JP2010280572A5

JP2010280572A5 -

Info

Publication number: JP2010280572A5
Application number: JP2009132792A
Authority: JP
Filing date: 2009-06-02
Publication date: 2012-07-05

Description

本発明は、キク科ノコギリソウ属（Achillea sp.）の植物体の抽出物を含有する、グルタチオン産生促進剤に関する。 The present invention contains an extract of a plant of the Asteraceae yarrow genus (Achillea sp.), Relates to glutathione production enhancer.

本発明は、この様な状況下為されたものであり、グルタチオン産生促進剤を提供することを課題とする。 The present invention has been made under such circumstances, and an object thereof is to provide a glutathione production enhancer.

この様な状況に鑑みて、本発明者らはグルタチオン産生促進作用を有する物質を求めて、鋭意研究努力を重ねた結果、キク科ノコギリソウ属（Achillea sp.）の植物体の抽出物にその様な作用を見出し、発明を完成させるに至った。即ち、本発明は以下に示すとおりである。
（１）キク科ノコギリソウ属（Achillea sp.）の植物体の抽出物であることを特徴とする、グルタチオン産生促進剤。
（２）前記キク科ノコギリソウ属（Achillea sp.）の植物がセイヨウノコギリソウ（Achillea millefolium）であることを特徴とする、（１）に記載のグルタチオン産生促進剤。（３）前記グルタチオンの産生促進剤に於いて、γ-グルタミルシステイン合成酵素の発現の促進を機作に含むことを特徴とする、（１）又は（２）に記載のグルタチオン産生促進剤。
（４）（１）〜（３）の何れかに記載のグルタチオン産生促進剤を含有する、グルタチオン産生促進用の経口投与組成物。
（５）次に示す疾患予防及び／又は改善のための、（４）に記載のグルタチオン産生促進用の経口投与組成物。
（疾患）しみ、にきび、アトピー性皮膚炎、動脈硬化、糖尿病、薬物中毒、妊娠中毒、白内障、慢性肝疾患、腎疾患、肺疾患
（６）（１）〜（３）の何れかに記載のグルタチオン産生促進剤の製造方法であって、前記キク科ノコギリソウ属（Achillea sp.）の植物体を、冷アルコール溶液に２４時間浸漬し、濾過後凍結乾燥して抽出物を得ることを特徴とする、製造方法。 In view of such circumstances, the present inventors have sought for a substance having a glutathione production-promoting action and made extensive research efforts. As a result, the extract of the plant of the genus Achillea sp. The present inventors have found an effective action and have completed the invention. That is, the present invention is as follows.
(1) Asteraceae yarrow genus (Achillea sp.), Characterized in that an extract of the plant body, glutathione production enhancer.
(2), wherein the plant of the Asteraceae yarrow genus (Achillea sp.) Are yarrow (Achillea millefolium), glutathione production enhancer according to (1). (3) In the production promoter of the glutathione, .gamma. characterized in that it comprises Guru promotion of expression of Tamil cysteine synthase The mechanisms, glutathione production enhancer according to (1) or (2).
(4) (1) to contain glutathione production enhancer of any crab described (3), an oral dosage composition for glutathione production enhancer.
(5) The composition for oral administration for promoting glutathione production according to ( 4 ), for preventing and / or improving the following diseases.
(Disease) Blot, Acne, Atopic dermatitis, Arteriosclerosis, Diabetes, Drug addiction, Pregnancy poisoning, Cataract, Chronic liver disease, Kidney disease, Lung disease (6) (1)-(3) a method of manufacturing a glutathione production enhancer, and characterized in that the plant of the Asteraceae yarrow genus (Achillea sp.), were immersed for 24 hours in the cold alcoholic solution to obtain an extract extract was lyophilized after filtration to, manufacturing method.

本発明によれば、グルタチオン産生促進剤を提供することができる。 According to the present invention, it is possible to provide a glutathione production enhancer.

＜１＞本発明のグルタチオン産生促進剤の必須成分であるキク科ノコギリソウ属（Achillea sp.）の植物体抽出物
本発明のグルタチオン産生促進剤は、キク科ノコギリソウ属（Achillea sp.）の植物体抽出物を有効成分として含有することを特徴とする。前記キク科ノコギリソウ属の植物としては、ノコギリソウ（Achillea alpina L.）、セイヨウノコギリソウ（Achillea millefolium）等が挙げられるが、セイヨウノコギリソウ（Achillea millefolium）が特に好適に
例示できる。抽出物の作製に用いる植物部位としては、地上部が好適に例示できる。
抽出に際して、植物体乃至はその乾燥物は予め、粉砕或いは細切して抽出効率を向上させるように加工することが好ましい。抽出は、植物体乃至はその乾燥物１質量部に対して、溶媒を１〜３０質量部加えて室温以下で２４時間浸漬することが好ましいが、４℃前後で２４時間浸漬することが特に好ましい。前記抽出溶媒としては、極性溶媒が好ましく、水、エタノ−ル、イソプロピルアルコ−ル、ブタノ−ル等のアルコ−ル類、アセトン、メチルエチルケトン等のケトン類、ジエチルエ−テル、テトラヒドロフラン等のエ−テル類から選択される１種乃至は２種以上が好適に例示でき、３０〜８０質量％、より好ましくは３０質量％エタノ−ル含有水溶液が特に好適に例示できる。浸漬後は室温に戻し、析出した不溶物を濾過により除去した後、溶媒を減圧濃縮等により除去することができるが、凍結乾燥による除去が特に好ましい。溶媒を除去した粉末組成物は、このまま粉末の状態で使用しても良いが、しかる後に、水と酢酸エチル、水とブタノ−ル等の液液抽出や、シリカゲルやイオン交換樹脂を充填したカラムクロマトグラフィ−等で分画精製しても良い。この様な抽出方法により得られた抽出物で、生体内のグルタチオン合成酵素の発現を促進することによりグルタチオン産生を促進し、活性酸素種の関与する多様な疾患に対する治療効果をもたらすことが期待される。 <1> glutathione production enhancer of the plant extract present invention Asteraceae Achillea sp is an essential component of glutathione production enhancer of the present invention (Achillea sp.), A plant of the Asteraceae yarrow genus (Achillea sp.) It contains an extract as an active ingredient. Examples of the plant belonging to the genus Achillea alpina L. and Achillea millefolium include Achillea millefolium, and Achillea millefolium can be particularly preferably exemplified. The plant part used for the production of the extract is preferably exemplified by the above-ground part.
Upon exiting extraction, plant or its dried product in advance, it is preferably processed so as to improve the extraction efficiency and pulverized or chopped. The extraction is preferably performed by adding 1 to 30 parts by mass of a solvent to 1 part by mass of the plant or its dried product and immersing it at room temperature or lower for 24 hours, but it is particularly preferable to immerse at around 4 ° C. for 24 hours. . The extraction solvent is preferably a polar solvent, alcohols such as water, ethanol, isopropyl alcohol and butanol, ketones such as acetone and methyl ethyl ketone, ethers such as diethyl ether and tetrahydrofuran. One to two or more selected from the above can be exemplified, and an aqueous solution containing 30 to 80% by mass, more preferably 30% by mass ethanol can be particularly preferably exemplified. After immersion, the temperature is returned to room temperature, and the precipitated insoluble matter is removed by filtration, and then the solvent can be removed by vacuum concentration or the like, but removal by freeze-drying is particularly preferred. The powder composition from which the solvent has been removed may be used as it is, but after that, liquid-liquid extraction of water and ethyl acetate, water and butanol, etc., or a column packed with silica gel or ion exchange resin It may be fractionated and purified by chromatography or the like. The extract obtained by such an extraction method is expected to promote glutathione production by promoting the expression of glutathione synthetase in vivo, and to have therapeutic effects on various diseases involving reactive oxygen species. The

＜２＞本発明のグルタチオン産生促進剤を含有する経口投与組成物
本発明のグルタチオン産生促進剤を含有する組成物は、生体内に於いてグルタチオンの産生を高める目的で経口投与組成物として投与するもの、皮膚内のグルタチオン産生量を高める目的で経皮的に投与されるものが存する。経口投与組成物としては、例えば、菓子やパン、麺等の一般食品、カプセル剤や、錠剤の形態を取る、健康増進の目的を有する食品群（例えば、特定保健用食品等）、顆粒剤、粉末剤、カプセル剤や、錠剤の形態を取る、経口投与医薬等が例示でき、それぞれの製剤で許容される任意成分を含有することができる。この様な任意成分としては、食品であれば、塩、砂糖、グルタミン酸ナトリウム、イノシン酸ナトリウム、酢等の調味成分、着色成分、フレーバー等の矯臭成分、増粘剤、乳化・分散剤、保存料、安定剤等が好適に例示でき、健康増進の目的を有する食品群や医薬であれば、結晶セルロース、乳糖等の賦形剤、アラビヤガムやヒドロキシプロピルセルロース等の結合剤、クロスカルメロースナトリウム、デンプン等の崩壊剤、ステアリン酸マグネシウム等の滑沢剤、矯味、矯臭剤、着色剤等が好ましく例示できる。これらを常法に従って処理することにより、本発明の経口投与組成物は製造することができる。この時、キク科ノコギリソウ属の植物体の抽出物は、総量で、組成物全量に対して、１〜１０質量、より好ましくは３〜８質量％含有されることが好ましい。斯くして得られた、本発明の経口投与組成物は、それを飲用することにより、生体内のグルタチオン量を増加させる作用に優れる。この様な作用を発揮させるためには、前記植物の抽出物を総量で、１日あたり、１０〜１０００ｍｇを１回乃至は数回に分けて飲用することが好ましい。 <2> An oral administration composition containing the glutathione production promoter of the present invention
The composition containing the glutathione production promoter of the present invention is administered as an oral administration composition for the purpose of increasing the production of glutathione in vivo, or administered transdermally for the purpose of increasing the amount of glutathione production in the skin. There is something to be done. The oral composition, if example embodiment, confectionery, bread, general foods such as noodles, or capsules, take the form of tablets, food groups having the desired health promotion (e.g., specified health food, etc.), granules Oral administration medicines in the form of powders, capsules, tablets, etc. can be exemplified, and any component acceptable in each preparation can be contained. Examples of such optional ingredients include foods such as salt, sugar, sodium glutamate, sodium inosinate, vinegar and other flavoring ingredients, coloring ingredients, flavoring ingredients such as flavors, thickeners, emulsifying / dispersing agents, and preservatives. Stabilizers and the like can be preferably exemplified, and if it is a food group or a medicine having the purpose of promoting health, excipients such as crystalline cellulose and lactose, binders such as arabic gum and hydroxypropylcellulose, croscarmellose sodium, starch Preferred examples include disintegrating agents such as magnesium stearate, lubricants such as magnesium stearate, flavoring agents, flavoring agents, and coloring agents. By treating these according to a conventional method, the composition for oral administration of the present invention can be produced. At this time, the extract of the plant belonging to the genus Asteraceae is preferably 1 to 10% by mass, more preferably 3 to 8% by mass, based on the total amount of the composition. The oral administration composition of the present invention thus obtained is excellent in the action of increasing the amount of glutathione in the living body by drinking it. In order to exert such an action, it is preferable to drink 10 to 1000 mg of the plant extract in a total amount once or several times a day.

＜試験例１＞グルタチオン産生量測定試験
ヒト肝ガン由来細胞株ＨｅｐＧ２（ＡＴＣＣ；ＣＲＬ-１１９９７）を用いてグルタチオン産生量の測定試験を実施した。１０容量％牛胎児血清（ＧＩＢＣＯ社製）を含むＲＰＭ
Ｉ１６４０培地（ＧＩＢＣＯ社製）に細胞を懸濁し、培養プレートに播種し、ＣＯ_２インキュベーター（９５容量％空気、５容量％二酸化炭素）内、３７℃の条件下で２４時間培養した。２４時間培養後、ジメチルスルホキシドにて懸濁した各抽出物を乾燥固形物量として培地中に２０μｇ／ｍｌの濃度で含む試料添加培地に交換し、さらに２４時間培養した。細胞を回収し、その後−３０℃で凍結、融解することで細胞を破砕し、細胞内のグルタチオンを溶出させた。１０００ｘｇで１５分間、２０℃で遠心し、上清を測定試料とした。ＧＳＨ／ＧＳＳＧ−４１２キット（ＯＸＩＳＲｅｓｅａｒｃｈ社製）を用いてグルタチオン量を測定した。即ち、グルタチオン標準品、ブランク、測定試料２００μlを、各々のキュベットへ入れ、キット付属のクロモジェン２００μlと酵素液２００μlを各キュベットに加え、室温で５分間インキュベートした。その後各キュベットにキット付属のＮＡＤＰＨ液２００μlを添加し、４１２ｎｍの吸光度の変化を３分間測定した。グルタチオン量は、同時に設定したグルタチオン標準品にて作成した検量線から計算した。これらを元に、グルタチオン産生促進値（検体存在下のグルタチオン産生量／検体非存在下のグルタチオン産生量）を求めた。この値が１．４以上の場合（４０％促進）、その検体はグルタチオン産生促進作用を有すると判断した。 <Test Example 1> Glutathione production measurement test A glutathione production measurement test was performed using a human liver cancer-derived cell line HepG2 (ATCC; CRL-11997). RPM containing 10% fetal bovine serum (GIBCO)
Cells were suspended in I1640 medium (GIBCO), seeded on a culture plate, and cultured in a CO ₂ incubator (95 vol% air, 5 vol% carbon dioxide) at 37 ° C. for 24 hours. After culturing for 24 hours, each extract suspended in dimethyl sulfoxide was replaced with a sample-added medium containing 20 μg / ml in the medium as a dry solid amount, and further cultured for 24 hours. The cells were collected and then frozen and thawed at −30 ° C. to disrupt the cells and elute intracellular glutathione. The sample was centrifuged at 1000 × g for 15 minutes at 20 ° C., and the supernatant was used as a measurement sample. The amount of glutathione was measured using a GSH / GSSG-412 kit (manufactured by OXIS Research). Specifically, 200 μl of glutathione standard, blank, and measurement sample were placed in each cuvette, 200 μl of chromogen and 200 μl of enzyme solution included in the kit were added to each cuvette, and incubated at room temperature for 5 minutes. Thereafter, 200 μl of NADPH solution attached to the kit was added to each cuvette, and the change in absorbance at 412 nm was measured for 3 minutes. The amount of glutathione was calculated from a calibration curve prepared with a glutathione standard set at the same time. Based on these, the glutathione production promotion value (glutathione production in the presence of specimen / glutathione production in the absence of specimen) was determined. When this value was 1.4 or more (40% promotion), it was judged that the sample had a glutathione production promoting action.

＜試験例２＞γ-グルタミルシステインシンセターゼ（ＧＣＳ）遺伝子の定量試験
上記試験例１と同様の方法でＨｅｐＧ２を培養し、ジメチルスルホキシドにて懸濁した各抽出物を乾燥固形物量として培地中に２０μｇ／ｍｌの濃度で含む試料添加培地に交換し、さらに６時間培養した。その後細胞を回収し、ＲＮＡｓｐｉｎＭｉｎｉＲＮＡｉｓｏｌａｔｉｏｎｋｉｔ（ＧＥヘルスケアバイオサイエンス社製）を用いて全ＲＮＡを抽出した。得られた全ＲＮＡを用いて、以下に示すようにリアルタイムＲＴ-ＰＣＲ法によりγ-グルタミルシステインシンセターゼ（ＧＣＳ）遺伝子の定量試験を実施した。即ち、１μｇのｔｏｔａｌＲＮＡを鋳型とし、ＧＣＳ合成プライマー（ＳＩＧＭＡＧＥＮＯＳＹＳ社製、配列番号１、配列番号２）及びＰｒｉｍｅＳｃｒｉｐｔ１ｓｔｓｔｒａｎｄｃＤＮＡｓｙｎｔｈｅｓｉｓｋｉｔ（タカラバイオ社製）を用いて逆転写反応を行った。逆転写反応によって得られたＧＣＳのｃＤＮＡを滅菌水にて１００倍希釈し、鋳型ｃＤＮＡとした。ＥｘＴａｑｋｉｔ（タカラバイオ社製）を用いてＰＣＲ反応を行った後に電気泳動を行った。得られたバンドは画像ソフトを用いて画像として取り込み、ＩｍａｇｅＪ（アメリカ国立衛生研究所製）を用いてバンドの濃淡を数値化した。ＧＣＳ遺伝子の発現量は、β-アクチン遺伝子についても同様に検討した発現量（合成プライマーはＳＩＧＭＡＧＥＮＯＳＹＳ社製、配列番号３、配列番号４）の相対値として表した。設計した合成プライマーの塩基配列及びＰＣＲ反応条件は以下の通りである。 <Test Example 2> Quantitative test of γ-glutamylcysteine synthetase (GCS) gene HepG2 was cultured in the same manner as in Test Example 1 above, and each extract suspended in dimethyl sulfoxide was used as a dry solid amount in the medium. The medium was replaced with a sample-added medium containing a concentration of 20 μg / ml, and further cultured for 6 hours. Thereafter, the cells were collected, and total RNA was extracted using RNAspinMini RNA isolation kit (GE Healthcare Bioscience). Using the obtained total RNA, a quantitative test of γ-glutamylcysteine synthetase (GCS) gene was performed by real-time RT-PCR method as shown below. That is, 1 μg of total RNA was used as a template, and reverse transcription reaction was performed using GCS synthesis primers (manufactured by SIGMA GENOSYS , SEQ ID NO: 1, SEQ ID NO: 2 ) and PrimeScript 1st strand cDNA synthesis kit (manufactured by Takara Bio Inc.). The GCS cDNA obtained by the reverse transcription reaction was diluted 100 times with sterilized water to obtain a template cDNA. After performing PCR reaction using Ex Taq kit (manufactured by Takara Bio Inc.), electrophoresis was performed. The obtained band was captured as an image using image software, and the density of the band was quantified using ImageJ (manufactured by National Institutes of Health). The expression level of the GCS gene was expressed as a relative value of the expression level similarly studied for the β-actin gene (synthetic primers are SIGMA GENOSYS , SEQ ID NO: 3, SEQ ID NO: 4 ). The base sequence of the designed synthetic primer and the PCR reaction conditions are as follows.

Claims

Characterized in that it is an extract of a plant of the Asteraceae yarrow genus (Achillea sp.), Glutathione production enhancer.

The plants of the Asteraceae yarrow genus (Achillea sp.) Is characterized in that it is a yarrow (Achillea millefolium), glutathione production enhancer according to claim 1.

In production promoter of the glutathione, .gamma. Guru Tamil promotion of expression of cysteine synthase, characterized in that it comprises a machine operation, glutathione production enhancer according to claim 1 or 2.

Containing glutathione production enhancer according to any one of 請 Motomeko 1-3, oral compositions for glutathione production enhancer.

The orally administered composition for promoting glutathione production according to claim 4, for preventing and / or improving the following diseases.
(Disease) Blot, Acne, Atopic dermatitis, Arteriosclerosis, Diabetes, Drug addiction, Pregnancy poisoning, Cataract, Chronic liver disease, Kidney disease, Lung disease

The method for producing a glutathione production promoter according to any one of claims 1 to 3, wherein the plant of the genus Achillea sp. Is immersed in a cold alcohol solution for 24 hours and filtered. and wherein the obtaining the extract extract was lyophilized, manufacturing methods.