯ−ｄ６）：＞
60.6(6''-C)，69.6(4''-C)，73.1(2''-C)，76.5(3''-C)，77.0(5''-C)，99.9(1''-C)，102.4(3'-C)，108.0(5'-C)，112.8(1'-C)，116.3(3',5'-C)，119.0(α-C)，128.2(1-C)，130.4(2',6'-C)，132.7(6'-C)，143.2(β-C)，159.2(4'-C)，164.9(2'-C)，165.5(4'-C)，191.2(C=O)
< 13 C-NMR chemical shift δ of sample 3 (assigned carbon, DMSO-d6):>
60.6 (6 ''-C), 69.6 (4 ''-C), 73.1 (2 ''-C), 76.5 (3 ''-C), 77.0 (5 ''-C), 99.9 (1 '' -C), 102.4 (3'-C), 108.0 (5'-C), 112.8 (1'-C), 116.3 (3 ', 5'-C), 119.0 (α-C), 128.2 (1- C), 130.4 (2 ', 6'-C), 132.7 (6'-C), 143.2 (β-C), 159.2 (4'-C), 164.9 (2'-C), 165.5 (4'- C), 191.2 (C = O)
＜試料４の１３Ｃ−ＮＭＲケミカルシフトδ（帰属炭素，ＤＭＳＯ−ｄ６）：＞
102.4(3'-C)，107.9(5'-C)，112.8(1'-C)，115.6(3',5'-C)，117.3(α-C)，125.6(1-C)，130.9(2',6'-C)，132.5(6'-C)，143.9(β-C)，156.0(4'-C)，164.6(2'-C)，165.4(4'-C)，191.2(C=O)
< 13 C-NMR chemical shift δ of sample 4 (assigned carbon, DMSO-d6):>
102.4 (3'-C), 107.9 (5'-C), 112.8 (1'-C), 115.6 (3 ', 5'-C), 117.3 (α-C), 125.6 (1-C), 130.9 (2 ', 6'-C), 132.5 (6'-C), 143.9 (β-C), 156.0 (4'-C), 164.6 (2'-C), 165.4 (4'-C), 191.2 (C = O)
＜試料５の１３Ｃ−ＮＭＲケミカルシフトδ（帰属炭素，ＣＤＣｌ３）：＞
27.1(4''',5'''-C)，39.8(1'''-C)，55.6(OMe)，101.1(3-C)，113.8(3'''-C)，115.6(3',5'-C)，116.3(1-C)，120.0(α-C)，124.6(5-C)，128.8(6-C)，130.8(1'-C)，131.2(2',6'-C)，141.4(β-C)，147.6(2'''-C)，158.3(2-C)，159.5(4-C)，160.9(4'-C)，190.8(C=O)
< 13 C-NMR chemical shift δ of sample 5 (assigned carbon, CDCl3):>
27.1 (4 ''',5'''-C), 39.8 (1 '''-C), 55.6 (OMe), 101.1 (3-C), 113.8 (3'''-C), 115.6 (3 ', 5'-C), 116.3 (1-C), 120.0 (α-C), 124.6 (5-C), 128.8 (6-C), 130.8 (1'-C), 131.2 (2', 6 '-C), 141.4 (β-C), 147.6 (2'''-C), 158.3 (2-C), 159.5 (4-C), 160.9 (4'-C), 190.8 (C = O)
以上の結果から、得られた各精製物のそれぞれが、リクイリチン（試料１）、リクイリチゲニン（試料２）、イソリクイリチン（試料３）、イソリクイリチゲニン（試料４）及びリコカルコンＡ（試料５）であることが確認された。 From the above results, each of the obtained purified products was liquiritin (sample 1), liquiritigenin (sample 2), isoliquiritin (sample 3), isoliquiritigenin (sample 4) and lycochalcone A (sample 5). It was confirmed that there was.
〔試験例１〕表皮角化細胞におけるグルタチオン産生促進作用試験
上記のようにして得られたリクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡ（試料１〜５）について、以下のようにして表皮角化細胞におけるグルタチオン産生促進作用を試験した。
[Test Example 1] Glutathione production promoting action test in epidermal keratinocytes About liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A (samples 1 to 5) obtained as described above, as follows. The glutathione production promoting effect in epidermal keratinocytes was tested.
正常ヒト新生児包皮表皮角化細胞（Normal Human Epidermal Keratinocyte：ＮＨＥＫ）を、正常ヒト表皮角化細胞長期培養用増殖培地（EpiLife-KG2）を用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞を１．０×１０５ｃｅｌｌｓ／ｍＬの細胞密度になるようにEpiLife-KG2で希釈した後、４８ウェルプレートに１ウェル当たり２００μＬずつ播種し、一晩培養した。 Normal human epidermal keratinocytes (NHEK) were cultured using normal human epidermal keratinocyte long-term culture growth medium (EpiLife-KG2), and then cells were collected by trypsin treatment. The collected cells were diluted with EpiLife-KG2 to a cell density of 1.0 × 10 5 cells / mL, seeded in a 48-well plate at 200 μL per well, and cultured overnight.
培養後、EpiLife-KG2で溶解した試料溶液（試料１〜５，試料濃度は下記表１を参照）を各ウェルに２００μＬ添加し、２４時間培養した。培養終了後、各ウェルから培地を抜き、４００μＬのＰＢＳ(−)にて洗浄後、１５０μＬのＭ−ＰＥＲ（ＰＩＥＲＣＥ社製）を使用して細胞を溶解した。 After culturing, 200 μL of a sample solution dissolved in EpiLife-KG2 (samples 1 to 5, refer to Table 1 below for the sample concentration) was added to each well and cultured for 24 hours. After completion of the culture, the medium was removed from each well, washed with 400 μL of PBS (−), and cells were lysed using 150 μL of M-PER (PIERCE).
このうちの１００μＬを使用して総グルタチオンの定量を行った。すなわち、９６ウェルプレートに溶解した細胞抽出液１００μＬ、０．１Ｍのリン酸緩衝液５０μＬ、２ｍＭのＮＡＤＰＨ２５μＬ及びグルタチオンレダクターゼ２５μＬ（終濃度１７．５ｕｎｉｔ／ｍＬ）を加え３７℃で１０分間加温した後、１０ｍＭの5,5'-dithiobis(2-nitrobenzoic acid)２５μＬを加え、５分後までの波長４１２ｎｍにおける吸光度を測定し、ΔＯＤ／ｍｉｎを求めた。総グルタチオン濃度は、酸化型グルタチオン（和光純薬社製）を使用して作成した検量線をもとに算出した。得られた値を総タンパク量当たりのグルタチオン量に補正した後、下記式に基づいてグルタチオン産生促進率（％）を算出した。 Of these, 100 μL was used to quantify total glutathione. That is, after adding 100 μL of cell extract dissolved in 96-well plate, 50 μL of 0.1 M phosphate buffer, 25 μL of 2 mM NADPH and 25 μL of glutathione reductase (final concentration 17.5 units / mL), the mixture was heated at 37 ° C. for 10 minutes. 25 μL of 10 mM 5,5′-dithiobis (2-nitrobenzoic acid) was added, and the absorbance at a wavelength of 412 nm until 5 minutes was measured to determine ΔOD / min. The total glutathione concentration was calculated based on a calibration curve prepared using oxidized glutathione (manufactured by Wako Pure Chemical Industries, Ltd.). After correcting the obtained value to the amount of glutathione per total protein amount, the glutathione production promotion rate (%) was calculated based on the following formula.
グルタチオン産生促進率(％)＝Ｂ／Ａ×１００
式中、Ａは「試料無添加時の細胞中における総タンパク量当たりのグルタチオン量（対照）」を表し、Ｂは「試料添加時の細胞中における総タンパク量当たりのグルタチオン量」を表す。
結果を表１に示す。
Glutathione production promotion rate (%) = B / A × 100
In the formula, A represents “the amount of glutathione per total protein in the cell when no sample is added (control)”, and B represents “the amount of glutathione per total amount of protein in the cell when added to the sample”.
The results are shown in Table 1.
表１に示すように、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡは、いずれも表皮角化細胞においてグルタチオンの産生を効果的に促進し得ることが確認された。 As shown in Table 1, it was confirmed that all of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A can effectively promote the production of glutathione in epidermal keratinocytes.
〔試験例２〕皮膚線維芽細胞におけるグルタチオン産生促進作用試験
上記のようにして得られたリクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡ（試料１〜５）について、以下のようにして皮膚線維芽細胞におけるグルタチオン産生促進作用を試験した。
[Test Example 2] Glutathione production promoting action test in skin fibroblasts About liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A (samples 1 to 5) obtained as described above, as follows. The effect of promoting glutathione production in dermal fibroblasts was examined.
ヒト正常皮膚線維芽細胞（ＮＢ１ＲＧＢ）を、１０％ＦＢＳ含有α−ＭＥＭを用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞を２．０×１０５ｃｅｌｌｓ／ｍＬの細胞密度になるように１０％ＦＢＳ含有α−ＭＥＭで希釈した後、４８ウェルプレートに１ウェルあたり２００μＬずつ播種し、一晩培養した。 Human normal skin fibroblasts (NB1RGB) were cultured using α-MEM containing 10% FBS, and then cells were collected by trypsin treatment. The collected cells were diluted with α-MEM containing 10% FBS so as to have a cell density of 2.0 × 10 5 cells / mL, then seeded at 200 μL per well in a 48-well plate and cultured overnight.
培養後、１％ＦＢＳ含有Ｄ−ＭＥＭで溶解した試料溶液（試料１〜５，試料濃度は下記表２を参照）を各ウェルに２００μＬ添加し、２４時間培養した。培養終了後、各ウェルから培地を抜き、４００μＬのＰＢＳ(−)にて洗浄後、１５０μＬのＭ−ＰＥＲ（ＰＩＥＲＣＥ社製）を用いて細胞を溶解した。 After culturing, 200 μL of a sample solution dissolved in D-MEM containing 1% FBS (Samples 1 to 5, see Table 2 below for the sample concentration) was added to each well and cultured for 24 hours. After completion of the culture, the medium was removed from each well, washed with 400 μL of PBS (−), and the cells were lysed using 150 μL of M-PER (PIERCE).
このうちの１００μＬを使用して総グルタチオンの定量を行った。すなわち、９６ウェルプレートに溶解した細胞抽出液１００μＬ、０．１Ｍのリン酸緩衝液５０μＬ、２ｍＭのＮＡＤＰＨ２５μＬ及びグルタチオンレダクターゼ２５μＬ（終濃度１７．５ｕｎｉｔ／ｍＬ）を加え３７℃で１０分間加温した後、１０ｍＭの5,5'-dithiobis(2-nitrobenzoic acid)２５μＬを加え、５分後までの波長４１２ｎｍにおける吸光度を測定し、ΔＯＤ／ｍｉｎを求めた。総グルタチオン濃度は、酸化型グルタチオン（和光純薬社製）を使用して作成した検量線をもとに算出した。得られた値を総タンパク量当たりのグルタチオン量に補正した後、下記式に基づいてグルタチオン産生促進率（％）を算出した。 Of these, 100 μL was used to quantify total glutathione. That is, after adding 100 μL of cell extract dissolved in 96-well plate, 50 μL of 0.1 M phosphate buffer, 25 μL of 2 mM NADPH and 25 μL of glutathione reductase (final concentration 17.5 units / mL), the mixture was heated at 37 ° C. for 10 minutes. 25 μL of 10 mM 5,5′-dithiobis (2-nitrobenzoic acid) was added, and the absorbance at a wavelength of 412 nm until 5 minutes was measured to determine ΔOD / min. The total glutathione concentration was calculated based on a calibration curve prepared using oxidized glutathione (manufactured by Wako Pure Chemical Industries, Ltd.). After correcting the obtained value to the amount of glutathione per total protein amount, the glutathione production promotion rate (%) was calculated based on the following formula.
グルタチオン産生促進率(％)＝Ｂ／Ａ×１００
式中、Ａは「試料無添加時の細胞中における総タンパク量当たりのグルタチオン量（対照）」を表し、Ｂは「試料添加時の細胞中における総タンパク量当たりのグルタチオン量」を表す。
結果を表２に示す。
Glutathione production promotion rate (%) = B / A × 100
In the formula, A represents “the amount of glutathione per total protein in the cell when no sample is added (control)”, and B represents “the amount of glutathione per total amount of protein in the cell when added to the sample”.
The results are shown in Table 2.
表２に示すように、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡは、いずれも皮膚線維芽細胞においてグルタチオンの産生を効果的に促進し得ることが確認された。 As shown in Table 2, it was confirmed that all of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A can effectively promote the production of glutathione in dermal fibroblasts.
〔試験例３〕Ｂ１６メラノーマ細胞におけるグルタチオン産生促進作用試験
上記のようにして得られたリクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡ（試料１〜５）について、以下のようにしてＢ１６メラノーマ細胞におけるグルタチオン産生促進作用を試験した。
[Test Example 3] Glutathione production promoting action test in B16 melanoma cells About the liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A (samples 1 to 5) obtained as described above, B16 was prepared as follows. The effect of promoting glutathione production in melanoma cells was examined.
Ｂ１６メラノーマ細胞を、１０％ＦＢＳ含有Ｄ−ＭＥＭ培地を用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞を１．０×１０５ｃｅｌｌｓ／ｍＬの細胞密度になるように１０％ＦＢＳ含有Ｄ−ＭＥＭ培地で希釈した後、４８ウェルプレートに１ウェル当たり２００μＬずつ播種し、一晩培養した。 B16 melanoma cells were cultured using D-MEM medium containing 10% FBS, and then cells were collected by trypsin treatment. The collected cells were diluted with 10% FBS-containing D-MEM medium to a cell density of 1.0 × 10 5 cells / mL, then seeded at 200 μL per well in a 48-well plate and cultured overnight.
培養後、１％ＦＢＳ含有Ｄ−ＭＥＭ培地で溶解した試料溶液（試料１〜５，試料濃度は下記表３を参照）を各ウェルに２００μＬ添加し、２４時間培養した。培養終了後、各ウェルから培地を抜き、４００μＬのＰＢＳ(−)にて洗浄後、１５０μＬのＭ−ＰＥＲ（ＰＩＥＲＣＥ社製）を使用して細胞を溶解した。 After culturing, 200 μL of a sample solution dissolved in 1% FBS-containing D-MEM medium (Samples 1 to 5, see Table 3 below for sample concentration) was added to each well and cultured for 24 hours. After completion of the culture, the medium was removed from each well, washed with 400 μL of PBS (−), and cells were lysed using 150 μL of M-PER (PIERCE).
このうちの１００μＬを使用して総グルタチオンの定量を行った。すなわち、９６ウェルプレートに溶解した細胞抽出液１００μＬ、０．１Ｍのリン酸緩衝液５０μＬ、２ｍＭのＮＡＤＰＨ２５μＬ及びグルタチオンレダクターゼ２５μＬ（終濃度１７．５ｕｎｉｔ／ｍＬ）を加え３７℃で１０分間加温した後、１０ｍＭの5,5'-dithiobis(2-nitrobenzoic acid)２５μＬを加え、５分後までの波長４１２ｎｍにおける吸光度を測定し、ΔＯＤ／ｍｉｎを求めた。総グルタチオン濃度は、酸化型グルタチオン（和光純薬社製）を使用して作成した検量線をもとに算出した。得られた値を総タンパク量当たりのグルタチオン量に補正した後、下記式に基づいてグルタチオン産生促進率（％）を算出した。 Of these, 100 μL was used to quantify total glutathione. That is, after adding 100 μL of cell extract dissolved in 96-well plate, 50 μL of 0.1 M phosphate buffer, 25 μL of 2 mM NADPH and 25 μL of glutathione reductase (final concentration 17.5 units / mL), the mixture was heated at 37 ° C. for 10 minutes. 25 μL of 10 mM 5,5′-dithiobis (2-nitrobenzoic acid) was added, and the absorbance at a wavelength of 412 nm until 5 minutes was measured to determine ΔOD / min. The total glutathione concentration was calculated based on a calibration curve prepared using oxidized glutathione (manufactured by Wako Pure Chemical Industries, Ltd.). After correcting the obtained value to the amount of glutathione per total protein amount, the glutathione production promotion rate (%) was calculated based on the following formula.
グルタチオン産生促進率(％)＝Ｂ／Ａ×１００
式中、Ａは「試料無添加時の細胞中における総タンパク量当たりのグルタチオン量（対照）」を表し、Ｂは「試料添加時の細胞中における総タンパク量当たりのグルタチオン量」を表す。
結果を表３に示す。
Glutathione production promotion rate (%) = B / A × 100
In the formula, A represents “the amount of glutathione per total protein in the cell when no sample is added (control)”, and B represents “the amount of glutathione per total amount of protein in the cell when added to the sample”.
The results are shown in Table 3.
表３に示すように、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡは、いずれもＢ１６メラノーマ細胞においてグルタチオンの産生を効果的に促進し得ることが確認された。 As shown in Table 3, it was confirmed that all of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A can effectively promote glutathione production in B16 melanoma cells.
〔試験例４〕肝細胞におけるグルタチオン産生促進作用試験
上記のようにして得られたリクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡ（試料１〜５）について、以下のようにして肝細胞におけるグルタチオン産生促進作用を試験した。
[Test Example 4] Test for promoting glutathione production in hepatocytes About the liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A (samples 1 to 5) obtained as described above, hepatocytes were obtained as follows. Were tested for their glutathione production promoting effect.
正常ヒト肝細胞（Cell System-Hc Cells，セルシステムズ社）を、ＣＳ−Ｃ無血清培地（セルシステムズ社製）を用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞を１．０×１０５ｃｅｌｌｓ／ｍＬの細胞密度になるようにＣＳ−Ｃ無血清培地で希釈した後、２４ウェルプレートに１ウェルあたり４００μＬずつ播種し、一晩培養した。 Normal human hepatocytes (Cell System-Hc Cells, Cell Systems) were cultured using CS-C serum-free medium (Cell Systems), and then cells were collected by trypsin treatment. The collected cells were diluted with CS-C serum-free medium to a cell density of 1.0 × 10 5 cells / mL, then seeded in a 24-well plate at 400 μL per well and cultured overnight.
培養後、ＣＳ−Ｃ無血清培地で溶解した試料溶液（試料１〜５，試料濃度は下記表４を参照）を各ウェルに４００μＬ添加し、２４時間培養した。培養終了後、各ウェルから培地を抜き、４００μＬのＰＢＳ(−)にて洗浄後、２５０μＬのＭ−ＰＥＲ（ＰＩＥＲＣＥ社製）を用いて細胞を溶解した。 After the culture, 400 μL of a sample solution dissolved in a CS-C serum-free medium (samples 1 to 5, see the following Table 4 for sample concentration) was added to each well and cultured for 24 hours. After completion of the culture, the medium was removed from each well, washed with 400 μL of PBS (−), and cells were lysed using 250 μL of M-PER (manufactured by PIERCE).
このうちの１００μＬを用いて総グルタチオンの定量を行った。すなわち、９６ウェルプレートに溶解した細胞抽出液１００μＬ、０．１Ｍのリン酸緩衝液５０μＬ、２ｍＭのＮＡＤＰＨ２５μＬ及びグルタチオンレダクターゼ２５μＬ（終濃度１７．５ｕｎｉｔ／ｍＬ）を加え３７℃で１０分間加温した後、１０ｍＭの5,5'-dithiobis(2-nitrobenzoic acid)２５μＬを加え、５分後までの波長４１２ｎｍにおける吸光度を測定し、ΔＯＤ／ｍｉｎを求めた。総グルタチオン濃度は、酸化型グルタチオン（和光純薬社製）を使用して作成した検量線をもとに算出した。得られた値を総タンパク量当たりのグルタチオン量に補正した後、下記式に基づいてグルタチオン産生促進率（％）を算出した。 The total glutathione was quantified using 100 μL of this. That is, after adding 100 μL of cell extract dissolved in 96-well plate, 50 μL of 0.1 M phosphate buffer, 25 μL of 2 mM NADPH and 25 μL of glutathione reductase (final concentration 17.5 units / mL), the mixture was heated at 37 ° C. for 10 minutes. 25 μL of 10 mM 5,5′-dithiobis (2-nitrobenzoic acid) was added, and the absorbance at a wavelength of 412 nm until 5 minutes was measured to determine ΔOD / min. The total glutathione concentration was calculated based on a calibration curve prepared using oxidized glutathione (manufactured by Wako Pure Chemical Industries, Ltd.). After correcting the obtained value to the amount of glutathione per total protein amount, the glutathione production promotion rate (%) was calculated based on the following formula.
グルタチオン産生促進率(％)＝Ｂ／Ａ×１００
式中、Ａは「試料無添加時の細胞中における総タンパク量当たりのグルタチオン量（対照）」を表し、Ｂは「試料添加時の細胞中における総タンパク量当たりのグルタチオン量」を表す。
結果を表４に示す。
Glutathione production promotion rate (%) = B / A × 100
In the formula, A represents “the amount of glutathione per total protein in the cell when no sample is added (control)”, and B represents “the amount of glutathione per total amount of protein in the cell when added to the sample”.
The results are shown in Table 4.
表４に示すように、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡは、いずれも肝細胞においてグルタチオンの産生を促進し得ることが確認された。 As shown in Table 4, it was confirmed that liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A can all promote the production of glutathione in hepatocytes.
〔試験例５〕表皮角化細胞におけるγ−グルタミルシステインシンテターゼｍＲＮＡ発現促進作用試験
上記のようにして得られたリクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡ（試料１〜５）について、以下のようにして表皮角化細胞におけるγ−グルタミルシステインシンテターゼｍＲＮＡ発現促進作用を試験した。
[Test Example 5] γ-glutamylcysteine synthetase mRNA expression promoting action test in epidermal keratinocytes About liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A (samples 1 to 5) obtained as described above, The γ-glutamylcysteine synthetase mRNA expression promoting action in epidermal keratinocytes was tested as follows.
正常ヒト新生児表皮角化細胞（Normal Human Epidermal Keratinocyte：ＮＨＥＫ）を、正常ヒト表皮角化細胞長期培養増殖培地（EpiLife-KG2）を用いた培養した後、トリプシン処理により細胞を回収した。回収した細胞を１．０×１０５ｃｅｌｌｓ／ｍＬの細胞密度になるようにEpiLife-KG2で希釈した後、３５ｍｍシャーレに２．５ｍＬずつ播種し、一晩培養した。 Normal human epidermal keratinocytes (NHEK) were cultured using normal human epidermal keratinocyte long-term culture growth medium (EpiLife-KG2), and then cells were collected by trypsin treatment. The collected cells were diluted with EpiLife-KG2 to a cell density of 1.0 × 10 5 cells / mL, and then seeded at 2.5 mL in a 35 mm petri dish and cultured overnight.
培養後、EpiLife-KG2で溶解した試料溶液（試料１〜５，試料濃度は下記表５を参照）を２．５ｍＬ添加し、さらに１６時間培養した。培養終了後、培養液を捨て、ＩＳＯＧＥＮ（ニッポンジーン社製，Cat.No.311-02501）にて総ＲＮＡを抽出し、それぞれのＲＮＡ量を分光光度計にて測定し、２００μｇ／ｍＬになるように総ＲＮＡを調整した。 After the culture, 2.5 mL of a sample solution dissolved with EpiLife-KG2 (samples 1 to 5, see the following Table 5 for the sample concentration) was added, and further cultured for 16 hours. After completion of the culture, the culture solution is discarded, total RNA is extracted with ISOGEN (Nippon Gene, Cat. No. 311-02501), and the amount of each RNA is measured with a spectrophotometer so that it becomes 200 μg / mL. Total RNA was prepared.
この総ＲＮＡを鋳型とし、γ−グルタミルシステインシンテターゼ及び内部標準であるＧＡＰＤＨのｍＲＮＡの発現量を測定した。検出はリアルタイムＰＣＲ装置Smart Cycler（Cepheid社製）を用いて、TaKaRa SYBR PrimeScriptTM RT-PCR Kit（Perfect Real Time，code No. RR063A）によるリアルタイム２ＳｔｅｐＲＴ−ＰＣＲ反応により行った。γ−グルタミルシステインシンテターゼのｍＲＮＡの発現量は、同一サンプルにおけるＧＡＰＤＨのｍＲＮＡの発現量の値で補正を行った後、さらに「試料無添加時」の補正値を１００としたときの「試料添加時」の補正値を算出した後に、下記式に基づいてγ−グルタミルシステインシンテターゼのｍＲＮＡの発現促進率（％）を算出した。 Using this total RNA as a template, the expression levels of γ-glutamylcysteine synthetase and GAPDH mRNA as an internal standard were measured. Detection was performed by real-time 2 Step RT-PCR reaction using TaKaRa SYBR PrimeScript ™ RT-PCR Kit (Perfect Real Time, code No. RR063A) using a real-time PCR device Smart Cycler (Cepheid). The expression level of γ-glutamylcysteine synthetase mRNA is corrected with the value of the expression level of GAPDH mRNA in the same sample, and then the correction value of “when no sample is added” is set to 100 ”Was calculated, and then the expression promotion rate (%) of γ-glutamylcysteine synthetase mRNA was calculated based on the following formula.
ｍＲＮＡ発現促進率(％)＝Ｂ／Ａ×１００
式中、Ａは「試料無添加時のｍＲＮＡ発現量（対照）」を表し、Ｂは「試料添加時のｍＲＮＡ発現量」を表す。
結果を表５に示す。
mRNA expression promotion rate (%) = B / A × 100
In the formula, A represents “mRNA expression level when no sample is added (control)” and B represents “mRNA expression level when sample is added”.
The results are shown in Table 5.
表５に示すように、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡは、いずれも表皮角化細胞においてγ−グルタミルシステインシンテターゼｍＲＮＡの発現を効果的に促進し得ることが確認された。 As shown in Table 5, it was confirmed that all of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A can effectively promote the expression of γ-glutamylcysteine synthetase mRNA in epidermal keratinocytes. .
〔試験例６〕皮膚線維芽細胞におけるγ−グルタミルシステインシンテターゼｍＲＮＡ発現促進作用試験
上記のようにして得られたリクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡ（試料１〜５）について、以下のようにして皮膚線維芽細胞におけるγ−グルタミルシステインシンテターゼｍＲＮＡ発現促進作用を試験した。
[Test Example 6] γ-glutamylcysteine synthetase mRNA expression promoting action test in dermal fibroblasts About liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A (samples 1 to 5) obtained as described above, The γ-glutamylcysteine synthetase mRNA expression promoting effect in skin fibroblasts was tested as follows.
ヒト正常皮膚線維芽細胞（ＮＢ１ＲＧＢ）を、１０％ＦＢＳ含有α−ＭＥＭを用いた培養した後、トリプシン処理により細胞を回収した。回収した細胞を２．０×１０５ｃｅｌｌｓ／ｍＬの細胞密度になるように１０％ＦＢＳ含有アルファ−ＭＥＭで希釈した後、３５ｍｍシャーレに２．５ｍＬずつ播種し、一晩培養した。 Human normal skin fibroblasts (NB1RGB) were cultured using α-MEM containing 10% FBS, and then cells were collected by trypsin treatment. The collected cells were diluted with 10% FBS-containing alpha-MEM so as to have a cell density of 2.0 × 10 5 cells / mL, then seeded with 2.5 mL each in a 35 mm petri dish, and cultured overnight.
培養後、１％ＦＢＳ含有Ｄ−ＭＥＭで溶解した試料溶液（試料１〜５，試料濃度は下記表６を参照）を２．５ｍＬ添加し、さらに１６時間培養した。培養終了後、培養液を捨て、ＩＳＯＧＥＮ（ニッポンジーン社製，Cat.No.311-02501）にて総ＲＮＡを抽出し、それぞれのＲＮＡ量を分光光度計にて測定し、２００μｇ／ｍＬになるように総ＲＮＡを調整した。 After culturing, 2.5 mL of a sample solution (Samples 1 to 5, see Table 6 below for sample concentration) dissolved in D-MEM containing 1% FBS was added, and further cultured for 16 hours. After completion of the culture, the culture solution is discarded, total RNA is extracted with ISOGEN (Nippon Gene, Cat. No. 311-02501), and the amount of each RNA is measured with a spectrophotometer so that it becomes 200 μg / mL. Total RNA was prepared.
この総ＲＮＡを鋳型とし、γ−グルタミルシステインシンテターゼ及び内部標準であるＧＡＰＤＨのｍＲＮＡの発現量を測定した。検出はリアルタイムＰＣＲ装置Smart Cycler（Cepheid社製）を用いて、TaKaRa SYBR PrimeScriptTM RT-PCR Kit（Perfect Real Time，code No. RR063A）によるリアルタイム２ＳｔｅｐＲＴ−ＰＣＲ反応により行った。γ−グルタミルシステインシンテターゼのｍＲＮＡの発現量は、同一サンプルにおけるＧＡＰＤＨのｍＲＮＡの発現量の値で補正を行った後、さらに「試料無添加時」の補正値を１００としたときの「試料添加時」の補正値を算出した後に、下記式に基づいてγ−グルタミルシステインシンテターゼのｍＲＮＡの発現促進率（％）を算出した。 Using this total RNA as a template, the expression levels of γ-glutamylcysteine synthetase and GAPDH mRNA as an internal standard were measured. Detection was performed by real-time 2 Step RT-PCR reaction using TaKaRa SYBR PrimeScript ™ RT-PCR Kit (Perfect Real Time, code No. RR063A) using a real-time PCR device Smart Cycler (Cepheid). The expression level of γ-glutamylcysteine synthetase mRNA is corrected with the value of the expression level of GAPDH mRNA in the same sample, and then the correction value of “when no sample is added” is set to 100 ”Was calculated, and then the expression promotion rate (%) of γ-glutamylcysteine synthetase mRNA was calculated based on the following formula.
ｍＲＮＡ発現促進率(％)＝Ｂ／Ａ×１００
式中、Ａは「試料無添加時のｍＲＮＡ発現量（対照）」を表し、Ｂは「試料添加時のｍＲＮＡ発現量」を表す。
結果を表６に示す。
mRNA expression promotion rate (%) = B / A × 100
In the formula, A represents “mRNA expression level when no sample is added (control)” and B represents “mRNA expression level when sample is added”.
The results are shown in Table 6.
表６に示すように、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡは、いずれも皮膚線維芽細胞においてγ−グルタミルシステインシンテターゼｍＲＮＡの発現を効果的に促進し得ることが確認された。 As shown in Table 6, it was confirmed that all of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A can effectively promote the expression of γ-glutamylcysteine synthetase mRNA in skin fibroblasts. .
〔試験例７〕肝細胞におけるγ−グルタミルシステインシンテターゼｍＲＮＡ発現促進作用試験
上記のようにして得られたリクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡ（試料１〜５）について、以下のようにして肝細胞におけるγ−グルタミルシステインシンテターゼｍＲＮＡ発現促進作用を試験した。
[Test Example 7] γ-glutamylcysteine synthetase mRNA expression promoting action test in hepatocytes About liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A (samples 1 to 5) obtained as described above, Thus, the γ-glutamylcysteine synthetase mRNA expression promoting action in hepatocytes was tested.
正常ヒト肝細胞（Cell System-Hc Cells，セルシステムズ社）を、ＣＳ−Ｃ無血清培地（セルシステムズ社製）を用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞を１．０×１０５ｃｅｌｌｓ／ｍＬの細胞密度になるようにＣＳ−Ｃ無血清培地で希釈した後、３５ｍｍシャーレに２．５ｍＬずつ播種し、一晩培養した。 Normal human hepatocytes (Cell System-Hc Cells, Cell Systems) were cultured using CS-C serum-free medium (Cell Systems), and then cells were collected by trypsin treatment. The collected cells were diluted with CS-C serum-free medium to a cell density of 1.0 × 10 5 cells / mL, then seeded at 2.5 mL in a 35 mm dish, and cultured overnight.
培養後、ＣＳ−Ｃ無血清培地で溶解した試料溶液（試料１〜５，試料濃度は下記表７を参照）を２．５ｍＬ添加し、さらに１６時間培養した。培養終了後、培養液を捨て、ＩＳＯＧＥＮ（ニッポンジーン社製，Cat.No.311-02501）にて総ＲＮＡを抽出し、それぞれのＲＮＡ量を分光光度計にて測定し、２００μｇ／ｍＬになるように総ＲＮＡを調整した。 After culturing, 2.5 mL of a sample solution dissolved in CS-C serum-free medium (Samples 1 to 5, see Table 7 below for sample concentration) was added, and further cultured for 16 hours. After completion of the culture, the culture solution is discarded, total RNA is extracted with ISOGEN (Nippon Gene, Cat. No. 311-02501), and the amount of each RNA is measured with a spectrophotometer so that it becomes 200 μg / mL. Total RNA was prepared.
この総ＲＮＡを鋳型とし、γ−グルタミルシステインシンテターゼ及び内部標準であるＧＡＰＤＨのｍＲＮＡの発現量を測定した。検出はリアルタイムＰＣＲ装置Smart Cycler（Cepheid社製）を用いて、TaKaRa SYBR PrimeScript RT-PCR Kit（Perfect Real Time，code No. RR063A）によるリアルタイム２ＳｔｅｐＲＴ−ＰＣＲ反応により行った。γ−グルタミルシステインシンテターゼのｍＲＮＡの発現量は、同一サンプルにおけるＧＡＰＤＨのｍＲＮＡの発現量の値で補正を行った後、さらに「試料無添加時」の補正値を１００としたときの「試料添加時」の補正値を算出した後に、下記式に基づいてγ−グルタミルシステインシンテターゼのｍＲＮＡの発現促進率（％）を算出した。 Using this total RNA as a template, the expression levels of γ-glutamylcysteine synthetase and GAPDH mRNA as an internal standard were measured. Detection was performed by real-time 2 Step RT-PCR reaction using TaKaRa SYBR PrimeScript RT-PCR Kit (Perfect Real Time, code No. RR063A) using a real-time PCR device Smart Cycler (Cepheid). The expression level of γ-glutamylcysteine synthetase mRNA is corrected with the value of the expression level of GAPDH mRNA in the same sample, and then the correction value of “when no sample is added” is set to 100 ”Was calculated, and then the expression promotion rate (%) of γ-glutamylcysteine synthetase mRNA was calculated based on the following formula.
ｍＲＮＡ発現促進率(％)＝Ｂ／Ａ×１００
式中、Ａは「試料無添加時のｍＲＮＡ発現量（対照）」を表し、Ｂは「試料添加時のｍＲＮＡ発現量」を表す。
結果を表７に示す。
mRNA expression promotion rate (%) = B / A × 100
In the formula, A represents “mRNA expression level when no sample is added (control)” and B represents “mRNA expression level when sample is added”.
The results are shown in Table 7.
表７に示すように、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡは、いずれも肝細胞においてγ−グルタミルシステインシンテターゼｍＲＮＡの発現を効果的に促進し得ることが確認された。 As shown in Table 7, it was confirmed that all of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A can effectively promote the expression of γ-glutamylcysteine synthetase mRNA in hepatocytes.
上述した試験例１〜４から明らかなように、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡは、いずれも優れたグルタチオン産生促進作用を有している。また、試験例５〜７から明らかなように、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡは、いずれも優れたγ−グルタミルシステインシンテターゼｍＲＮＡ発現促進作用を有している。このことから、リクイリチン、リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡは、γ−グルタミルシステインシンテターゼｍＲＮＡ発現促進作用を通じて、グルタチオンの前駆体であるγ−グルタミルシステインの生合成を促進し、それにより生体内でのグルタチオンの産生を促進しているものと推察される。 As is clear from Test Examples 1 to 4 described above, all of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A have an excellent glutathione production promoting action. Further, as is clear from Test Examples 5 to 7, all of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A have an excellent γ-glutamylcysteine synthetase mRNA expression promoting action. From this, liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A promote the biosynthesis of γ-glutamylcysteine, which is a precursor of glutathione, through the action of promoting γ-glutamylcysteine synthetase mRNA expression. It is presumed that the production of glutathione in vivo is promoted.
〔配合例１〕
常法により、以下の組成を有する錠剤を製造した。
リクイリチン（試料１） ７．５ｍｇ
イソリクイリチゲニン（試料４） ３．５ｍｇ
リコカルコンＡ（試料５） ４．０ｍｇ
乳精ミネラル（カルシウム２５〜３０％含有） １００．０ｍｇ
ビタミンＫ２（１％含有粉末） １．５ｍｇ
マルチトール １７１．０ｍｇ
グリセリン脂肪酸エステル １２．５ｍｇ
[Formulation Example 1]
A tablet having the following composition was produced by a conventional method.
Liquiritin (sample 1) 7.5mg
Isoliquiritigenin (Sample 4) 3.5mg
Lycochalcone A (Sample 5) 4.0 mg
Milky mineral (containing 25-30% calcium) 100.0mg
Vitamin K 2 (1% powder) 1.5mg
Maltitol 171.0mg
Glycerin fatty acid ester 12.5mg
〔配合例２〕
常法により、以下の組成を有する錠剤を製造した。
リクイリチゲニン（試料２） ３．０ｍｇ
イソリクイリチゲニン（試料４） ２．０ｍｇ
ドロマイト（カルシウム２０％、マグネシウム１０％含有） ８３．４ｍｇ
カゼインホスホペプチド １６．７ｍｇ
ビタミンＣ ３３．４ｍｇ
マルチトール １３６．８ｍｇ
コラーゲン １２．７ｍｇ
ショ糖脂肪酸エステル １２．０ｍｇ
[Formulation Example 2]
A tablet having the following composition was produced by a conventional method.
Liquiritigenin (sample 2) 3.0mg
Isoliquiritigenin (Sample 4) 2.0mg
Dolomite (containing 20% calcium and 10% magnesium) 83.4mg
Casein phosphopeptide 16.7mg
Vitamin C 33.4mg
Maltitol 136.8mg
Collagen 12.7mg
Sucrose fatty acid ester 12.0mg
〔配合例３〕
常法により、以下の組成を有するカプセル剤を製造した。なお、カプセルとしては、１号ハードゼラチンカプセルを使用した。
＜１カプセル（１錠２００ｍｇ）中の組成＞
イソリクイリチン（試料３） ４．０ｍｇ
イソリクイリチゲニン（試料４） ４．０ｍｇ
リコカルコンＡ（試料５） ２．０ｍｇ
コーンスターチ ７０．０ｍｇ
乳糖 １００．０ｍｇ
乳酸カルシウム １０．０ｍｇ
ヒドロキシプロピルセルロース（ＨＰＣ−Ｌ） １０．０ｍｇ
[Composition Example 3]
Capsules having the following composition were produced by a conventional method. As the capsule, No. 1 hard gelatin capsule was used.
<Composition in 1 capsule (1 tablet 200 mg)>
Isoliquiritin (Sample 3) 4.0mg
Isoliquiritigenin (Sample 4) 4.0mg
Lycochalcone A (Sample 5) 2.0mg
Corn starch 70.0mg
Lactose 100.0mg
Calcium lactate 10.0mg
Hydroxypropylcellulose (HPC-L) 10.0mg
〔配合例４〕
常法により、以下の組成を有する経口液状製剤を製造した。
＜１アンプル（１本１００ｍＬ）中の組成＞
リクイリチン（試料１） ０．２質量％
イソリクイリチゲニン（試料４） ０．１質量％
ソルビット １２．０質量％
安息香酸ナトリウム ０．１質量％
香料 １．０質量％
硫酸カルシウム ０．５質量％
精製水 残部（１００質量％）
[Formulation Example 4]
By an ordinary method, an oral liquid preparation having the following composition was produced.
<Composition in one ampoule (one 100 mL)>
Liquiritin (Sample 1) 0.2% by mass
Isoliquiritigenin (Sample 4) 0.1% by mass
Sorbit 12.0% by mass
Sodium benzoate 0.1% by mass
Fragrance 1.0% by mass
Calcium sulfate 0.5% by mass
Purified water balance (100% by mass)
本発明のグルタチオン産生促進剤は、皮膚の老化、シミ、紫外線の照射による酸化ストレスに対する傷害、グルタチオンの欠乏に起因する疾患（例えば、活性酸素種等の酸化ストレスによって起こる疾患；肝炎、肝機能障害等の肝臓疾患；慢性腎不全；特発性肺線維症、成人呼吸器障害症候群等の呼吸器系疾患；白内障；虚血性心疾患；パーキンソン病；アルツハイマー病：胃潰瘍等の消化器系疾患；癌；骨髄形成不全；後天性免疫不全症候群；潜伏性ウイルス感染症等）、酸化型グルタチオンの欠乏に起因する不眠症等の睡眠障害等の予防、治療又は改善に大きく貢献できる。 The glutathione production promoter of the present invention is used for skin aging, spots, damage to oxidative stress caused by ultraviolet irradiation, diseases caused by deficiency of glutathione (for example, diseases caused by oxidative stress such as reactive oxygen species; hepatitis, liver dysfunction Liver diseases such as: chronic renal failure; idiopathic pulmonary fibrosis, respiratory diseases such as adult respiratory syndrome; cataract; ischemic heart disease; Parkinson's disease; Alzheimer's disease: gastrointestinal diseases such as gastric ulcer; Bone marrow dysplasia; acquired immune deficiency syndrome; latent viral infection, etc.), and sleep disorders such as insomnia due to lack of oxidized glutathione.
Claims (3)
リクイリチゲニン、イソリクイリチン、イソリクイリチゲニン及びリコカルコンＡからなる群より選ばれる１種又は２種以上を有効成分として含有することを特徴とするグルタチオンの欠乏に起因する疾患の予防・治療剤。 Diseases caused by oxidative stress, liver disease, chronic renal failure, respiratory disease, cataract, ischemic heart disease, Parkinson's disease, Alzheimer's disease, digestive system disease, cancer, myelogenesis failure, acquired immune deficiency syndrome, latency A preventive / therapeutic agent for diseases caused by deficiency of glutathione selected from the group consisting of viral infections and sleep disorders,
A prophylactic / therapeutic agent for diseases caused by glutathione deficiency, comprising as an active ingredient one or more selected from the group consisting of liquiritigenin, isoliquiritin, isoliquiritigenin and lycochalcone A.
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|---|---|---|---|
| JP2008123940A JP5403942B2 (en) | 2008-05-09 | 2008-05-09 | Glutathione production promoter and preventive / therapeutic agent for diseases caused by glutathione deficiency |
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| KR101208115B1 (en) * | 2010-08-12 | 2012-12-05 | 건국대학교 산학협력단 | A composition for anti-aging or wrinkle improvement of skin comprising sativanone, medicarpin, dalbergin, isoliquiritigenin, or pharmaceutically acceptable salts thereof |
| JP6482930B2 (en) * | 2015-03-31 | 2019-03-13 | 丸善製薬株式会社 | Skin cosmetics and foods and drinks |
| CN104873481A (en) * | 2015-05-22 | 2015-09-02 | 杭州紫金医药科技有限公司 | Application of licochalcone A in preparation of pulmonary fibrosis preventing and treating medicine |
| JP2016222621A (en) * | 2015-06-02 | 2016-12-28 | 学校法人九州文化学園 | Melanin synthesis promoting composition |
| CN106420902B (en) * | 2016-10-13 | 2020-01-03 | 北京大学 | Liver protection activity of glycyrrhiza inflata extract and licochalcone A and new pharmaceutical application |
| CN108514556B (en) * | 2018-07-09 | 2020-11-13 | 南京农业大学 | Application of licochalcone A in preparation of medicine for resisting porcine epidemic diarrhea virus |
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