JP2007308417A - Tetrahydrobiopterin production promoter - Google Patents
Tetrahydrobiopterin production promoter Download PDFInfo
- Publication number
- JP2007308417A JP2007308417A JP2006138628A JP2006138628A JP2007308417A JP 2007308417 A JP2007308417 A JP 2007308417A JP 2006138628 A JP2006138628 A JP 2006138628A JP 2006138628 A JP2006138628 A JP 2006138628A JP 2007308417 A JP2007308417 A JP 2007308417A
- Authority
- JP
- Japan
- Prior art keywords
- tetrahydrobiopterin
- acid
- chlorogenic acids
- production promoter
- production
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
Description
本発明は、高フェニルアラニン血症等のテトラヒドロビオプテリン量低下に起因する疾患の予防治療剤として有用なテトラヒドロビオプテリン産生促進剤に関する。 The present invention relates to a tetrahydrobiopterin production promoter useful as a prophylactic / therapeutic agent for diseases caused by a decrease in the amount of tetrahydrobiopterin such as hyperphenylalaninemia.
テトラヒドロビオプテリン（ＢＨ4）は、フェニルアラニン水酸化酵素、チロシン水酸化酵素、トリプトファン水酸化酵素など芳香族アミノ酸モノオキゲナーゼの補酵素であり、カテコールアミンやセロトニン合成に重要な役割を果たしている。ＢＨ4の生体内濃度が低下すると、高フェニルアラニン血症、パーキンソン病、うつ病等の疾患が生じることが知られている。実際に、テトラヒドロビオプテリン自体は、先天性代謝異常の一つである高フェニルアラニン血症の治療薬として広く用いられている。 Tetrahydrobiopterin (BH 4 ) is a coenzyme of aromatic amino acid monooxygenase such as phenylalanine hydroxylase, tyrosine hydroxylase, tryptophan hydroxylase, and plays an important role in catecholamine and serotonin synthesis. It is known that when the in vivo concentration of BH 4 decreases, diseases such as hyperphenylalaninemia, Parkinson's disease, and depression occur. In fact, tetrahydrobiopterin itself is widely used as a therapeutic agent for hyperphenylalaninemia, which is one of inborn errors of metabolism.
ＢＨ4自体を投与するのでなく、生体内でＢＨ4の産生を促進させてＢＨ4の低下に起因する疾患を治療しようとする試みもされている（特許文献１及び２）。
しかしこれらのＢＨ4産生促進剤は、いずれもＢＨ4誘導体又はその類縁化合物であり、合成が極めて困難であるとともに安全性の点でも十分満足できるものではない。
従って、本発明の目的は、容易に入手可能であって、安全性も高い、ＢＨ4産生促進剤を提供することにある。
However, any of these BH 4 production promoters is a BH 4 derivative or a related compound, and is extremely difficult to synthesize and is not sufficiently satisfactory in terms of safety.
Therefore, an object of the present invention is to provide a BH 4 production promoter that is easily available and highly safe.
そこで本発明者は、植物由来の成分からＢＨ4産生促進剤を見出すべく種々検索した結果、コーヒー等に含まれるクロロゲン酸類に有意なＢＨ4産生促進作用があることを見出した。 Therefore, the present inventor conducted various searches to find BH 4 production promoters from plant-derived components, and as a result, found that chlorogenic acids contained in coffee and the like have a significant BH 4 production promoting action.
すなわち、本発明はクロロゲン酸類又はその薬学的に許容される塩を有効成分とするＢＨ4産生促進剤及びＢＨ4低下に基づく疾患の予防治療剤を提供するものである。 That is, the present invention provides a BH 4 production promoter comprising a chlorogenic acid or a pharmaceutically acceptable salt thereof as an active ingredient, and a prophylactic / therapeutic agent for a disease based on a decrease in BH 4 .
また、本発明は、クロロゲン酸類又はその薬学的に許容される塩を有効成分として含有し、テトラヒドロビオプテリンを産生促進する効果を有することを特徴とし、テトラヒドロビオプテリンの低下に伴う疾患の予防または改善をする為に用いるものである旨の表示をした食品又は飲料を提供するものである。 In addition, the present invention contains chlorogenic acids or pharmaceutically acceptable salts thereof as an active ingredient, and has an effect of promoting production of tetrahydrobiopterin, thereby preventing or improving a disease associated with a decrease in tetrahydrobiopterin. The present invention provides food or beverages with a display indicating that they are used for the purpose.
クロロゲン酸類は、優れたＢＨ4産生促進作用を有し、安全性も高く、入手も容易である。従って、本発明のＢＨ4産生促進剤は、長期摂取可能であり、ＢＨ4低下に基づく疾患、例えば高フェニルアラニン血症、パーキンソン病、うつ病、うつ状態等の症状の改善、治療に有用である。 Chlorogenic acids have an excellent BH 4 production promoting action, are highly safe, and are easily available. Thus, BH 4 production promoter of the present invention is a long-ingestible, diseases based on BH 4 decreases, for example, hyperphenylalaninemia, Parkinson's disease, depression, improvement of symptoms such as depression, are useful in the treatment .
本発明で用いるクロロゲン酸類は、これを含有する天然物、特に植物から抽出することもでき、化学合成により工業的に製造することもできる。 The chlorogenic acids used in the present invention can be extracted from natural products containing them, particularly from plants, and can also be industrially produced by chemical synthesis.
本発明におけるクロロゲン酸類には、立体異性体が存在し、本発明では、純粋な立体異性体又はそれらの混合物を用いることができる。本発明におけるクロロゲン酸類には、具体的には、３−カフェイルキナ酸、４−カフェイルキナ酸、５−カフェイルキナ酸、３，４−ジカフェイルキナ酸、３，５−ジカフェイルキナ酸、４，５−ジカフェイルキナ酸、３−フェルリルキナ酸、４−フェルリルキナ酸、５−フェルリルキナ酸及び３−フェルリル−４−カフェイルキナ酸等が含まれる（中林ら，コーヒー焙煎の化学と技術，弘学出版株式会社，ｐ１６６−１６７）。 The chlorogenic acids in the present invention have stereoisomers, and in the present invention, pure stereoisomers or a mixture thereof can be used. Specific examples of the chlorogenic acids in the present invention include 3-caffeylquinic acid, 4-caffeylquinic acid, 5-caffeylquinic acid, 3,4-dicaffeylquinic acid, 3,5-dicaffeylquinic acid, 4,5- Dicaffeylquinic acid, 3-ferrylquinic acid, 4-ferrylquinic acid, 5-ferrylquinic acid, 3-ferryl-4-caffeylquinic acid, etc. are included (Nakabayashi et al., Chemistry and Technology of Coffee Roasting, Kogaku Publishing Co., Ltd. , P166-167).
クロロゲン酸類は、塩にすることにより水溶性を向上させ、生理学的有効性を増大させることができる。これらの塩としては、薬学的に許容される塩であればよい。このような塩形成用の塩基物質としては、例えば、水酸化リチウム、水酸化ナトリウム、水酸化カリウム等のアルカリ金属の水酸化物；水酸化マグネシウム、水酸化カルシウム等のアルカリ土類金属の水酸化物；水酸化アンモニウム等の無機塩基、アルギニン、リジン、ヒスチジン、オルニチン等の塩基性アミノ酸；モノエタノールアミン、ジエタノールアミン、トリエタノールアミン等の有機塩基が用いられるが、特にアルカリ金属又はアルカリ土類金属の水酸化物が好ましい。本発明においては、これらの塩を調製してから、その他の成分からなる組成物中に添加したものでもよいし、クロロゲン酸類等と塩形成成分とを別々に該組成物中に添加して、この中で塩を形成せしめたものでもよい。 Chlorogenic acids can be salted to improve water solubility and increase physiological effectiveness. These salts may be pharmaceutically acceptable salts. Examples of such basic substances for salt formation include hydroxides of alkali metals such as lithium hydroxide, sodium hydroxide and potassium hydroxide; hydroxides of alkaline earth metals such as magnesium hydroxide and calcium hydroxide. Products; inorganic bases such as ammonium hydroxide, basic amino acids such as arginine, lysine, histidine, ornithine; organic bases such as monoethanolamine, diethanolamine, and triethanolamine are used, particularly alkali metals or alkaline earth metals Hydroxides are preferred. In the present invention, these salts may be prepared and then added to the composition comprising other components, or chlorogenic acids and the salt-forming component may be separately added to the composition, In this, a salt may be formed.
クロロゲン酸類を含有する天然物抽出物としては、例えば、コーヒー、キャベツ、レタス、アーチチョーク、トマト、ナス、ジャガイモ、ニンジン、リンゴ、ナシ、プラム、モモ、アプリコット、チェリー、ヒマワリ、モロヘイヤ、カンショ、南天の葉、ブルーベリー、小麦などの植物から抽出したものが好ましい。 Examples of natural product extracts containing chlorogenic acids include coffee, cabbage, lettuce, arch chalk, tomato, eggplant, potato, carrot, apple, pear, plum, peach, apricot, cherry, sunflower, moroheiya, sweet potato, southern sky What was extracted from plants, such as a leaf, a blueberry, and wheat, is preferable.
クロロゲン酸類は、コーヒー生豆、南天の葉、リンゴ未熟果等の植物体から抽出したものが好ましく、さらにアカネ科コーヒー（Ｃｏｆｆｅｅ ａｒａｂｉｃａ ＬＩＮＮＥ）の種子より、温時アスコルビン酸、クエン酸酸性水溶液又は熱水で抽出して得たものがより好ましい。具体的には、生コーヒー豆抽出物としては、長谷川香料（株）「フレーバーフォールダー」、リンゴ抽出物としては、ニッカウヰスキー（株）「アップルフェノン」、ヒマワリ種抽出物としては、大日本インキ化学工業（株）「ヘリアント」などが挙げられる。 The chlorogenic acids are preferably extracted from plants such as green coffee beans, southern leaves, and unripe apples. Further, ascorbic acid, citric acid aqueous solution or heat from seeds of Rubiaceae coffee (Coffee arabica LINENE) Those obtained by extraction with water are more preferred. Specifically, Hasegawa Fragrance Co., Ltd. “Flavor Folder” as raw coffee bean extract, Nikka Whiskey Co., Ltd. “Applephenon” as apple extract, Dainippon Ink Chemical Co., Ltd. as sunflower seed extract Industrial Co., Ltd. “Heliant” and the like.
後記実施例に示すように、クロロゲン酸類は、血中ビオプテリン濃度が低下した状態を回復させる効果を有し、かつＢＨ4の産生に関与する酵素の発現を増強させる効果を有することから、ＢＨ4産生促進剤として有用である。従って、クロロゲン酸類は、ＢＨ4低下に基づく疾患、例えば高フェニルアラニン血症、パーキンソン病、うつ病、うつ状態等の予防及び治療用医薬、食品および飲料として使用することができる。また、本発明のＢＨ4産生促進剤は高フェニルアラニン血症、パーキンソン病、うつ病、うつ状態等の予防及び治療用医薬、食品および飲料の製造のために使用することができる。 As shown in Examples below, chlorogenic acids, since it has the effect of having the effect of restoring the state of blood biopterin concentration is decreased, and enhanced the expression of enzymes involved in the production of BH 4, BH 4 It is useful as a production promoter. Therefore, chlorogenic acids can be used as pharmaceuticals, foods and beverages for the prevention and treatment of diseases based on BH 4 lowering, such as hyperphenylalaninemia, Parkinson's disease, depression, and depression. Moreover, BH 4 production promoter of the present invention can be used hyperphenylalaninemia, Parkinson's disease, depression, prophylactic and therapeutic medicament, such as depression, for the manufacture of food and beverages.
本発明のＢＨ4産生促進剤の有効成分であるクロロゲン酸類等は、そのまま服用してもよいが、好ましくは薬学的に許容されうる塩、例えば塩酸塩の形で、賦形剤、担体等の薬品及び食品分野で慣用の補助成分、例えば乳糖、ショ糖、液糖、蜂蜜、ステアリン酸マグネシウム、ヒドロキシプロピルセルロース、各種ビタミン類、クエン酸、リンゴ酸、香料、無機塩などとともに、カプセル剤、錠剤、粉末剤、顆粒剤、ドリンク剤、注射剤、点滴剤等にすることができる。 Chlorogenic acids and the like as an active ingredient of the BH 4 production enhancer of the present invention may be taken as it is, preferably pharmaceutically acceptable salts, for example in the form of hydrochloride salt, excipients, carriers, etc. Auxiliary ingredients commonly used in the pharmaceutical and food fields, such as lactose, sucrose, liquid sugar, honey, magnesium stearate, hydroxypropylcellulose, various vitamins, citric acid, malic acid, fragrance, inorganic salts, capsules, tablets , Powders, granules, drinks, injections, drops, etc.
ドリンク剤及び食品の場合、必要に応じ、他の生理活性成分、ミネラル、ビタミン、ホルモン、栄養成分、香味剤等を混合することも可能である。また、緑茶系飲料、烏龍茶系飲料、紅茶系飲料、コーヒー系飲料、アイソトニック系飲料とすることもできる。 In the case of drinks and foods, other physiologically active ingredients, minerals, vitamins, hormones, nutritional ingredients, flavoring agents, and the like can be mixed as necessary. Moreover, it can also be set as a green tea type | system | group drink, oolong tea type | system | group drink, tea type | system | group drink, a coffee type | system | group drink, and an isotonic type | system | group drink.
本発明の食品及び飲料は、ＢＨ4が低下している方へ、ＢＨ4の低下に伴う疾患をお持ちの方へ、ＢＨ4の低下に伴う疾患を予防又は改善したい方へ等と表示して使用することができる。 Food and beverage of the present invention, towards the BH 4 has decreased, the person who has a disease associated with decrease in BH 4, display and the like to those who wish to prevent or ameliorate diseases associated with decrease in BH 4 Can be used.
本発明のＢＨ4産生促進剤の服用量は成人１日あたりクロロゲン酸類又はその薬学的に許容される塩として３０〜１４０００ｍｇ、より好ましくは５０〜１００００ｍｇ、さらに好ましくは２００〜７６００ｍｇ、特に２５０〜３０００ｍｇが好ましい。効果をより有効に発現させるためには、毎日継続して服用することが好ましいと考えられる。 30~14000mg Salts dose of BH 4 production promoter that is acceptable chlorogenic acids or pharmaceutically daily adult present invention, more preferably 50～10000Mg, more preferably 200～7600Mg, especially 250~3000mg Is preferred. In order to exhibit the effect more effectively, it is considered preferable to take the medicine continuously every day.
実施例１（クロロゲン酸（ＣＱＡ）摂取時における血漿中総ビオプテリン（ＢＰ）量の比較）
Ｗｉｓｔｅｒ系ラット（ＳＨＲ：１２週齢、♂；日本ＳＬＣより購入）を用いた。ラットは、室温２３±２℃、湿度５５±１０％、１２時間の明暗サイクル（明期；ＡＭ７：００〜ＰＭ７：００）下で飼育した。
食餌として標準粉末飼料（ＣＥ−２）、及び、１％ＣＱＡ（Ｃａｙｍａｎ社）含有標準粉末飼料（ＣＥ−２）を１０週間、自由摂食させ、飲料は、水道水を自動給水により自由飲水させた。摂取終了後、ラットにペントバルビタール注射液（４０ｍｇ／ｋｇ）を腹腔投与し、深麻酔状態を確認後、腹部大動脈から全採血した。全血は抗凝固剤（終濃度２ｍＭ ＥＤＴＡ）を含む採血管に採取し、４℃で、２０００ｘｇで２０分遠心して得られた上清を血漿とした。採血後、ラットから、腎臓組織を摘出し、ＲＮＡ ｌａｔｅｒ（ＱＩＡＧＥＮ社）に浸漬した。血漿及び組織は、使用時まで−８０℃に保存した。
Example 1 (Comparison of plasma total amount of biopterin (BP) upon ingestion of chlorogenic acid (CQA))
Wister rats (SHR: 12 weeks old, rabbit; purchased from Japan SLC) were used. Rats were housed under a room-temperature 23 ± 2 ° C., humidity 55 ± 10%, 12 hour light-dark cycle (light period; AM7: 00 to PM7: 00).
The standard powdered feed (CE-2) and 1% CQA (Cayman) -containing standard powdered feed (CE-2) were allowed to eat freely for 10 weeks, and the beverage was allowed to drink tap water by automatic water supply. It was. After completion of ingestion, pentobarbital injection solution (40 mg / kg) was intraperitoneally administered to the rat, and after deep anesthesia was confirmed, whole blood was collected from the abdominal aorta. Whole blood was collected in a blood collection tube containing an anticoagulant (final concentration 2 mM EDTA), and the supernatant obtained by centrifugation at 4 ° C. and 2000 × g for 20 minutes was used as plasma. After blood collection, kidney tissue was removed from the rat and immersed in RNA later (QIAGEN). Plasma and tissues were stored at −80 ° C. until use.
（１）血漿中総ＢＰ量の測定
ＢＨ4濃度の測定は、Ｆｕｋｕｓｈｉｍａ ＆ Ｎｉｘｏｎの方法（１）に準じて行った。
詳しくは、２００μＬの採取した血漿に、４０μＬ １Ｎ ＨＣｌ、及び、１００μＬヨウ素酸化液（１％ Ｉ2、２％ ＫＩ）を添加後、混合し、室温、暗所にて１時間静置した。静置後、２０μＬ ５％アスコルビン酸、及び、４０μＬ蒸留水を加え、攪拌し、限外ろ過（Ｍｉｃｒｏｃｏｎ １０ＮＭＷＬミリポア社使用）後、ヨウ素酸化物を含むろ液を、ＨＰＬＣを用い解析した。得られた解析結果は、全て平均値（Ａｖｅ）±標準誤差（ＳＥ）を用いてグラフにし、ｓｔｕｄｅｎｔ’ｓ ｔ−ｔｅｓｔにより有意差検定を行った。
(1) Measurement of plasma total BP amount BH 4 concentration was measured according to the method (1) of Fukushima & Nixon.
Specifically, 40 μL of 1N HCl and 100 μL of iodine oxidation solution (1% I 2 , 2% KI) were added to 200 μL of collected plasma, mixed, and allowed to stand at room temperature in the dark for 1 hour. After standing, 20 μL 5% ascorbic acid and 40 μL distilled water were added, stirred, and after ultrafiltration (using Microcon 10NMWL Millipore), the filtrate containing iodine oxide was analyzed using HPLC. The obtained analysis results were all graphed using the average value (Ave) ± standard error (SE), and the significant difference test was performed by student's t-test.
（２）ＨＰＬＣ分析条件
分析機器はＨＰＬＣ（ＳＨＩＭＡＤＺＵ ＳＣＬ−１０Ａｖｐ）を使用した。
装置の構成ユニットは、次の通りである。
ディテクター：分光蛍光検出器（ＲＦ−１０ＡｘＬ）
カラムオーブン：ＣＴＯ−１０ＡＣｖｐ
ポンプ：ＬＣ１０ＡＤｖｐ
オートサンプラー：ＳＩＬ−１０ＡＤｖｐ
カラム：Ｈｙｐｅｒｓｉｌ ＯＤＳ（Ｃ１８）内径４．６ｍｍ×長さ２５０ｍｍ、粒径５μｍ（カラム温度；３５℃）
(2) HPLC analysis conditions HPLC (SHIMADZU SCL-10Avp) was used as an analytical instrument.
The structural units of the apparatus are as follows.
Detector: Spectral fluorescence detector (RF-10AxL)
Column oven: CTO-10ACvp
Pump: LC10ADvp
Autosampler: SIL-10ADvp
Column: Hypersil ODS (C18) inner diameter 4.6 mm × length 250 mm, particle size 5 μm (column temperature: 35 ° C.)
分析条件は次の通りである。サンプル注入量：５０μＬ、流量：０．８ｍＬ／ｍｉｎ、溶離は１５ｍｍｏｌ／Ｌリン酸カリウム緩衝液（ｐＨ６．０）：ｍｅｔｈａｎｏｌ（９２％：８％）で行い、検出は蛍光検出器（蛍光波長：４５０ｎｍ、励起波長：３５０ｎｍ）を用いた。 The analysis conditions are as follows. Sample injection volume: 50 μL, flow rate: 0.8 mL / min, elution is performed with 15 mmol / L potassium phosphate buffer (pH 6.0): methanol (92%: 8%), and detection is performed with a fluorescence detector (fluorescence wavelength: fluorescence wavelength: 450 nm, excitation wavelength: 350 nm).
（３）ｔｏｔａｌ ＲＮＡの採取
凍結した組織を、室温にて溶解後、ＩＳＯＧＥＮ（ＮＩＰＰＯＮ ＧＥＮＥ社）中で、ホモジナイザーを用い破砕した。破砕液にクロロホルムを添加し、混合後、４℃、２００００ｘｇで１５分間遠心分離を行い、上清を回収した。次に、上清に含まれるｔｏｔａｌ ＲＮＡをイソプロパノールを用い沈殿させ（４℃、２００００ｘｇで１５分間遠心分離）、沈殿物を７０％エタノールを用い洗浄後、滅菌蒸留水に溶解させ、ＲＮＡサンプルとした。
(3) Collecting total RNA Frozen tissue was lysed at room temperature and then disrupted in ISOGEN (NIPPON GENE) using a homogenizer. Chloroform was added to the crushed liquid, and after mixing, centrifuged at 4 ° C. and 20000 × g for 15 minutes, and the supernatant was collected. Next, the total RNA contained in the supernatant is precipitated using isopropanol (centrifugation at 4 ° C., 20000 × g for 15 minutes), and the precipitate is washed with 70% ethanol and dissolved in sterilized distilled water to obtain an RNA sample. .
（４）ｃＤＮＡの合成
熱変性（６５℃、１０分間）させた１μｇのＲＮＡサンプルを用い、ＯｍｎｉｓｃｒｉｐｔTM Ｒｅｖｅｒｓｅ Ｔｒａｎｓｃｒｉｐｔａｓｅ（ＱＩＡＧＥＮ社）により、以下の条件で逆転写反応を行いｃＤＮＡサンプルを合成した。
反応は３７℃で６０分間行い、その後９３℃で５分間処理し、酵素反応を停止した。そ
の後、４℃で急冷した。
(4) Synthesis of cDNA A 1 μg RNA sample that had been heat-denatured (65 ° C., 10 minutes) was subjected to reverse transcription reaction under the following conditions using Omniscript ™ Reverse Transcriptase (QIAGEN) to synthesize a cDNA sample.
The reaction was carried out at 37 ° C. for 60 minutes and then treated at 93 ° C. for 5 minutes to stop the enzyme reaction. Thereafter, it was rapidly cooled at 4 ° C.
（５）遺伝子量の定量（Ｔａｑｍａｎ ＰＣＲ）
ＴａｑＭａｎTM １０００ ＲＸＮ Ｇｏｌｄ ｗｉｔｈ Ｂｕｆｆｅｒ Ａ Ｐａｃｋ（アプライドバイオシステムズ社）を用い、ｃＤＮＡサンプルにおける、ＢＨ4合成酵素遺伝子の定量的ＰＣＲを行った。詳細を以下に記す。
解析にはＡＢＩ ＰＲＩＳＭ ７０００ Ｓｅｑｕｅｎｃｅ Ｄｅｔｅｃｔｏｒ及びＡＢＩ ＰＲＩＳＭ ７７００ Ｓｅｑｕｅｎｃｅ Ｄｅｔｅｃｔｏｒ（アプライドバイオシステムズ社）を用いた。
定量的ＰＣＲに使用したプライマー・プローブは表１に示した。反応液組成は下に記す。
(5) Quantification of gene dosage (Taqman PCR)
Using TaqMan ™ 1000 RXN Gold with Buffer A Pack (Applied Biosystems), quantitative PCR of BH 4 synthase gene in cDNA samples was performed. Details are described below.
For analysis, ABI PRISM 7000 Sequence Detector and ABI PRISM 7700 Sequence Detector (Applied Biosystems) were used.
The primers and probes used for quantitative PCR are shown in Table 1. The reaction solution composition is described below.
今回、解析を行った遺伝子は、３６Ｂ４、ＧＴＰ ｃｙｃｌｏｈｙｄｒｏｌａｓｅ １（ＧＣＨ １：ＢＨ4の産生に作用する酵素）、ｑｕｉｎｏｉｄ ｄｉｈｙｄｒｏｐｔｅｒｉｄｉｎｅ ｒｅｄｕｃｔａｓｅ（ＱＤＰＲ：Ｑ−ＢＨ2からＢＨ4を産生する酵素）である。
尚、今回のＰＣＲに用いたＰＣＲプライマーとプローブは、Ｐｒｉｍｅｒ Ｅｘｐｒｅｓｓ Ｓｏｆｔｗａｒｅ Ｖｅｒｓｉｏｎ ２．００を用いて設計した。また、ＰＣＲプライマーとプローブの塩基配列の遺伝子特異性は、Ｗｅｂｓｉｔｅ のＮＣＢＩ Ｂｌａｓｔで確認した。なお、蛍光標識プローブは、表３のプローブの５’側にＦＡＭ（レポーター色素）を、３’側にＴＡＭＲＡ（クエンチャー色素）を結合させて用いた。
The genes analyzed this time are 36B4, GTP cyclohydrolase 1 (an enzyme that acts on the production of GCH 1: BH 4 ), and quinoid dihydropteridine reductase (QDPR: an enzyme that produces BH 4 from Q-BH 2 ).
The PCR primers and probes used in this PCR were designed using Primer Express Software Version 2.00. The gene specificity of the PCR primer and probe base sequences was confirmed by NCBI Blast of Website. The fluorescently labeled probe was used by binding FAM (reporter dye) on the 5 ′ side of the probe shown in Table 3 and TAMRA (quencher dye) on the 3 ′ side.
得られた解析結果は内部標準として３６Ｂ４の発現量を用い補正し、相対的ｍＲＮＡ発現量として表した。得られた解析結果は、全て平均値（Ａｖｅ）±標準誤差（ＳＥ）を用いてグラフにし、ｓｔｕｄｅｎｔ’ｓ ｔ−ｔｅｓｔにより有意差検定を行った。 The obtained analysis results were corrected using the expression level of 36B4 as an internal standard, and expressed as a relative mRNA expression level. The obtained analysis results were all graphed using the average value (Ave) ± standard error (SE), and the significant difference test was performed by student's t-test.
（６）結果１
ＣＱＡ摂取群及び対照群の血漿中総ＢＰ量を測定した結果、ＣＱＡ摂取群の血漿中総ＢＰ量（２４．２±１．５ｎｇ）は、対照群の血漿中総ＢＰ量（１８．７±１．２ｎｇ）に比較し、統計学的有意（ｐ＜０．０５）に多かった（図１）。
(6) Result 1
As a result of measuring the total plasma BP amount in the CQA intake group and the control group, the total plasma BP amount (24.2 ± 1.5 ng) in the CQA intake group was equal to the total plasma BP amount (18.7 ± in the control group). Compared to 1.2 ng), it was more statistically significant (p <0.05) (FIG. 1).
（７）結果２
ＣＱＡ摂取群及び対照群のＧＣＨ１遺伝子発現量を測定した結果、ＣＱＡ摂取群のＧＣＨ１遺伝子発現量（１２１±９．２％）は、対照群（１００±８．８％）のＧＣＨ１遺伝子発現量と比較し、統計学的有意（ｐ＜０．０５）に増加していた（図２）。
また、ＣＱＡ摂取群及び対照群のＱＤＰＲ遺伝子発現量を測定した結果、ＣＱＡ摂取群のＱＤＰＲ遺伝子発現量（１５５±８．７％）は、対照群のＱＤＰＲ遺伝子発現量（１００±１２％）と比較し、統計学的有意（ｐ＜０．０５）に増加していた（図２）。
配合例
製剤例
（１）果汁飲料
クロロゲン酸 ３００ｍｇ
ビタミンＣ ３００ｍｇ
葡萄果汁 ３００ｍＬ
水 ２００ｍＬ
香料 若干量
ブドウ糖 ２ｇ
（２）錠剤
クロロゲン酸 １０（質量％）
乳糖 １０
麦芽糖 １０
ブドウ糖 ２０
グルタミン １０
ビタミンＣ １５
セルロース １０
カフェイン ４
キシリトール ９
ビタミンＥ １
香料 １
(7) Result 2
As a result of measuring the GCH1 gene expression level of the CQA intake group and the control group, the GCH1 gene expression level (121 ± 9.2%) of the CQA intake group is equal to the GCH1 gene expression level of the control group (100 ± 8.8%). In comparison, it increased statistically (p <0.05) (FIG. 2).
In addition, as a result of measuring the QDPR gene expression level of the CQA intake group and the control group, the QDPR gene expression level (155 ± 8.7%) of the CQA intake group is equal to the QDPR gene expression level (100 ± 12%) of the control group. In comparison, it increased statistically (p <0.05) (FIG. 2).
Formulation Example Formulation Example (1) Fruit juice drink Chlorogenic acid 300mg
Vitamin C 300mg
Strawberry juice 300mL
200 mL water
Fragrance Some amount Glucose 2g
(2) Tablet chlorogenic acid 10 (mass%)
Lactose 10
Maltose 10
Glucose 20
Glutamine 10
Vitamin C 15
Cellulose 10
Caffeine 4
Xylitol 9
Vitamin E 1
Fragrance 1
Claims (3)
It contains chlorogenic acids or pharmaceutically acceptable salts thereof as an active ingredient and has the effect of promoting the production of tetrahydrobiopterin. It is used to prevent or ameliorate diseases associated with a decrease in tetrahydrobiopterin. A food or beverage with a certain indication.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2006138628A JP4856471B2 (en) | 2006-05-18 | 2006-05-18 | Tetrahydrobiopterin production promoter |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2006138628A JP4856471B2 (en) | 2006-05-18 | 2006-05-18 | Tetrahydrobiopterin production promoter |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2007308417A true JP2007308417A (en) | 2007-11-29 |
| JP4856471B2 JP4856471B2 (en) | 2012-01-18 |
Family
ID=38841614
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2006138628A Active JP4856471B2 (en) | 2006-05-18 | 2006-05-18 | Tetrahydrobiopterin production promoter |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP4856471B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2164483A2 (en) * | 2007-06-11 | 2010-03-24 | National Cancer Center | Transglutaminase inhibitor comprising chlorogenic acid and a method for producing thereof |
| JP2017148804A (en) * | 2017-05-10 | 2017-08-31 | 国立研究開発法人農業・食品産業技術総合研究機構 | Fenton reaction catalyst using reducing organic substance as raw material |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001076580A1 (en) * | 2000-04-11 | 2001-10-18 | Takara Bio Inc. | Remedies |
| WO2002060462A2 (en) * | 2001-01-30 | 2002-08-08 | Laboratórios Biosintética Ltda. | Waay to obtain a extract with antidepressant and opioid antagonists properties and pharmaceutical products obtained from it |
| JP2006016311A (en) * | 2004-06-30 | 2006-01-19 | Kao Corp | Hyperhomocysteinemia improving agent |
| WO2006014028A1 (en) * | 2004-08-06 | 2006-02-09 | Mochida Pharmaceutical Co., Ltd. | Sweet potato stem extract and use thereof |
-
2006
- 2006-05-18 JP JP2006138628A patent/JP4856471B2/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001076580A1 (en) * | 2000-04-11 | 2001-10-18 | Takara Bio Inc. | Remedies |
| WO2002060462A2 (en) * | 2001-01-30 | 2002-08-08 | Laboratórios Biosintética Ltda. | Waay to obtain a extract with antidepressant and opioid antagonists properties and pharmaceutical products obtained from it |
| JP2006016311A (en) * | 2004-06-30 | 2006-01-19 | Kao Corp | Hyperhomocysteinemia improving agent |
| WO2006014028A1 (en) * | 2004-08-06 | 2006-02-09 | Mochida Pharmaceutical Co., Ltd. | Sweet potato stem extract and use thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2164483A2 (en) * | 2007-06-11 | 2010-03-24 | National Cancer Center | Transglutaminase inhibitor comprising chlorogenic acid and a method for producing thereof |
| JP2010529188A (en) * | 2007-06-11 | 2010-08-26 | ナショナル キャンサー センター | Transglutaminase inhibitor containing chlorogenic acid and method for producing the same |
| EP2164483A4 (en) * | 2007-06-11 | 2013-08-28 | Nat Cancer Ct | Transglutaminase inhibitor comprising chlorogenic acid and a method for producing thereof |
| JP2017148804A (en) * | 2017-05-10 | 2017-08-31 | 国立研究開発法人農業・食品産業技術総合研究機構 | Fenton reaction catalyst using reducing organic substance as raw material |
Also Published As
| Publication number | Publication date |
|---|---|
| JP4856471B2 (en) | 2012-01-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| ES2851177T3 (en) | Tetrahydrobiopterin for the treatment of conditions associated with elevated phenylalanine levels | |
| JP6603133B2 (en) | Treatment of DNA damage and mitochondrial dysfunction using palm fruit | |
| TWI402069B (en) | Quercetin-containing compositions | |
| EP2408460B1 (en) | Sceletium extract and uses thereof | |
| AU2018203677A1 (en) | Modified polyphenol compositions | |
| JP2012501974A (en) | Methods and compositions for the treatment of cancer | |
| AU2015275507A1 (en) | Cyclic dipeptide-containing composition | |
| EP3308781B1 (en) | Antihypertensive agent | |
| JP2022031929A (en) | Ampelopsin-containing composition for reducing urine acid level in blood | |
| JP4856471B2 (en) | Tetrahydrobiopterin production promoter | |
| JP6599592B2 (en) | α-Glucosidase activity inhibitor | |
| JP2017048185A (en) | Xanthine oxidase inhibitor | |
| KR102059885B1 (en) | Hydroxymethylfurfural derivative | |
| JP2007246478A (en) | Acetaldehyde metabolism promoter | |
| JP2006016340A (en) | Blood uric acid level reduction agent having extract of punica granatum l. as active ingredient | |
| CA2459240C (en) | Pharmaceutical composition for treating mood disorders | |
| JP2019163234A (en) | Xanthine oxidase inhibitor and method for producing the same | |
| TWI810657B (en) | Use of a marigold extract for preparing a composition for reducing blood uric acid concentration | |
| KR102187956B1 (en) | Pharmaceutical composition for preventing or treating male sexual dysfunction | |
| JP2007045784A (en) | New ellagic acid derivative and xanthin-oxydase inhibitor | |
| JP2021014415A (en) | Method for producing composition having uric acid level reducing effect | |
| JP2007290977A (en) | Activity improver of active oxygen scavenging enzyme | |
| KR20200141012A (en) | Pharmaceutical composition for preventing or treating male sexual dysfunction | |
| JPWO2003068215A1 (en) | Mitochondrial drugs | |
| CN116782944A (en) | Compositions for enhancing adrenomedullin gene expression |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20101207 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20110201 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20110614 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20110727 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20111025 |
|
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20111028 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20141104 Year of fee payment: 3 |
|
| R151 | Written notification of patent or utility model registration |
Ref document number: 4856471 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R151 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20141104 Year of fee payment: 3 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |