JP2013253071A - Oph activity enhancer - Google Patents
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Abstract
Description
本発明は、酸化蛋白質を分解する生体内酵素である酸化蛋白質分解酵素(oxidized protein hydrolase:OPH)の活性を増強させるOPH活性増強剤、及び、当該OPH活性増強剤を含有する飲食品や医薬品などに関する。 The present invention relates to an OPH activity enhancer that enhances the activity of oxidized protein hydrolase (OPH), which is an in vivo enzyme that degrades oxidized protein, and foods and beverages and pharmaceuticals containing the OPH activity enhancer. About.
生体内における酸化反応が細胞や組織に悪影響を及ぼすことが知られている。例えば、組織コラーゲンなどの加齢変化、アルツハイマー病、白内障、皮膚老化などさまざまな疾患や老化の原因の一つとなる。 It is known that an oxidation reaction in a living body adversely affects cells and tissues. For example, aging changes such as tissue collagen, Alzheimer's disease, cataract, skin aging, and other diseases and aging.
酸化蛋白質はOPHによって分解除去される。OPHは生体組織中に広く分布し、酸化蛋白質を優先的に分解するセリンプロテアーゼの一種であり、蛋白質のN末端アシル化アミノ酸を遊離する酵素であるアシルアミノ酸遊離酵素(Acylamino-acid-releasing enzyme:AARE)として知られている。このOPHは加齢とともにその活性が低下することが知られている。 Oxidized protein is decomposed and removed by OPH. OPH is a kind of serine protease that is widely distributed in living tissues and preferentially degrades oxidized proteins. Acylamino-acid-releasing enzyme: an enzyme that releases N-terminal acylated amino acids of proteins. AARE). It is known that the activity of OPH decreases with age.
OPH活性は糖尿病ラットの血清中において顕著に上昇することや、OPH活性が上昇した血清ではカルボニル修飾蛋白質量が低いことが報告されており、OPHが糖尿病や老化進展抑制に関わる可能性があるとされている。 It has been reported that OPH activity is significantly increased in the serum of diabetic rats, and that the amount of carbonyl-modified protein is low in serum with increased OPH activity, and that OPH may be related to the suppression of diabetes and aging progression. Has been.
OPHの活性を促進させることにより、疾患や老化を予防することが期待されているものの、OPH活性を促進する作用物質としては、ドクダミ、セイヨウサンザシ、ローマカミツレ、ブドウが知られているに過ぎない(特許文献1)。 Although it is expected to prevent diseases and aging by promoting the activity of OPH, only dodami, hawthorn, roman chamomile, and grape are known as agents that promote OPH activity. (Patent Document 1).
上記の事情を鑑み、本発明は、OPHの活性を増強させるOPH活性増強剤を提供することを課題とする。 In view of the above circumstances, an object of the present invention is to provide an OPH activity enhancer that enhances the activity of OPH.
上記課題を解決するための手段として、以下の発明などを提供する。すなわち、第一の発明として、ゴボウ、ウーロン茶、ほうじ茶、レモングラス、キダチアロエ、クマザサ、トウガラシ、オオムギ、アマチャヅル、ギムネマ、エビスグサ、ベニバナ、クワ、レイシ、プーアール茶、番茶、ドクダミ、ハトムギ、ハブソウの抽出物を1種類以上含む組み合わせからなり、酸化蛋白質分解酵素(OPH)の酸化蛋白質分解活性を1.2倍以上活性化するOPH活性増強剤を提供する。 As means for solving the above problems, the following inventions and the like are provided. That is, as the first invention, the extract of burdock, oolong tea, roasted tea, lemongrass, yellow tea aloe vera, pepper, barley, barley, macaques, gymnema, shrimp, safflower, mulberry, litchi, pu-erh tea, bancha, dokudami, pearl barley, hawthorn And an OPH activity enhancer that activates the oxidized proteolytic activity of oxidized proteolytic enzyme (OPH) by a factor of 1.2 or more.
第二の発明として、ゴボウ、ウーロン茶、ほうじ茶の抽出物を1種類以上含む組み合わせからなり、OPH活性を3倍以上活性化するOPH活性増強剤を提供する。 As a second invention, there is provided an OPH activity enhancer comprising a combination containing at least one extract of burdock, oolong tea, and hoji tea and activating OPH activity three times or more.
第三の発明として、ウーロン茶、ほうじ茶、レモングラス、番茶、プーアール、ベニバナの抽出物を1種類以上含む組み合わせからなり、OPHの酸化蛋白質分解活性を1.2倍以上活性化するとともにOPHのAGEs分解を活性化するOPH活性増強剤を提供する。 The third invention consists of a combination of one or more extracts of oolong tea, roasted tea, lemongrass, bancha, pu-eru, safflower, which activates the oxidized proteolytic activity of OPH by 1.2 times or more and decomposes AGEs of OPH Provided is an OPH activity enhancer that activates.
第四の発明としては、第一の発明から第三の発明に記載のOPH活性増強剤を1種類以上含む組み合わせを含有する飲食品、健康食品、食品添加物、医薬品、化粧品、医薬部外品を提供する。 As 4th invention, the food / beverage products, health food, food additive, pharmaceutical, cosmetics, quasi-drug containing the combination containing 1 or more types of OPH activity enhancer as described in 1st invention from 3rd invention are included. I will provide a.
本発明により、OPHの酸化蛋白質分解活性を増強させるOPH活性増強剤を提供することが可能となる。 According to the present invention, an OPH activity enhancer that enhances the oxidized proteolytic activity of OPH can be provided.
以下、本発明の実施の形態について説明する。なお、本発明は、これらの実施形態に何ら限定されるべきものではなく、その要旨を逸脱しない範囲において、種々なる態様で実施し得る。
<実施形態1>
<実施形態1 概要>
Embodiments of the present invention will be described below. In addition, this invention should not be limited to these embodiments at all, and can be implemented in various modes without departing from the gist thereof.
<
<Summary of
本実施形態は、酸化蛋白質の分解酵素であるOPHの酸化蛋白質分解活性(以下、単にOPH活性)の増強作用について、コントロール(サンプル無添加)と比較して、OPH活性増強作用が試験により認められた17種類の植物の抽出物を含むOPH活性増強剤に関する。
<実施形態1 構成>
In this embodiment, OPH activity enhancing action of OPH, which is an oxidized protein degrading enzyme (hereinafter simply referred to as OPH activity), was confirmed by tests compared to control (no sample added). The present invention also relates to an OPH activity enhancer comprising 17 kinds of plant extracts.
<Configuration of
本実施形態に係るOPH活性増強剤は、ゴボウ、ウーロン茶、ほうじ茶、レモングラス、キダチアロエ、クマザサ、トウガラシ、オオムギ、アマチャヅル、ギムネマ、エビスグサ、ベニバナ、クワ、レイシ、プーアール茶、番茶、ドクダミ、ハトムギ、ハブソウの抽出物を1種類以上含む組み合わせからなる。ここに挙げられた17種のサンプルは、後述するOPH酸化蛋白質分解活性増強作用測定試験を行った100種を超えるサンプルの中で、サンプルを添加しないコントロールと比較して1.2倍以上のOPH酸化蛋白質分解活性増強作用が認められたものである。 The OPH activity enhancer according to the present embodiment includes burdock, oolong tea, roasted tea, lemongrass, yellow tea aloe vera, kumazasa, chili pepper, barley, macaque, gymnema, shrimp, safflower, mulberry, litchi, pu-erh tea, bancha, dokudami, pearl barley, hawthorn It consists of a combination containing one or more types of extracts. The 17 types of samples listed here are more than 1.2 times more OPH than the control to which no sample is added, among the over 100 types of samples that have been subjected to the OPH oxidation proteolytic activity enhancement test described later. An action of enhancing oxidized protein degradation activity was observed.
本実施形態における植物の抽出物は、植物のどの部位から抽出したものであってもよく、例えば、全草、花、種子、果実、枝、茎、樹皮、根などから抽出したものであってよい。また、抽出物の性状を限定するものではない。以下に、本実施形態で用いられる植物を説明する。 The plant extract in this embodiment may be extracted from any part of the plant, for example, extracted from whole grass, flowers, seeds, fruits, branches, stems, bark, roots, etc. Good. Moreover, the property of the extract is not limited. Below, the plant used by this embodiment is demonstrated.
「ゴボウ」Arctium lappaは、キク科ゴボウ属の多年草である。 The "burdock" Arctium lappa is a perennial plant belonging to the genus Burdock.
「ウーロン茶」は、茶葉(Camellia sinensisの葉)を発酵途中で加熱して発酵を止め、半発酵させた茶である。 "Oolong tea" is a tea that is semi-fermented by heating tea leaves (Camellia sinensis leaves) during fermentation to stop fermentation.
「ほうじ茶」は、番茶を焙じた茶である。 "Hojicha" is a tea roasted from bancha.
「レモングラス」Cymbopogon citratusは、イネ科オガルカヤ属の多年草植物である。 “Lemongrass” Cymbopogon citratus is a perennial plant belonging to the genus Ogarkaya.
「キダチアロエ」Aloe arborescensは、ユリ科アロエ属の多肉植物である。 "Kidachi aloe" Aloe arborescens is a succulent plant of the genus Aloe.
「クマザサ」Sasa veitchiiは、イネ科ササ属の植物である。 "Kumazasa" Sasa veitchii is a plant belonging to the genus Sasa genus.
「トウガラシ」Capsicum annuumは、ナス科トウガラシ属の植物である。 “Capsicum” Capsicum annuum is a plant belonging to the genus Capsicum.
「オオムギ」Hordeum vulgareは、イネ科オオムギ属の植物である。 "Barley" Hordeum vulgare is a plant of the genus Barley.
「アマチャヅル」Gynostemma pentaphyllumは、ウリ科アマチャヅル属の植物である。 “Amateur” Gynostemma pentaphyllum is a plant belonging to the genus Acuchar.
「ギムネマ」Gymnema sylvestreは、ガガイモ科ホウライアオカズラ属の植物である。 "Gymnema" Gymnema sylvestre is a plant belonging to the genus Houraiaokazura.
「エビスグサ」Senna obtusifoliaは、ジャケツイバラ科センナ属の植物である。 "Ebisugusa" Senna obtusifolia is a plant belonging to the genus Senna genus Jackaceae.
「ベニバナ」Carthamus tinctoriusは、キク科ベニバナ属の植物である。 “Safflower” Carthamus tinctorius is a plant belonging to the genus Asteraceae.
「クワ」Morus lhou、M. bombycis、M. alba、M. nigra、M. serrata、M. laevigata、M. tiliaefolia、M. boninensisは、クワ科クワ属の植物である。 "Mulberry" Morus lhou, M. bombycis, M. alba, M. nigra, M. serrata, M. laevigata, M. tiliaefolia, M. boninensis are plants of the genus Mulberry family.
「レイシ」Ganoderma lucidumは、マンネンタケ科マンネンタケ属の菌類である。 “Reishi” Ganoderma lucidum is a fungus of the genus Mannentake.
「プーアール茶」は、茶葉(Camellia sinensisの葉)の酸化醗酵を加熱によって止め、麹などの微生物とともに熟成させて作る後発酵茶である。 “Puer tea” is a post-fermented tea made by oxidative fermentation of tea leaves (Camellia sinensis leaves) by heating and aging with microorganisms such as strawberries.
「番茶」は、夏以降に摘む二番茶以降のかたい茶葉(Camellia sinensisの葉)を茎とともに刈り取って製茶したものである。 “Bancha” is made by harvesting hard tea leaves (leaf of Camellia sinensis) after 2nd tea, which are picked after summer, together with stems.
「ドクダミ」Houttuynia cordataは、ドクダミ科ドクダミ属の植物である。 "Dokudami" Houttuynia cordata is a plant of the genus Dokudami family.
「ハトムギ」Coix lacryma-jobi var. frumentaceaは、イネ科ジュズダマ属の植物である。 "Betley" Coix lacryma-jobi var. Frumentacea is a plant belonging to the genus Giusedama.
「ハブソウ」Senna occidentalisは、ジャケツイバラ科センナ属の植物である。 “Husou” Senna occidentalis is a plant belonging to the genus Senna genus.
上述した19種の植物抽出物の一種又は二種以上の組み合わせからなるOPH活性増強剤は、これを含有する飲食品、健康食品、食品添加物、医薬品、化粧品、医薬部外品などとして応用することが可能である(実施形態2についても同様である)。
<実施形態1 試験>
The OPH activity enhancer comprising one or a combination of two or more of the 19 kinds of plant extracts described above is applied as a food / beverage product, health food, food additive, pharmaceutical product, cosmetic product, quasi drug, etc. containing the same. (The same applies to the second embodiment).
<
上述した19種の植物抽出物は、以下の試験において、OPH活性増強作用が認められたものである。
<試験1>
The 19 plant extracts mentioned above were confirmed to have an effect of enhancing OPH activity in the following tests.
<
(1)サンプルの抽出 (1) Sample extraction
各乾燥サンプル3.75gを恒温水槽中で80℃に加温した蒸留水150mL中に加えて1時間インキュベートした。その後、抽出液を4500rpmで15分間遠心分離して上清を回収し、サンプル抽出液とした。 3.75 g of each dried sample was added to 150 mL of distilled water heated to 80 ° C. in a constant temperature water bath and incubated for 1 hour. Thereafter, the extract was centrifuged at 4500 rpm for 15 minutes, and the supernatant was collected to obtain a sample extract.
(2)反応試薬の調製 (2) Preparation of reaction reagent
OPHとして0.5Uのアシルアミノ酸遊離酵素(AARE; タカラバイオ)を入れたチューブに50mmol/Lリン酸ナトリウム緩衝液を1mL入れて希釈した。さらに200mmol/L Tris-HCl(pH7.4)で10倍希釈して0.05U/mL OPHとした。次に、50mmol/L のN-acetyl-L-alanine p-nitroanilide(AAPA)基質(フナコシ)50%エタノール水溶液を調製した。調製した各反応液を下記の表1の通りに混合して、反応試薬A〜Dを調製した。
(3)OPH活性増強作用の測定 (3) Measurement of OPH activity enhancing action
調製した反応試薬(AAPA基質を除く)は、96ウェルマイクロプレートの各ウェルに入れた後、AAPA基質を添加して反応を開始した。測定時のマイクロプレートリーダーの設定温度は37℃とし、405nmの測定波長における吸光度を10分毎に測定し、90分間測定を続けた。 The prepared reaction reagent (excluding AAPA substrate) was placed in each well of a 96-well microplate, and then AAPA substrate was added to initiate the reaction. The set temperature of the microplate reader at the time of measurement was 37 ° C., the absorbance at a measurement wavelength of 405 nm was measured every 10 minutes, and the measurement was continued for 90 minutes.
OPH活性増強作用は、まず反応試薬A〜Dの各吸光度を求めた。そして、求められた各反応試薬の吸光度を下記の式に当てはめて得られた値をOPH活性増強率とした。OPH活性増強率が100を超えているサンプルはOPH活性を増強したことを示し、100を下回るサンプルはOPH活性を低下させたことを示す。
(式1)OPH活性増強率= (反応試薬B−C) / (反応試薬A−D) × 100
For the OPH activity enhancing action, first, the absorbance of each of the reaction reagents A to D was determined. The value obtained by applying the obtained absorbance of each reaction reagent to the following formula was defined as the OPH activity enhancement rate. A sample with an OPH activity enhancement rate exceeding 100 indicates that the OPH activity is enhanced, and a sample below 100 indicates that the OPH activity is decreased.
(Formula 1) OPH activity enhancement rate = (reaction reagent B−C) / (reaction reagent A−D) × 100
(4)結果 (4) Results
図1は、19種のサンプルのうちハトムギとハブソウを除く17種について、測定開始から30分後、60分後、90分後のOPH活性増強率の推移をグラフとして表したものである。 FIG. 1 is a graph showing the transition of the OPH activity enhancement rate after 30 minutes, 60 minutes, and 90 minutes after the start of measurement for 17 types of 19 samples excluding pearl barley and yellowtail.
図2は、19種のサンプルについて測定開始から90分後のOPH活性増強率を上位から並べて表したものである。 FIG. 2 shows the OPH activity enhancement rates 90 minutes after the start of measurement for 19 samples, arranged from the top.
以上の通り、本実施形態に係る19種の植物抽出物は、いずれも無添加のコントロールと比較して、1.2倍以上のOPH活性増強作用が認められた。とくに、ゴボウは599%、ウーロン茶は457%、ほうじ茶は326%という測定結果を得ることができ、いずれも無添加のコントロールと比較して、3倍以上のOPH活性増強作用が認められた。
<実施形態1 効果>
As described above, all of the 19 plant extracts according to the present embodiment were observed to have an effect of enhancing OPH activity by 1.2 times or more compared to the control without addition. In particular, the measurement results of 599% for burdock, 457% for oolong tea, and 326% for houji tea were obtained, and in all cases, the effect of enhancing OPH activity by 3 times or more was observed compared to the control without addition.
<
本実施形態に係る植物抽出物は、いずれも無添加のコントロールと比較して、1.2倍以上のOPH活性増強作用が認められ、とくにゴボウ、ウーロン茶、ほうじ茶については、3倍以上のOPH活性増強作用が認められた。
<実施形態2>
<実施形態2 概要>
The plant extract according to this embodiment has an OPH activity enhancing effect of 1.2 times or more as compared with the control without any addition, and in particular, burdock, oolong tea, and hoji tea have 3 times or more OPH activity. A potentiating effect was observed.
<
<Overview of
本実施形態は、まず、酸化蛋白質を分解する酵素であるOPHに、糖化蛋白質や糖化最終生成物(AGEs)に対する分解作用が備わるのか否かの検証を行う。そして、OPHが分解作用を有する場合に、OPH活性増強作用が認められた実施形態1に係る19種の植物抽出物が、OPHの糖化蛋白質やAGEsに対する分解作用をより活性化させることができるか否かについて示すものである。
In this embodiment, first, it is verified whether OPH, which is an enzyme that degrades oxidized protein, has a degradation action on glycated proteins and glycated end products (AGEs). And, when OPH has a degrading action, can the 19 plant extracts according to
糖化蛋白質やAGEsは、生体内の細胞や組織と生命維持に不可欠な糖質との反応により生成される。この糖化蛋白質やAGEsが、皮膚老化、認知症、癌、高血圧、動脈硬化症などといった悪影響を及ぼすことはよく知られている。 Glycated proteins and AGEs are produced by the reaction of in vivo cells and tissues with carbohydrates essential for life support. It is well known that glycated proteins and AGEs have adverse effects such as skin aging, dementia, cancer, hypertension, arteriosclerosis and the like.
ところで、酸化蛋白質を分解する酵素としてプロテアソーム(proteasome)が知られているが、このプロテアソームは糖化蛋白質やAGEsを分解することはできない。したがって、酸素と糖質を摂取して生命活動を行っている限り不可避的に生じる蛋白質の酸化及び糖化反応のいずれをも抑制する効果をプロテアソームに求めることはできない。しかし、OPHが糖化蛋白質をも分解することができれば、OPHを摂取することにより効率的に酸化及び糖化反応のいずれをも抑制することが可能となる。 By the way, a proteasome is known as an enzyme that degrades oxidized proteins, but this proteasome cannot degrade glycated proteins and AGEs. Therefore, as long as life activity is carried out by ingesting oxygen and sugar, the proteasome cannot be sought for the effect of suppressing both protein oxidation and saccharification reactions that are unavoidable. However, if OPH can also degrade glycated proteins, it is possible to efficiently suppress both oxidation and saccharification reactions by ingesting OPH.
さらに、OPHの酸化蛋白質に対する分解作用を活性化する物質が、OPHの糖化蛋白質やAGEsに対する分解作用をも活性化することができれば、OPHの効果をより増大させることができ、ヒトが健康で若々しくいられることに大きく寄与することが期待される。
<実施形態2 試験>
<試験1>
Furthermore, if a substance that activates the degradation action of OPH on oxidized proteins can also activate the degradation action of OPH on glycated proteins and AGEs, the effect of OPH can be further increased, and humans are healthy and young. It is expected to make a great contribution to being able to live.
<
<
本試験では、OPHが、糖化蛋白質やAGEsに対する分解作用を有するか否かについて測定試験を行う。なお、本試験で用いられるサンプルは、実施形態1に係る19種の植物から抽出されたサンプルと同様である。
In this test, a measurement test is conducted to determine whether OPH has a degrading action on glycated proteins and AGEs. In addition, the sample used by this test is the same as the sample extracted from 19 types of plants which concern on
(1)AGEsの調整 (1) Adjustment of AGEs
100mmol/Lリン酸緩衝液(pH7.4) 500μL、2mol/Lグルコース(Glc) 100μL、40mg/mLヒト血清アルブミン(HSA) 200μL、蒸留水200μLを混合し、GlcとHSAの混合液(Glc-HSA G+)を調製した。次に、100mmol/Lリン酸緩衝液500μL、40mg/mL HSA 200μL、蒸留水300μLを混合し、Glcを含まない溶液(Glc-HSA G-)を調製した。そして各混合液を60℃、40時間インキュベートした。 100 mmol / L phosphate buffer (pH 7.4) 500 μL, 2 mol / L glucose (Glc) 100 μL, 40 mg / mL human serum albumin (HSA) 200 μL, distilled water 200 μL, mixed with Glc and HSA (Glc- HSA G +) was prepared. Next, 500 μL of 100 mmol / L phosphate buffer, 200 μL of 40 mg / mL HSA, and 300 μL of distilled water were mixed to prepare a solution containing no Glc (Glc-HSA G-). Each mixture was incubated at 60 ° C. for 40 hours.
(2)OPHと糖化蛋白質の反応 (2) Reaction between OPH and glycated protein
インキュベート後のGlc-HSA G+とGlc-HSA G-を使用し、下記の表2のA〜Eの反応液を調製し37℃で90分インキュベートした。インキュベート後の反応液には、PCAを100μL加えて遠心分離し、蛋白を沈澱させ上清を除去した。さらに沈殿物にTris-HCl(pH7.4) 350μLを加えて再溶解した。
(3)蛍光性AGEsの測定 (3) Measurement of fluorescent AGEs
再溶解後の反応液A〜Eを96ウェルマイクロプレートに250μL添加し、マイクロプレートリーダーで励起波長370nm、検出波長440nmにおける蛍光を測定した。 250 μL of the re-dissolved reaction solutions A to E were added to a 96-well microplate, and fluorescence at an excitation wavelength of 370 nm and a detection wavelength of 440 nm was measured with a microplate reader.
(4)ペントシジンの測定 (4) Pentosidine measurement
再溶解後の反応試薬A〜Eに6N 塩酸1mLを加え、ブロックインキュベーターで100℃、18時間加水分解した。その後、反応液サンプルを乾固し蒸留水1mLで溶解した.次に、各反応液サンプル500μLをイオン交換カラム(Oasis MCX)に注入し、0.1N 塩酸 3mLを通液後、7% NH3水溶液 4.5mLでペントシジンを溶出した。
1 mL of 6N hydrochloric acid was added to the reaction reagents A to E after redissolving, followed by hydrolysis with a block incubator at 100 ° C. for 18 hours. Thereafter, the reaction solution sample was dried and dissolved with 1 mL of distilled water. Next, 500 μL of each reaction solution sample was injected into an ion exchange column (Oasis MCX), and after passing 3 mL of 0.1N hydrochloric acid, pentosidine was eluted with 4.5 mL of 7
ペントシジン溶出液を蒸発乾固し、そこに10%アセトニトリル(ACN)を含む0.2%HFBAを300μL加え溶解した。次いで本サンプルを逆相HPLCにて分析した。 The pentosidine eluate was evaporated to dryness, and 300 μL of 0.2% HFBA containing 10% acetonitrile (ACN) was added and dissolved therein. The sample was then analyzed by reverse phase HPLC.
カラムはYMC Triart C18/S-5μm/12nm 150 x 4.6mm使用し、分析条件は流速1.0ml/min、カラム温度40℃、蛍光検出(励起波長335nm,蛍光波長385nm)とした。インジェクション量は50μLとし,溶離液条件はA) 0.2% HFBA, B) ACNとして、20%B (0-9min),100%B (9-17min),のグラジェント分析とした。 The column used was YMC Triart C18 / S-5 μm / 12 nm 150 × 4.6 mm, and the analysis conditions were a flow rate of 1.0 ml / min, a column temperature of 40 ° C., and fluorescence detection (excitation wavelength 335 nm, fluorescence wavelength 385 nm). The injection volume was 50 μL, and eluent conditions were A) 0.2% HFBA, B) ACN, and 20% B (0-9 min), 100% B (9-17 min) gradient analysis.
(5)CMLの測定 (5) CML measurement
再溶解後の反応試薬A〜E中のCMLを、CircuLex CML/Nε-(carboxymethyl) Lysine ELISA Kit (サイクレックス)を使用して測定した。 CML in the reaction reagents A to E after re-dissolution was measured using a CircuitLex CML / Nε- (carboxymethyl) Lysine ELISA Kit (Cyclex).
(6)OPHによるAGEs分解作用の測定 (6) Measurement of AGE degradation by OPH
蛍光性AGEs、ペントシジン、CMLの各測定値は下記式2(OPHを含む蛍光値)及び式3(OPHを含まない蛍光値)より求めた。
(式2)OPH+= 反応液A−C−E
(式3)OPH-= 反応液B−D
Each measured value of fluorescent AGEs, pentosidine, and CML was obtained from the following formula 2 (fluorescence value including OPH) and formula 3 (fluorescence value not including OPH).
(Formula 2) OPH + = Reaction solution A-C-E
(Formula 3) OPH- = Reaction solution BD
(7)結果 (7) Results
図3は、蛍光性AGEs、ペントシジン、CMLの各測定結果を示すものである。図示するように、いずれにおいても、OPHを含まない「OPH-」よりもOPH を含む「OPH+」の方が低い蛍光値を示していることから、OPHはGlc-HSA G+に含有されるAGEsを分解したものと認められる。
<試験2>
FIG. 3 shows the measurement results of fluorescent AGEs, pentosidine, and CML. As shown in the figure, since OPH + containing OPH shows a lower fluorescence value than OPH- without OPH, OPH contains AGEs contained in Glc-HSA G +. Permitted to have been disassembled.
<
上記試験1において、OPHがAGEsを分解することが認められた。そこで、OPH活性増強作用が認められた実施形態1に係る19種の植物抽出物が、OPHのAGEsに対する分解作用をより活性化させることができるか否かについて試験を行った。
In
(1)AGEs分解活性増強作用の測定 (1) Measurement of AGEs degradation activity enhancing action
100mmol/Lリン酸緩衝液(pH7.4) 500μL、2mol/Lグルコース(Glc)100μL、40mg/mLヒト血清アルブミン(HSA)200μL、蒸留水200μLを混合し、GlcとHSAの混合液(Glc-HSA G+)を調製した。同様にGlcを添加しない溶液(Glc-HSA G-)を調製した。 100 mmol / L phosphate buffer (pH 7.4) 500 μL, 2 mol / L glucose (Glc) 100 μL, 40 mg / mL human serum albumin (HSA) 200 μL, distilled water 200 μL, mixed with Glc and HSA (Glc- HSA G +) was prepared. Similarly, a solution (Glc-HSA G-) to which Glc was not added was prepared.
この各混合液を60℃、40時間インキュベートして得られた反応液を含む下記の表A〜Dの反応試薬を調製した。調製したA〜Dの反応試薬は37℃、90分インキュベートし、その後70%PCAを100μL加え遠心分離し、反応液中の蛋白を沈澱させた。
沈殿した蛋白質は50mmol/L Tris-HCl(pH7.4)を加えて溶解して再溶解させた後、反応試薬A〜Dを96ウェルマイクロプレートに250μLずつ添加した。マイクロプレートリーダーで励起波長370nm、検出波長440nmにおけるAGEs由来の蛍光を測定した。AGEs分解活性増強率は反応試薬A〜Dを下記の式4に当てはめて蛍光値から求めた。
(式4)AGEs分解活性増強率(%)=100−(反応試薬B-C-D)/(反応試薬A-C-D)×100
The precipitated protein was dissolved and re-dissolved by adding 50 mmol / L Tris-HCl (pH 7.4), and then 250 μL of reaction reagents A to D were added to each 96-well microplate. AGEs-derived fluorescence at an excitation wavelength of 370 nm and a detection wavelength of 440 nm was measured with a microplate reader. The AGEs degradation activity enhancement rate was obtained from the fluorescence value by applying the reaction reagents A to D to the following formula 4.
(Formula 4) AGEs degradation activity enhancement rate (%) = 100− (reaction reagent BCD) / (reaction reagent ACD) × 100
(2)結果 (2) Results
図4は、AGEs分解活性増強率の測定結果のうち、プラスの結果が得られたサンプルを示すものである。 FIG. 4 shows a sample from which a positive result was obtained among the measurement results of the AGEs degradation activity enhancement rate.
図示するように、番茶、レモングラス、ほうじ茶、ウーロン茶、プーアール、ベニバナについて、OPHのAGEsに対する分解作用をより活性化させることが示された。したがって、この6種の植物抽出物がOPHの酸化蛋白質の分解作用を活性化させるだけでなく、AGEsに対する分解作用をも活性化させることが示された。
<実施形態2 効果>
As shown in the figure, it was shown that bancha, lemongrass, roasted green tea, oolong tea, pu-eru and safflower are more active in decomposing OPH to AGEs. Therefore, it was shown that these six kinds of plant extracts not only activate the degradation action of oxidized protein of OPH but also activate the degradation action on AGEs.
<
本実施形態により、ウーロン茶、ほうじ茶、レモングラス、番茶、プーアール、ベニバナの抽出物を1種類以上含有させることにより、OPHのAGEs分解をも活性化するOPH活性増強剤を提供することが可能となる。 According to this embodiment, it becomes possible to provide an OPH activity enhancer that also activates AGE decomposition of OPH by containing one or more extracts of oolong tea, hoji tea, lemongrass, bancha, pu-eru, safflower. .
Claims (9)
Priority Applications (1)
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JP2017178970A (en) * | 2012-05-08 | 2017-10-05 | 株式会社アンチエイジングコミュニケーション | OPH activity enhancer |
JP2018171044A (en) * | 2017-03-31 | 2018-11-08 | 株式会社東洋新薬 | Oral composition |
JP2018177749A (en) * | 2017-04-21 | 2018-11-15 | 花王株式会社 | Agent for activating glucose metabolism of astrocyte |
JP2019521076A (en) * | 2016-07-18 | 2019-07-25 | アモーレパシフィック コーポレーションAmorepacific Corporation | Composition for Improving cognitive function comprising 3-O-galoyl-3,3 ', 5,5', 7-pentahydroxyflavone |
JP2019187354A (en) * | 2018-04-27 | 2019-10-31 | 株式会社アンチエイジングコミュニケーション | OPH activity enhancer |
JP2019535652A (en) * | 2016-10-18 | 2019-12-12 | アモーレパシフィック コーポレーションAmorepacific Corporation | Composition for improving cognitive function comprising a novel quercetin compound |
JP2021031472A (en) * | 2019-08-28 | 2021-03-01 | 日本メナード化粧品株式会社 | APEH production promoter |
JP2021054750A (en) * | 2019-09-30 | 2021-04-08 | 丸善製薬株式会社 | Ages formation inhibitor and method for producing the same |
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JP2017178970A (en) * | 2012-05-08 | 2017-10-05 | 株式会社アンチエイジングコミュニケーション | OPH activity enhancer |
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JP7116682B2 (en) | 2016-07-18 | 2022-08-10 | アモーレパシフィック コーポレーション | Composition for improving cognitive function containing 3-O-galloyl-3,3',5,5',7-pentahydroxyflavone |
JP7106531B2 (en) | 2016-10-18 | 2022-07-26 | アモーレパシフィック コーポレーション | COMPOSITION FOR IMPROVING COGNITIVE FUNCTION CONTAINING NOVEL QUERCETIN COMPOUND |
JP2019535652A (en) * | 2016-10-18 | 2019-12-12 | アモーレパシフィック コーポレーションAmorepacific Corporation | Composition for improving cognitive function comprising a novel quercetin compound |
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JP2021031472A (en) * | 2019-08-28 | 2021-03-01 | 日本メナード化粧品株式会社 | APEH production promoter |
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JP2021054750A (en) * | 2019-09-30 | 2021-04-08 | 丸善製薬株式会社 | Ages formation inhibitor and method for producing the same |
JP7411205B2 (en) | 2019-09-30 | 2024-01-11 | 丸善製薬株式会社 | AGEs formation inhibitor and its manufacturing method |
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