JP2015184230A - Screening method of anti-aging material - Google Patents
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Abstract
Description
本発明は、抗老化物質のスクリーニング方法に関する。 The present invention relates to a screening method for anti-aging substances.
従来、美容又は健康を目的とした食品組成物の配合成分として、コラーゲンペプチド、ヒアルロン酸、N−アセチルグルコサミンなど種々の成分が利用されている(特許文献1〜3)。 Conventionally, various components such as collagen peptides, hyaluronic acid, N-acetylglucosamine have been used as ingredients of food compositions for beauty or health purposes (Patent Documents 1 to 3).
これら成分は、美容と健康に対し有用な効果を有すると認識されているが、その効果に関しては満足のいくものではなかった。 Although these ingredients are recognized as having useful effects on beauty and health, their effects have not been satisfactory.
生体内で産生される活性酸素は、体内のタンパク質、脂質、遺伝子を酸化し、癌や生活習慣病などの種々の疾患、そして生体老化の原因にもなるとされている。これに対して、生体内には抗酸化物質が含まれており、活性酸素の発生を抑制したり、発生した活性酸素を除去したりする効果があり、この中には実際に老化を抑える効果を示すものも知られている。 Active oxygen produced in the body is said to oxidize proteins, lipids and genes in the body, causing various diseases such as cancer and lifestyle-related diseases, and bioaging. In contrast, antioxidants are contained in the living body, which has the effect of suppressing the generation of active oxygen and removing the generated active oxygen. Among these, the effect of actually suppressing aging Some are also known.
これまでの抗酸化物質の評価は、候補物質の抗酸化能を試験管内で物理化学的に、たとえばイオンの還元反応などで測定することにより評価していた。 So far, antioxidant substances have been evaluated by measuring the antioxidant ability of candidate substances in a test tube physicochemically, for example, by ion reduction reaction.
しかしながら、このような評価系で抗酸化能を示した物質が、生体に対し実用的な抗酸化能を示すとは限らない。培養細胞や細菌を用いた抗酸化活性の検査法が用いられているが、抗酸化候補物質の吸収、代謝などの影響を評価することはできない。 However, a substance that exhibits antioxidant ability in such an evaluation system does not always exhibit practical antioxidant ability for a living body. Antioxidant activity testing methods using cultured cells and bacteria are used, but the effects of absorption and metabolism of antioxidant candidate substances cannot be evaluated.
抗酸化物質のスクリーニング法としてマウスやラットなどの実験動物を用いた生体老化評価法が望ましいが、この方法では、大量の候補物質を用いる必要があり、老化の評価になると動物の寿命に対応した年単位の期間が必要になる。 As a screening method for antioxidants, biological aging evaluation methods using experimental animals such as mice and rats are desirable, but this method requires the use of a large number of candidate substances. An annual period is required.
本発明は、短期間で信頼性の高い抗老化物質のスクリーニング方法を提供することを目的とする。 An object of the present invention is to provide a highly reliable screening method for anti-aging substances in a short period of time.
本発明は、以下のスクリーニング方法を提供するものである。
項1. 活性酸素の除去能が低下したショウジョウバエに候補物質を摂取させ、以下の(i)〜(iv)
(i)寿命
(ii)行動量、
(iii)脳内の運動神経数、
(iv)筋肉内の異常タンパク質の量
の少なくとも1つの項目について評価し、その評価結果に基づき抗老化物質を選別することを特徴とする、抗老化物質のスクリーニング方法。
項2. 活性酸素の除去能が低下したショウジョウバエが、Cu/Zn-SOD変異体、Mn-SOD変異体、Trxr-1突然変異体のいずれかである、項1に記載のスクリーニング方法。
項3. 評価項目が、(i)寿命と、(ii)〜(iv)の少なくとも1つの項目の組み合わせである、項1又は2に記載のスクリーニング方法。
The present invention provides the following screening methods.
Item 1. Drosophila with reduced ability to remove active oxygen are ingested with candidate substances, and (i) to (iv) below
(i) Life
(ii) Action amount,
(iii) number of motor nerves in the brain,
(iv) A method for screening an anti-aging substance, comprising evaluating at least one item of the amount of abnormal protein in muscle and selecting the anti-aging substance based on the evaluation result.
Item 2. Item 2. The screening method according to Item 1, wherein the fruit fly having reduced ability to remove active oxygen is any one of a Cu / Zn-SOD mutant, a Mn-SOD mutant, and a Trxr-1 mutant.
Item 3. Item 3. The screening method according to Item 1 or 2, wherein the evaluation item is a combination of (i) life and at least one item of (ii) to (iv).
本発明によれば、以下の効果が得られる:
1. 抗老化効果のある天然物を迅速、低コストで評価可能。
2. 抗老化効果のある化合物を迅速、低コストで評価可能。
3. 抗老化物質が効く生体内の組織(脳神経、筋肉系、消化管)の同定。
4. 活性酸素を産生させる酸化ストレス物質の探索にも利用可。
According to the present invention, the following effects can be obtained:
1. Quickly and inexpensively evaluate natural products with anti-aging effects.
2. Evaluate compounds with anti-aging effects quickly and at low cost.
3. Identification of in vivo tissues (cranial nerves, muscular system, digestive tract) where anti-aging substances are effective.
4. Can also be used to search for oxidative stress substances that produce active oxygen.
本発明で使用するショウジョウバエは、活性酸素の消去に関与する少なくとも1つの遺伝子の機能が遺伝子のアミノ酸置換およびトランスポゾン挿入突然変異、ノックダウンなどにより低下もしくは欠損し、それにより活性酸素消去能の低下したショウジョウバエである。このような遺伝子としては、Cu/Zn-SOD、Mn-SODなどのSOD(スーパーオキシドヂスムターゼ)、Trxr-1などのチオレドキシンレダクターゼ、カタラーゼなどが挙げられ、好ましくはSOD,Trxレダクターゼが挙げられる。具体的なショウジョウバエの系統としては、以下のものが挙げられる:
・sod1[n1]突然変異体
・sod2[n283]突然変異体
・UAS-sod1RNAi[F103](Cu/Zn-SODをノックダウンするのに必要な系統)
・UAS-sod1RNAi[IR4](Cu/Zn-SODをノックダウンするのに必要な系統)
・UAS-sod2RNAi[IR-15](Mn-SODをノックダウンするのに必要な系統)
・UAS-sod2RNAi[HMS00783](Mn-SODをノックダウンするのに必要な系統)
・P{Act5C-Gal4}25F01 (全身の細胞でノックダウンをおこなうのに必要な系統)
・P{wmW.hs=GawB}elavC155 (筋肉細胞でノックダウンをおこなうのに必要な系統)
・P{wmC=Gal4-Mef2.R}3 (神経細胞でノックダウンをおこなうのに必要な系統)
・Trxr-1[1]突然変異体
・P{Cat-LacZ}(Catalase遺伝子の発現調節領域の下流にLacZ遺伝子を置いた改変遺伝子をもつ系統)
・P{Gadd45-GFP} (Gadd45遺伝子の発現調節領域の下流にGFP遺伝子を置いた改変遺伝子をもつ系統)
In Drosophila used in the present invention, the function of at least one gene involved in scavenging reactive oxygen is reduced or deleted by amino acid substitution of the gene, transposon insertion mutation, knockdown, etc., thereby reducing the reactive oxygen scavenging ability. Drosophila. Examples of such genes include SOD (superoxide dismutase) such as Cu / Zn-SOD and Mn-SOD, thioredoxin reductase such as Trxr-1, and catalase, preferably SOD and Trx reductase. . Specific Drosophila strains include the following:
・ Sod1 [n1] mutant ・ sod2 [n283] mutant
・ UAS-sod1RNAi [F103] (Strain required for knocking down Cu / Zn-SOD)
・ UAS-sod1RNAi [IR4] (Strain required for knocking down Cu / Zn-SOD)
・ UAS-sod2RNAi [IR-15] (Strain required for knocking down Mn-SOD)
・ UAS-sod2RNAi [HMS00783] (Strain required for knocking down Mn-SOD)
・ P {Act5C-Gal4} 25F01 (Strain required for knocking down whole body cells)
・ P {w mW.hs = GawB} elav C155 (strain required for knockdown in muscle cells)
· P {w mC = Gal4- Mef2.R} 3 ( lines needed to perform the knockdown neurons)
・ Trxr-1 [1] mutant
・ P {Cat-LacZ} (Strain with a modified gene with the LacZ gene located downstream of the expression regulatory region of the Catalase gene)
・ P {Gadd45-GFP} (Strain having a modified gene with a GFP gene placed downstream of the Gadd45 gene expression regulatory region)
本発明のスクリーニング方法により抗老化作用が明らかにされた候補物質は、寿命を延長することができるので、医薬品、食品(健康食品、特定保健用食品、機能性食品、サプリメントなど)などとして有用である。このような候補物質としては、抗酸化作用、細胞賦活作用などを有し、老化抑制作用が期待される天然物(天然物自体、その抽出物あるいは抽出物の分画あるいは天然物から単離した物質)、医薬品または生理活性物質などが挙げられる。さらに医薬品や食品などの老化促進のような有害作用(副作用)の確認目的でも本発明のスクリーニング方法を利用可能である。 Candidate substances whose anti-aging action has been clarified by the screening method of the present invention can extend the life span, and are useful as pharmaceuticals, foods (health foods, foods for specified health use, functional foods, supplements, etc.), etc. is there. Such candidate substances include natural products that have anti-oxidant action, cell activation action, etc. and are expected to have anti-aging action (natural products themselves, extracts thereof, fractions of extracts or isolated from natural products) Substance), pharmaceuticals or physiologically active substances. Furthermore, the screening method of the present invention can be used for the purpose of confirming harmful effects (side effects) such as promotion of aging of pharmaceuticals and foods.
候補物質は、飼料の中に添加するのが好ましいが、常温で気体の物質はショウジョウバエの密閉されたケージ(容器)中に気体として添加しても良い。飼料は、固形でも液体でも良く、ペースト状などの半固形の状態であっても良い。 Although the candidate substance is preferably added to the feed, the gaseous substance at room temperature may be added as a gas in a Drosophila sealed cage (container). The feed may be solid or liquid, and may be in a semi-solid state such as a paste.
候補物質の飼料中の濃度は、例えば0.0001〜5質量%程度が挙げられる。 As for the density | concentration in the feed of a candidate substance, about 0.0001-5 mass% is mentioned, for example.
ショウジョウバエが老化すると(i)寿命が短くなり、(ii)行動量が低下し、(iii)脳内の運動神経数が減少し、(iv)筋肉内に以上タンパク質が蓄積するようになる。したがって、寿命の延長、行動量及び脳内運動神経数の維持、筋肉内の異常タンパク質の蓄積がないことにより、候補物質が抗老化物質であると評価され、選別されることになる。(v)また、医薬品や食品に含まれる老化促進物質を食べたときに、消化管の細胞に対する影響を検出することができるようになる。幼虫あるいは成虫の体内に吸収された後に体内の組織、細胞、遺伝子に与える影響を評価することができる。 As Drosophila ages, (i) the life span is shortened, (ii) the amount of behavior decreases, (iii) the number of motor nerves in the brain decreases, and (iv) the protein accumulates in the muscle. Therefore, the candidate substance is evaluated and selected as an anti-aging substance due to the prolongation of life span, the maintenance of the amount of behavior and the number of motor neurons in the brain, and the absence of abnormal protein accumulation in muscle. (V) In addition, when an aging-promoting substance contained in pharmaceuticals and foods is eaten, the influence on the cells of the digestive tract can be detected. After being absorbed into the body of larvae or adults, the effects on tissues, cells, and genes in the body can be evaluated.
寿命は測定が最も容易な評価項目であるので、本発明のスクリーニング方法で評価するのが好ましい。 Since the lifetime is the evaluation item that can be measured most easily, it is preferably evaluated by the screening method of the present invention.
行動量は、一定距離を移動できたショウジョウバエ個体の割合で評価することができる。例えば一定の長さ(距離)を有する円筒などの筒状体の底にショウジョウバエを置き、筒状体の上部に到達したショウジョウバエ数をカウントすることにより、行動量を評価することができる。行動量は、ショウジョウバエの動きをCCDカメラなどで撮影し、画像解析により評価しても良い。 The amount of action can be evaluated by the percentage of Drosophila individuals that have moved a certain distance. For example, the amount of action can be evaluated by placing Drosophila on the bottom of a cylindrical body such as a cylinder having a certain length (distance) and counting the number of Drosophila reaching the top of the cylindrical body. The amount of action may be evaluated by analyzing the Drosophila movement with a CCD camera or the like and analyzing the image.
老化すると行動量が低下するが、行動量の低下を抑制できる候補物質は、行動力を伴った寿命の延長を実現できるので好ましい。 Although the amount of behavior decreases when aging, a candidate substance that can suppress the decrease in the amount of behavior is preferable because it can achieve a long life with behavioral power.
脳内の運動神経数は、例えば脳内のドーパミン神経細胞をTH免疫染色法にて標識、共焦点レーザー顕微鏡にて観察し、6つのドーパミン神経クラスター内の細胞数を計測することにより行う。脳内の運動神経数は加齢に伴い減少するので、これを定量化することで抗老化作用の程度を評価することができる。運動神経数を評価することで、運動能力を維持しつつ寿命を延長する抗老化物質を選別することができる。 The number of motor nerves in the brain is determined by, for example, labeling dopamine neurons in the brain with TH immunostaining and observing with a confocal laser microscope and measuring the number of cells in six dopamine clusters. Since the number of motor nerves in the brain decreases with aging, the degree of anti-aging action can be evaluated by quantifying this. By evaluating the number of motor nerves, it is possible to select an anti-aging substance that extends life while maintaining motor ability.
筋肉内の異常タンパク質の蓄積は、成虫間接飛翔筋内にできた異常タンパク質凝集体を抗ポリユビキチン抗体による免疫染色法にて視覚化し、蛍光顕微鏡にて観察することで評価できる。加齢にともなう異常タンパク質の増加は、筋力の衰えと相関するので、筋肉内の異常タンパク質の蓄積を評価することで、加齢に伴う筋力の維持の程度を評価することができる。 Accumulation of abnormal proteins in muscle can be evaluated by visualizing abnormal protein aggregates formed in the adult indirect flight muscle by immunostaining with anti-polyubiquitin antibody and observing with a fluorescence microscope. Since an increase in abnormal protein with aging correlates with a decline in muscle strength, the degree of maintenance of muscle strength associated with aging can be evaluated by evaluating the accumulation of abnormal protein in muscle.
(ii)〜(iv)により加齢時の運動能力を評価することができ、本発明のスクリーニング方法により寿命を延ばし、かつ、運動能力や運動量を向上させる物質を選別できる。 The motor ability at the time of aging can be evaluated by (ii) to (iv), and the screening method of the present invention can select a substance that extends the life span and improves the motor ability and the amount of exercise.
(v) 老化促進物質が細胞に作用するとカタラーゼ遺伝子の発現が上昇する。成虫消化管におけるカタラーゼ遺伝子の発現を調べることにより、体内に吸収された物質の有害性を評価することができる。さらに老化促進物質の中には、体内の増殖細胞に作用して、遺伝子DNAを酸化損傷するものがある。遺伝子損傷が起きると損傷修復酵素Gadd45の遺伝子が発現誘導されるので、それを検出することにより、老化促進物質が遺伝子DNAに与える損傷効果を評価できる。 (v) When the aging promoting substance acts on cells, the expression of the catalase gene increases. By examining the expression of the catalase gene in the adult digestive tract, the harmfulness of the substance absorbed in the body can be evaluated. In addition, some aging promoting substances act on proliferating cells in the body to oxidize and damage genetic DNA. When gene damage occurs, the gene for damage repair enzyme Gadd45 is induced, and by detecting it, the damage effect of the aging promoting substance on the gene DNA can be evaluated.
以下、本発明を実施例に基づきより詳細に説明するが、本発明がこれら実施例に限定されないことはいうまでもない。 EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, it cannot be overemphasized that this invention is not limited to these Examples.
実施例1
アミノ酸置換により活性が低下したCu/Zn-SODをもつショウジョウバエの突然変異体( sod[n1] )を用い、直径25mmプラスチックチューブに成虫を入れて飼育し、ビタミンC(アスコルビン酸)を1mM、10mM、25mM、50mM含む餌を摂取させて、寿命を測定した。結果を試験に用いたショウジョウバエの数(n)とともに図1に示す。コントロールはビタミンCを添加していない餌を用いた。
Example 1
Using Drosophila mutants ( sod [n1] ) with Cu / Zn-SOD whose activity is reduced by amino acid substitution, adults are placed in plastic tubes with a diameter of 25 mm and vitamin C (ascorbic acid) is 1 mM and 10 mM. Life expectancy was measured by feeding with food containing 25 mM and 50 mM. The results are shown in FIG. 1 together with the number (n) of Drosophila used in the test. As a control, a bait not added with vitamin C was used.
図1に示すように、ショウジョウバエの寿命は、アスコルビン酸濃度が50mMにおいて顕著に増加した。 As shown in FIG. 1, the life span of Drosophila markedly increased when the ascorbic acid concentration was 50 mM.
この結果から、本発明のスクリーニング方法により、寿命を増加させる抗老化物質を選別できることが明らかになった。また、トランスポゾン挿入によりチオレドキシンレダクターゼ(Trx-1)の発現量が低下したショウジョウバエの突然変異体(Trxr-1[1])を用いても同様な結果が得られることを本発明者は確認した。 From this result, it became clear that the anti-aging substance that increases the lifespan can be selected by the screening method of the present invention. Furthermore, the present inventors have confirmed that similar results can be obtained using a Drosophila mutant ( Trxr-1 [1] ) in which the expression level of thioredoxin reductase (Trx-1) is reduced by transposon insertion.
実施例2
SODの活性低下したショウジョウバエの突然変異体(sod[n1])を用い、煎茶、ルイボスティ、玉露、ほうじ茶、ジャスミン茶の抽出液1mlで標準飼料を作製し、直径25mmプラスチックチューブに成虫20匹を入れて飼育し、24時間ごとの生存率を計測した。結果を試験に用いたショウジョウバエの数(n)とともに図2に示す。
Example 2
Using a Drosophila mutant with reduced SOD activity ( sod [n1] ), prepare a standard feed with 1 ml of sencha, rooibosti, gyokuro, hojicha, jasmine tea extract, and put 20 adults in a 25 mm diameter plastic tube The survival rate was measured every 24 hours. The results are shown in FIG. 2 together with the number (n) of Drosophila used in the test.
図2に示すように、各茶抽出物は、いずれも抽出物無添加のコントロールと比較して寿命延長効果を有しており、ルイボスティは成虫生存率を50%の日数が最も長く、最も寿命延長効果が大きいことが示された。 As shown in FIG. 2, each tea extract has an effect of extending the life compared to the control without any extract, and Rooibosti has the longest life span of 50% and the longest life span. It was shown that the extension effect is great.
また、図1,2の結果ともに2週間程度の短期間で結果が得られることが明らかになった。 Moreover, it became clear that the result of FIG. 1 and 2 was obtained in a short period of about 2 weeks.
実施例3
標準飼料1mlを入れた直径25mmプラスチックチューブに成虫(下記)20匹を入れて飼育し、24時間ごとの生存率を計測した。使用した成虫は野生型(コントロール)、SOD1ノックダウン系統の*1(Actin-Gal4/+; UAS-sod1[F103]/+)、SOD2ノックダウン系統の*2(Actin-Gal4/+; UAS-sod2[IR15]/+)の3種である。*1と*2の系統については既に報告があり、研究者ならインディアナ大学ならびにウイーン大学系統センターから入手可能である。
Example 3
Twenty adults (below) were placed in a 25 mm diameter plastic tube containing 1 ml of standard feed and reared, and the survival rate was measured every 24 hours. Adults used were wild type (control), SOD1 knockdown line * 1 (Actin-Gal4 / +; UAS-sod1 [F103] / +), SOD2 knockdown line * 2 (Actin-Gal4 / +; UAS- sod2 [IR15] / +). * 1 and * 2 strains have already been reported, and researchers can obtain them from Indiana University and the University of Vienna University Center.
SOD1またはSOD2をノックダウンすることで成虫の運動量はコントロールと比較して顕著に低下し、行動量を高める物質のスクリーニングに適していることが明らかになった(図3)。 Knocking down SOD1 or SOD2 significantly reduced the amount of adult motility compared to the control, indicating that it is suitable for screening for substances that increase the amount of behavior (FIG. 3).
実施例4
標準飼料1mlを入れた直径25mmプラスチックチューブに成虫20匹を入れて飼育し、羽化後5日目の脳内のドーパミン神経細胞をTH免疫染色法にて標識し、共焦点レーザー顕微鏡にて観察した。6つのドーパミン神経クラスター内の細胞数を計測し、加齢にともなう減少を定量化した。結果を図4に示す。使用した成虫は野生型(コントロール)、SOD1ノックダウン系統の*1(Actin-Gal4/+; UAS-sod1[F103]/+)、SOD2ノックダウン系統の*2(Actin-Gal4/+; UAS-sod2[IR15]/+)の3種である。
Example 4
Twenty adults were bred in a 25 mm diameter plastic tube containing 1 ml of standard feed. Dopamine neurons in the brain 5 days after emergence were labeled by TH immunostaining and observed with a confocal laser microscope. . The number of cells in six dopamine neuronal clusters was counted and the decrease with age was quantified. The results are shown in FIG. Adults used were wild type (control), SOD1 knockdown line * 1 (Actin-Gal4 / +; UAS-sod1 [F103] / +), SOD2 knockdown line * 2 (Actin-Gal4 / +; UAS- sod2 [IR15] / +).
SOD2をノックダウンした成虫では羽化して5日めの若い成虫で既にクラスター内の神経細胞の数が減少しており、正常成虫の左の図との違いは明らかである。これはパーキンソン病に似た老化症状であり、本発明により、パーキンソン病に罹患する確率を低下させる物質を選別できることが明らかになった。 In adults knocked down with SOD2, the number of neurons in the cluster is already reduced in young adults on the fifth day, and the difference from the left figure of normal adults is clear. This is an aging symptom similar to Parkinson's disease, and it has become clear that the present invention can select a substance that reduces the probability of suffering from Parkinson's disease.
実施例5
標準飼料1mlを入れた直径25mmプラスチックチューブに成虫20匹を入れて飼育し、羽化後5日目及び30日目の成虫間接飛翔筋内にできた異常タンパク質凝集体を抗ポリユビキチン抗体による免疫染色法にて視覚化し、蛍光顕微鏡にて観察して加齢に伴う異常タンパク質の増加を定量化した。結果を図5に示す。使用した成虫は野生型(コントロール)、SOD1ノックダウン系統の*1(Actin-Gal4/+; UAS-sod1[F103]/+)、SOD2ノックダウン系統の*2(Actin-Gal4/+; UAS-sod2[IR15]/+)の3種である。
Example 5
20 adults were placed in a 25 mm diameter plastic tube containing 1 ml of standard feed and reared, and abnormal protein aggregates formed in the adult indirect flight muscles on the 5th and 30th days after emergence were immunostained with anti-polyubiquitin antibodies. The increase in abnormal protein with aging was quantified by visualizing by a method and observing with a fluorescence microscope. The results are shown in FIG. Adults used were wild type (control), SOD1 knockdown line * 1 (Actin-Gal4 / +; UAS-sod1 [F103] / +), SOD2 knockdown line * 2 (Actin-Gal4 / +; UAS- sod2 [IR15] / +).
異常タンパク質は、30日後においてSOD1/2のノックダウンにより蓄積することが明らかになった。 Abnormal proteins were found to accumulate by knockdown of SOD1 / 2 after 30 days.
実施例6
老化促進物質を食べたときにそれが最初に吸収される消化管において、その物質の有害性を評価した。成虫消化管におけるカタラーゼ遺伝子の発現をLacZレポーターにより検出した。老化促進物質のパラコート農薬10mMを48時間摂食させた成虫を解剖して、消化管をLacZ染色した。消化管の細胞が酸化損傷されるとカタラーゼが強く誘導され、染色領域が広がった。これを指標にその有害性を評価することができる。さらに個体全体を用いてCatalase遺伝子の発現量をリアルタイムPCR法により定量化した。この方法により評価の精度をあげることができる。また、老化促進物質のパラコート農薬10mMを48時間摂食させた。体内の増殖細胞において遺伝子DNAが酸化損傷するとGadd45遺伝子が発現誘導される。その誘導はGFPレポーターを用いて検出できる。リアルタイムPCR法によりmRNA量を定量化すれば評価の精度をあげることができる。これらの方法で老化促進物質が体内の遺伝子に及ぼす影響を評価することができる。
Example 6
The toxicity of the aging-promoting substance was evaluated in the digestive tract where it was first absorbed when eaten. The expression of catalase gene in adult digestive tract was detected by LacZ reporter. Adults fed with 10 mM of paraquat pesticide as an aging promoting substance were dissected for 48 hours, and the digestive tract was stained with LacZ. When cells in the gastrointestinal tract were oxidatively damaged, catalase was strongly induced, and the stained area expanded. The harmfulness can be evaluated using this as an index. Furthermore, the expression level of the Catalase gene was quantified by real-time PCR using the whole individual. This method can increase the accuracy of evaluation. Moreover, 10 mM of paraquat pesticide as an aging promoting substance was fed for 48 hours. When the gene DNA is oxidatively damaged in proliferating cells in the body, expression of the Gadd45 gene is induced. The induction can be detected using a GFP reporter. If the amount of mRNA is quantified by a real-time PCR method, the accuracy of evaluation can be improved. By these methods, the effect of aging promoting substances on the genes in the body can be evaluated.
本発明のスクリーニング方法は、以下の用途に応用可能である。
(i)老化抑制効果のある天然物成分あるいは化合物入りの医薬品、健康食品の開発
(ii)老化抑制効果のある天然物成分あるいは化合物の作用機構の解明(商品の付加価値の向上)
(iii)活性酸素を産生させる有害医薬品、食品添加物の生体検査
The screening method of the present invention can be applied to the following uses.
(i) Development of pharmaceuticals and health foods containing natural products or compounds that have an anti-aging effect
(ii) Elucidation of the mechanism of action of natural product components or compounds with anti-aging effects (improvement of added value of products)
(iii) Biological examination of harmful drugs and food additives that produce active oxygen
Claims (3)
(i)寿命
(ii)行動量、
(iii)脳内の運動神経数、
(iv)筋肉内の異常タンパク質の量
の少なくとも1つの項目について評価し、その評価結果に基づき抗老化物質を選別することを特徴とする、抗老化物質のスクリーニング方法。 Drosophila with reduced ability to remove active oxygen are ingested with candidate substances, and (i) to (iv) below
(i) Life
(ii) Action amount,
(iii) number of motor nerves in the brain,
(iv) A method for screening an anti-aging substance, comprising evaluating at least one item of the amount of abnormal protein in muscle and selecting the anti-aging substance based on the evaluation result.
The screening method according to claim 1 or 2, wherein the evaluation item is a combination of (i) life and at least one item of (ii) to (iv).
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