JP6891364B2 - Fumaric acid derivative exhibiting gene repair action - Google Patents
Fumaric acid derivative exhibiting gene repair action Download PDFInfo
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- JP6891364B2 JP6891364B2 JP2017003840A JP2017003840A JP6891364B2 JP 6891364 B2 JP6891364 B2 JP 6891364B2 JP 2017003840 A JP2017003840 A JP 2017003840A JP 2017003840 A JP2017003840 A JP 2017003840A JP 6891364 B2 JP6891364 B2 JP 6891364B2
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Description
この発明は遺伝子修復作用を呈するフマル酸誘導体に関するものである。 The present invention relates to a fumaric acid derivative exhibiting a gene repair action.
遺伝子は日々の生活の中で障害を受けている。また、加齢に伴って遺伝子は酸化され、障害される。生体には傷ついた遺伝子を修復する遺伝子修復作用が備わっている。 Genes are impaired in our daily lives. In addition, genes are oxidized and damaged with aging. The living body has a gene repair action that repairs damaged genes.
遺伝子修復の働きには、数種類が認められている。たとえば、ＤＮＡポリメラーゼとリガーゼの組み合わせによる修復機能がある。また、活性酸素による塩基の障害に対してはＯＧＧ１、つまり、８ヒドロキシル−２−デオキシグアノシンの修復酵素である８−オキソグアニンＤＮＡグリコシラーゼである。 Several types of gene repair functions are recognized. For example, there is a repair function by a combination of DNA polymerase and ligase. For base damage caused by active oxygen, OGG1, that is, 8-oxoguanine DNA glycosylase, which is a repair enzyme of 8-hydroxyl-2-deoxyguanosine.
さらに、ＤＮＡポリメラーゼを介したＳＯＳ修復機能も存在し、いろいろな遺伝子障害に対する修復機能が存在している。これらの遺伝子修復機能を活性化することは加齢や化学物質による遺伝子の障害を修復し、疾患を遺伝子から回復させることから好ましい。この遺伝子修復機能を活性化する研究が行われている。 Furthermore, there is also an SOS repair function via DNA polymerase, and there is a repair function for various gene disorders. It is preferable to activate these gene repair functions because it repairs genetic damage caused by aging and chemical substances and recovers the disease from the gene. Research is being conducted to activate this gene repair function.
遺伝子修復機能に関する発明としては、たとえば、標的ＤＮＡのインビボ除去による遺伝子修復があるものの、細胞レベルでの治療であり生体には応用が限られる（例えば、特許文献１参照。）。また、ＤＮＡの標的改変の発明ではベクターによる遺伝子の調整が記載されている（例えば、特許文献２参照。）。 An invention relating to a gene repair function includes, for example, gene repair by in vivo removal of target DNA, but it is a treatment at the cellular level and its application to a living body is limited (see, for example, Patent Document 1). Further, in the invention of target modification of DNA, gene adjustment by a vector is described (see, for example, Patent Document 2).
既存の物質による遺伝子修復作用は軽度であり、産業上への利用が限定されるという課題があり、また、化学合成された物質では安全性に問題があり、利用が限られている。 The gene repair action of existing substances is mild, and there is a problem that industrial use is limited, and chemically synthesized substances have safety problems, and their use is limited.
そこで、副作用が弱く優れた遺伝子修復作用を呈する天然物が望まれている。 Therefore, a natural product having weak side effects and exhibiting an excellent gene repair action is desired.
上記の目的を達成するために、請求項１に記載の発明は下記の式（１）で示される遺伝子修復作用を呈するフマル酸誘導体に関するものである。 In order to achieve the above object, the invention according to claim 1 relates to a fumaric acid derivative exhibiting a gene repair action represented by the following formula (1).
この発明は、以上のように構成されているため、次のような効果を奏する。 Since the present invention is configured as described above, the following effects are obtained.
請求項１に記載のフマル酸誘導体は遺伝子修復作用に優れている。 The fumaric acid derivative according to claim 1 is excellent in gene repair action.
以下、この発明を具体化した実施形態について詳細に説明する。 Hereinafter, embodiments embodying the present invention will be described in detail.
遺伝子修復作用を呈するフマル酸誘導体とは、下記の式（１）で示される構造からなるものである。 The fumaric acid derivative exhibiting a gene repair action has a structure represented by the following formula (1).
前記の式（１）のように遺伝子修復作用を呈するフマル酸誘導体はフマル酸の２分子、イソラムネチンの１分子及びケイ素（Ｓｉ）の１元素から構成されている。 The fumaric acid derivative exhibiting a gene repair action as described in the above formula (1) is composed of two molecules of fumaric acid, one molecule of isorhamnetin and one element of silicon (Si).
これらの分子及びその結合はすべて自然界に存在する天然型であり、各分子間はケイ素を介したイオン結合及び共有結合を介して結合している。 These molecules and their bonds are all naturally occurring, and each molecule is bonded via a silicon-mediated ionic bond and a covalent bond.
このフマル酸誘導体はフマル酸、イソラムネチン及びケイ素を原料として化学合成により得ることができる。しかし、その化学的な合成では原料の損失が多く、製造コストが高くなるため、産業への利用は限定される。 This fumaric acid derivative can be obtained by chemical synthesis using fumaric acid, isorhamnetin and silicon as raw materials. However, its chemical synthesis results in a large loss of raw materials and high manufacturing costs, which limits its use in industry.
化学合成された純度の高いフマル酸誘導体は分析の標準品や微量な試供品を得るために用いられる。 Chemically synthesized high-purity fumaric acid derivatives are used to obtain analytical standards and trace amounts of free samples.
このフマル酸誘導体の構造を解析することは有効成分の特定ができる点から好ましい。また、製品や製剤に利用して販売する際の有効成分の含有量の指標として利用できることから好ましい。 It is preferable to analyze the structure of this fumaric acid derivative from the viewpoint that the active ingredient can be identified. In addition, it is preferable because it can be used as an index of the content of the active ingredient when it is used for products and formulations and sold.
このフマル酸誘導体の構造解析の一例としてフマル酸を原材料として化学合成された高純度（純度９５％以上）の標準品を用いて重水素化ジメチルスルホキシド中の２００ＭＨｚのＨ−ＮＭＲ（１Ｈ−ＮＭＲ）により解析した場合、ピークの位置は６．１７、６．２４、６．２５、６．３２、６．４８、６．６２、９．７２、９．８６、９．８７、９．９４、１０．１５及び１０．３８ｐｐｍに認められる。 As an example of structural analysis of this fumaric acid derivative, 200 MHz H-NMR (1H-NMR) in deuterated dimethyl sulfoxide is used using a high-purity (purity 95% or more) standard product chemically synthesized using fumaric acid as a raw material. The peak positions are 6.17, 6.24, 6.25, 6.32, 6.48, 6.62, 9.72, 9.86, 9.87, 9.94, 10 It is found at .15 and 10.38 ppm.
また、Ｃ−ＮＭＲ（１３Ｃ−ＮＭＲ）により解析した場合、ピークの位置は９４．５、９４．７、９５．９、９７．３、９９．３、９９．４、１２３．５、１２３．９、１２４．６、１２５．４、１３８．６、１４３．５、１４４．０、１４７．５、１４７．７、１５２．９、１５６．２、１５６．５、１６０．５及び１６２．１ｐｐｍに認められる。 When analyzed by C-NMR (13C-NMR), the peak positions are 94.5, 94.7, 95.9, 97.3, 99.3, 99.4, 123.5, 123.9. , 124.6, 125.4, 138.6, 143.5, 144.0, 147.5, 147.7, 152.9, 156.2, 156.5, 160.5 and 162.1 ppm. Be done.
さらに、このフマル酸誘導体は高速液体クロマトグラフィーや質量分析装置で解析され、その構造が同定される。また、その化学式はＣ２４Ｈ１６Ｏ１２Ｓｉ１である。つまり、炭素元素２４個、水素元素１６個、酸素元素１２個及びケイ素元素１個から構成されている。 Furthermore, this fumaric acid derivative is analyzed by high performance liquid chromatography or mass spectrometer to identify its structure. The chemical formula is C24H16O12Si1. That is, it is composed of 24 carbon elements, 16 hydrogen elements, 12 oxygen elements and 1 silicon element.
この構成成分であるフマル酸は天然に存在している化合物である。フマル酸の化学式はＣ４Ｈ４Ｏ４である。もともと、フマル酸はＴＣＡ回路の構成成分である有機酸の一種であり、エネルギーの源となる他に、有機酸としてガスの発生源となり、細胞機能を亢進させる働きがある。 This constituent, fumaric acid, is a naturally occurring compound. The chemical formula of fumaric acid is C4H4O4. Originally, fumaric acid is a kind of organic acid which is a component of the TCA cycle, and in addition to being a source of energy, it also becomes a source of gas as an organic acid and has a function of enhancing cell function.
このフマル酸誘導体ではフマル酸とイソラムネチンの結合の間にケイ素分子１分子が結合した構造を呈している。２分子のフマル酸の構造は同一であるが、１分子はイソラムネチンの２位にエーテル結合している。もう一つはイソラムネチンの７位の水酸基とフマル酸のカルボキシル基がエステル結合している。 This fumaric acid derivative has a structure in which one silicon molecule is bonded between the bonds of fumaric acid and isorhamnetin. The structure of the two molecules of fumaric acid is the same, but one molecule is ether-bonded to the 2-position of isorhamnetin. The other is an ester bond between the hydroxyl group at the 7-position of isorhamnetin and the carboxyl group of fumaric acid.
このフマル酸分子とケイ素の結合によりこのフマル酸誘導体は安定的に、かつ、吸収率と反応性が高くなる。つまり、ベンゼン環による疎水性と水酸基による水溶性が加わり、両親媒性を呈することにより吸収が高まる。腸管の吸収においてはフマル酸単体、ケイ素単体またはイソラムネチン単体に比して４倍程度の吸収が増加する。 The bond between the fumaric acid molecule and silicon makes the fumaric acid derivative stable and has a high absorption rate and reactivity. That is, hydrophobicity due to the benzene ring and water solubility due to the hydroxyl group are added, and the absorption is enhanced by exhibiting amphipathic properties. In the intestinal absorption, absorption is increased by about 4 times as compared with fumaric acid alone, silicon alone or isorhamnetin alone.
この誘導体の構成分子であるイソラムネチン（ｉｓｏｒｈａｍｎｅｔｉｎ）はポリフェノールの一種であり、メチル化フラボノールに分類され、植物や野菜に幅広く分布している。その化学式はＣ１６Ｈ１２Ｏ７であり、分子量は３１６．２６である。 Isorhamnetin, which is a constituent molecule of this derivative, is a kind of polyphenol, is classified into methylated flavonols, and is widely distributed in plants and vegetables. Its chemical formula is C16H12O7 and its molecular weight is 316.26.
イソラムネチンはフェノール性水酸基により抗酸化作用を呈し、物質を安定に維持して長時間作用させる働きがあることは好ましい。 It is preferable that isorhamnetin exhibits an antioxidant effect due to a phenolic hydroxyl group, and has a function of maintaining a stable substance and allowing it to act for a long time.
もう一つの構成成分であるケイ素は原始番号１４の元素であり、天然に存在する元素であり、二酸化ケイ素として安定化し、動物、ヒトや植物にも含まれる。ヒトでは爪、皮膚、髪をはじめ全身の臓器に存在している。特に、皮膚においては保湿作用を発揮している。 Another constituent, silicon, is the element of primitive number 14, a naturally occurring element that stabilizes as silicon dioxide and is also found in animals, humans and plants. In humans, it is present in organs throughout the body, including nails, skin, and hair. In particular, it exerts a moisturizing effect on the skin.
また、ケイ素は昆布、藻類、海藻、植物にも含有されており、植物ではスギナやナズナに多い。ケイ素の安全性は確認されている。 Silicon is also contained in kelp, algae, seaweed, and plants, and is abundant in horsetail and shepherd's purse. The safety of silicon has been confirmed.
このフマル酸誘導体はフマル酸部分によるエネルギー産生作用及びイソラムネチン部分による抗酸化作用の他に、ＤＮＡの分子を加齢、酸化、活性酸素や紫外線から防御して遺伝子を保護する働きがある。また、遺伝子の安定化作用に寄与している。さらに、ラジカルスカベンジャー作用及び活性酸素消去作用を呈してラジカルや活性酸素を消去し、遺伝子の障害を軽減する。 This fumaric acid derivative has an energy-producing action by the fumaric acid moiety and an antioxidant action by the isorhamnetin moiety, as well as a function of protecting DNA molecules from aging, oxidation, active oxygen and ultraviolet rays to protect genes. It also contributes to gene stabilization. Furthermore, it exerts a radical scavenger action and an active oxygen scavenging action to scavenge radicals and active oxygen, and alleviate gene damage.
さらに、フマル酸のカルボン酸部分は弱酸性に荷電していることから、耐酸性が強く、経口摂取された場合に、胃酸に対して抵抗性を示し、吸収率が高まることは、好ましい。また、弱酸性であるため、皮膚に塗布した場合、皮膚に対して刺激性がないことは好ましい。 Further, since the carboxylic acid portion of fumaric acid is weakly acidic, it is preferable that it has strong acid resistance, shows resistance to gastric acid when ingested orally, and has an increased absorption rate. Moreover, since it is weakly acidic, it is preferable that it is not irritating to the skin when applied to the skin.
また、このフマル酸誘導体は両親媒性であることにより核膜内に到達しやすく、障害された遺伝子に直接作用できることは好ましい。また、このフマル酸誘導体は疎水性的に核膜に浸透して障害された遺伝子の近傍に存在する遺伝子修復酵素を活性化する。標的になる遺伝子修復酵素はＤＮＡポリメラーゼ及び８−オキソグアニンＤＮＡグリコシラーゼまたは８−ヒドロキシルデオキシグアニンＤＮＡグリコシターゼ（いずれもＯＧＧ１と略す）である。 Further, it is preferable that this fumaric acid derivative is amphipathic so that it can easily reach the nuclear envelope and can act directly on the damaged gene. In addition, this fumaric acid derivative hydrophobically permeates the nuclear envelope and activates a gene repair enzyme existing in the vicinity of the damaged gene. Target gene repair enzymes are DNA polymerase and 8-oxoguanine DNA glycosylase or 8-hydroxyldeoxyguanine DNA glycosylase (both abbreviated as OGG1).
ＤＮＡポリメラーゼによる遺伝子の修復はＳＯＳ修復といわれ、塩基の変化や付加体に働き、ＤＮＡ鎖の切断と複製を行う工程からなる。このＤＮＡポリメラーゼによる修復は１日に１万回以上行われているため、随時、この誘導体を利用し続けることが好ましい。 Gene repair by DNA polymerase is called SOS repair, and consists of steps that act on base changes and adducts to cleave and replicate DNA strands. Since this repair with DNA polymerase is performed 10,000 times or more a day, it is preferable to continue using this derivative at any time.
ＯＧＧ１による遺伝子修復は８ＯＨｄＧのような塩基の酸化体の排除と正常な塩基の組み込みを行う工程である。このフマル酸誘導体はこれらの遺伝子修復酵素の働きを行うこと及び遺伝子を酸化物質や活性酸素から防御する働きの２つの作用により遺伝子修復作用を呈する。遺伝子修復酵素作用には酵素活性の活性化と酵素のｍＲＮＡ転写レベルでの活性化の両方による。 Gene repair by OGG1 is a step of eliminating an oxidized substance of a base such as 8OHdG and incorporating a normal base. This fumaric acid derivative exhibits a gene repair action by performing the functions of these gene repair enzymes and the function of protecting the gene from oxidizing substances and active oxygen. Gene repair enzyme action depends on both activation of enzyme activity and activation of the enzyme at the mRNA transcription level.
このフマル酸誘導体による遺伝子修復作用は核内に遺伝子が存在するすべての細胞に働き、遺伝子を修復する。また、活性酸素、フリーラジカル、紫外線、化学物質、医薬品の副作用、金属、加齢などすべての物質による遺伝子の障害に対応して遺伝子を修復させる。 The gene repair action of this fumaric acid derivative acts on all cells in which the gene exists in the nucleus and repairs the gene. In addition, it repairs genes in response to genetic damage caused by all substances such as active oxygen, free radicals, ultraviolet rays, chemical substances, side effects of medicines, metals, and aging.
また、このフマル酸誘導体は脂溶性と水溶性の両方の性質を呈することから動物の細胞膜及び植物や酵母の細胞壁を通過し、細胞内に吸収されやすい。また、水溶性溶媒と油溶性溶媒の両方に溶解することから幅広い溶媒を利用することができる点は好ましい。 In addition, since this fumaric acid derivative exhibits both fat-soluble and water-soluble properties, it passes through the cell membrane of animals and the cell wall of plants and yeasts, and is easily absorbed into cells. Further, it is preferable that a wide range of solvents can be used because it is soluble in both a water-soluble solvent and an oil-soluble solvent.
さらに、皮膚の角質細胞膜も通過しやすく、角質層のバリア機能を維持することは皮膚の健康や美容の点から好ましい。また、このフマル酸誘導体は細胞膜を通過し、皮膚細胞内で遺伝子修復を活性化して細胞の再生や機能を促進することから好ましい。また、ミトコンドリア膜に対してもこのフマル酸誘導体は通過してミトコンドリアのエネルギー産生を高める。 Furthermore, it easily passes through the stratum corneum cell membrane of the skin, and maintaining the barrier function of the stratum corneum is preferable from the viewpoint of skin health and beauty. Further, this fumaric acid derivative is preferable because it passes through the cell membrane, activates gene repair in the skin cells, and promotes cell regeneration and function. In addition, this fumaric acid derivative also passes through the mitochondrial membrane to enhance mitochondrial energy production.
植物に対してはこのフマル酸誘導体が植物の細胞壁と細胞膜を通過して植物細胞内に入り、障害された遺伝子の修復を促進し、花の開花や結実、葉の成長を促進して植物の寿命を延長することは好ましい。また、ケイ素部分は植物に働くミネラルとして成長を促進する。すなわち、このフマル酸誘導体には植物活性化剤としての働きがある。 For plants, this fumaric acid derivative passes through the cell wall and cell membrane of the plant and enters the plant cell, promoting the repair of damaged genes, promoting flowering, fruiting, and leaf growth of the plant. It is preferable to extend the life. In addition, the silicon part promotes growth as a mineral that works on plants. That is, this fumaric acid derivative has a function as a plant activator.
また、このフマル酸誘導体は粉末にした場合、水溶性溶媒と反応すると水素ガスを発生し、活性酸素を消去する。水素ガスの発生量は１，６ｐｐｍの飽和濃度であり、溶解した１分から２時間程度発生する。水素ガスはヒドロキシラジカルを消去する働きがあり、優れた活性酸素消去作用が確認されている。 When this fumaric acid derivative is powdered, it generates hydrogen gas when it reacts with a water-soluble solvent to eliminate active oxygen. The amount of hydrogen gas generated is a saturated concentration of 1.6 ppm, and it is generated for about 1 minute to 2 hours when it is dissolved. Hydrogen gas has a function of scavenging hydroxyl radicals, and an excellent active oxygen scavenging action has been confirmed.
このフマル酸誘導体はヒトの皮膚細胞に対して遺伝子の修復に働く以外に、細胞の増殖、コラーゲンやエラスチン産生を促進することにより皮膚細胞機能を促進することは好ましい。 In addition to acting on gene repair for human skin cells, this fumaric acid derivative preferably promotes skin cell function by promoting cell proliferation and collagen and elastin production.
神経細胞においても細胞内の遺伝子修復を活性化する。神経細胞は認知症、アルツハイマー症などで活性酸素やアミロイドβたんぱく質による遺伝子の障害を受けやすく、遺伝子は修復されにくいという弱点がある。そのため、このフマル酸誘導体による遺伝子修復は神経の働きを回復させ、かつ、神経疾患の防御と回復の目的で好ましい。 It also activates intracellular gene repair in nerve cells. Nerve cells are susceptible to gene damage due to active oxygen and amyloid β protein due to dementia, Alzheimer's disease, etc., and have the weakness that genes are difficult to repair. Therefore, gene repair with this fumaric acid derivative is preferable for the purpose of restoring nerve function and protecting and recovering from neurological diseases.
また、神経終末からの神経伝達物質の放出を促進して神経伝達を高めることは好ましい。さらに、発生する水素ガスは低分子で血液脳関門を通過して障害された脳細胞を修復する。 It is also preferable to promote the release of neurotransmitters from nerve endings to enhance neurotransmission. In addition, the hydrogen gas generated is a small molecule that crosses the blood-brain barrier and repairs damaged brain cells.
運動神経細胞の神経末端からのアセチルコリンの放出を高めることにより筋肉の収縮を高めて神経と筋肉の活動性を増すことは好ましい。 It is preferable to increase muscle contraction and increase nerve and muscle activity by increasing the release of acetylcholine from the nerve endings of motor neurons.
また、このフマル酸誘導体は皮膚細胞の遺伝子修復作用を呈し、かつ、コラーゲンやエラスチンの遺伝子を防御してこれらの産生を高めることは好ましい。化粧料としての利用が高まることから好ましい。 In addition, it is preferable that this fumaric acid derivative exhibits a gene repair action on skin cells and protects collagen and elastin genes to enhance their production. It is preferable because its use as a cosmetic is increased.
このフマル酸誘導体は心筋梗塞においては冠状動脈の梗塞や虚血状態でも心筋細胞の遺伝子修復作用により心臓の活動を活性化して強心作用を発揮することは好ましい。また、同時に発生する水素ガスは心筋における虚血再灌流による活性酸素の障害を改善する。 In myocardial infarction, it is preferable that this fumaric acid derivative activates the activity of the heart by the gene repair action of cardiomyocytes and exerts a cardiotonic action even in an infarcted coronary artery or an ischemic state. In addition, the hydrogen gas generated at the same time improves the damage of active oxygen due to ischemic reperfusion in the myocardium.
特に、梗塞部位の血管においてはこのフマル酸誘導体は血管新生を促進し、血流の改善し、血圧を低下させる。 In particular, in blood vessels at the infarct site, this fumaric acid derivative promotes angiogenesis, improves blood flow, and lowers blood pressure.
また、このフマル酸誘導体はアスリートの活動、一般人の運動時、また、筋肉を増強したい場合、筋肉細胞での脂肪の輸送を促進して遺伝子レベルでのエネルギー産生を活性化することから好ましい。また、筋肉の活動時にこの誘導体から発生する水素ガスが運動時の活性酸素を消去することから好ましい。 In addition, this fumaric acid derivative is preferable because it promotes fat transport in muscle cells and activates energy production at the gene level during athlete activity, exercise of ordinary people, and when muscles are to be strengthened. Further, hydrogen gas generated from this derivative during muscle activity is preferable because it eliminates active oxygen during exercise.
このフマル酸誘導体は生体内では腎臓や肝臓のエステラーゼにより分解され、尿中に排泄される。分解されて構成成分である安全性の高いフマル酸及びケイ素に分解される。したがって、このフマル酸誘導体は体内に蓄積されることはなく、分解も生体内酵素で行われ、分解物も天然物であることから安全性が高い。 This fumaric acid derivative is decomposed by renal and hepatic esterase in vivo and excreted in urine. It is decomposed into highly safe fumaric acid and silicon, which are constituents. Therefore, this fumaric acid derivative is not accumulated in the body, is decomposed by an enzyme in the living body, and the decomposed product is also a natural product, so that it is highly safe.
さらに、フマル酸部分には植物の生育を促進する植物活性化作用があることからこのフマル酸誘導体にも植物の生育を促進できる点は産業上の利用の点から好ましい。 Furthermore, since the fumaric acid moiety has a plant activating effect that promotes the growth of plants, it is preferable that this fumaric acid derivative can also promote the growth of plants from the viewpoint of industrial use.
また、植物が細菌やウイルスに感染した場合、遺伝子が障害を受ける場合がある。このような遺伝子の障害に対して遺伝子修復を活性化することは好ましい。 In addition, when a plant is infected with a bacterium or a virus, the gene may be damaged. It is preferable to activate gene repair against such gene disorders.
このフマル酸誘導体は天然にも存在しており、ナズナなどの植物やワカメなどの海藻類にも極微量認められる。また、フランスカイガンショウ、ヒメマツタケなどの植物やキノコ類、オキアミやプラセンタなどの組織にも認められる。 This fumaric acid derivative also exists in nature, and is found in trace amounts in plants such as shepherd's purse and seaweeds such as wakame seaweed. It is also found in plants such as Pinus pinaster and Pinus pinaster, mushrooms, and tissues such as krill and placenta.
このフマル酸誘導体を精製により上記の植物から抽出することは可能である。ただし、精製には大量の原料を必要とし、有機溶媒などを利用することから産業上への利用は制限される。 It is possible to extract this fumaric acid derivative from the above plants by purification. However, purification requires a large amount of raw materials and uses organic solvents, etc., which limits its industrial use.
このフマル酸誘導体はナズナの葉を発酵法などにより製造させることは好ましい。発酵法としては米糠と混合して納豆菌やベニコウジ菌により発酵させて得る。用いる菌体は食用に利用できるものであるため、安全性が高い。 It is preferable that the fumaric acid derivative is produced by fermenting the leaves of shepherd's purse. As a fermentation method, it is obtained by mixing it with rice bran and fermenting it with Bacillus natto or Monascus purpureus. Since the bacterial cells used are edible, they are highly safe.
この製造方法は食経験があり、フマル酸誘導体の産生量も多いことから好ましい。 This production method is preferable because it has eating experience and produces a large amount of fumaric acid derivative.
得られたフマル酸誘導体を医薬品素材として利用する場合、目的とするフマル酸誘導体を精製することは、目的とするフマル酸誘導体の純度が高まり、不純物を除去できる点から好ましい。 When the obtained fumaric acid derivative is used as a pharmaceutical material, it is preferable to purify the target fumaric acid derivative because the purity of the target fumaric acid derivative is increased and impurities can be removed.
医薬品としては注射剤または経口剤または塗布剤などの非経口剤として利用され、医薬部外品としては錠剤、カプセル剤、ドリンク剤、石鹸、塗布剤、ゲル剤、歯磨き粉等に配合されて利用される。 As a pharmaceutical product, it is used as an injection or an oral preparation or a parenteral preparation such as a coating agent, and as a quasi-drug, it is used by being blended in tablets, capsules, drinks, soaps, coating agents, gel agents, toothpaste, etc. Toothpaste.
経口剤としては錠剤、カプセル剤、散剤、シロップ剤、ドリンク剤等が挙げられる。前記の錠剤及びカプセル剤に混和される場合には、結合剤、賦形剤、膨化剤、滑沢剤、甘味剤、香味剤等とともに用いることができる。前記の錠剤はシェラックまたは砂糖などで被覆することもできる。 Examples of oral preparations include tablets, capsules, powders, syrups, and drinks. When mixed with the above-mentioned tablets and capsules, it can be used together with a binder, an excipient, a leavening agent, a lubricant, a sweetener, a flavoring agent and the like. The tablets can also be coated with shellac, sugar or the like.
また、前記のカプセル剤の場合には、上記の材料にさらに油脂等の液体担体を含有させることができる。前記のシロップ剤及びドリンク剤の場合には、甘味剤、防腐剤、色素香味剤等を添加することができる。 Further, in the case of the above-mentioned capsule, the above-mentioned material can further contain a liquid carrier such as oil and fat. In the case of the above-mentioned syrup and energy drinks, sweeteners, preservatives, pigment flavors and the like can be added.
非経口剤としては、軟膏剤、クリーム剤、水剤等の外用剤の他に、注射剤が挙げられる。外用剤の基材としては、ワセリン、パラフィン、油脂類、ラノリン、マクロゴールド等が用いられ、通常の方法によって軟膏剤やクリーム剤等とすることができる。 Examples of parenteral preparations include injections in addition to external preparations such as ointments, creams, and liquids. As the base material of the external preparation, petrolatum, paraffin, oils and fats, lanolin, macrogold and the like are used, and ointments, creams and the like can be prepared by a usual method.
注射剤には、液剤があり、その他、凍結乾燥剤がある。これは使用時、注射用蒸留水や生理食塩液等に無菌的に溶解して用いられる。 Injections include liquids and lyophilizers. When used, it is aseptically dissolved in distilled water for injection, physiological saline, or the like.
食品製剤としては遺伝子修復作用を呈するため、エナジードリンクや強壮性の食品に利用される。神経活動を促進することから神経細胞の遺伝子の障害を介した神経のリハビリ用食品や学習時の食事などに利用される。また、美容食品にも利用される。保健機能食品として栄養機能食品や特定保健用食品に利用することは好ましい。 As a food preparation, it exhibits a gene repair effect, so it is used for energy drinks and tonic foods. Since it promotes nerve activity, it is used as a food for nerve rehabilitation through a disorder of nerve cell genes and as a diet during learning. It is also used in beauty foods. It is preferable to use it as a food with a nutritional function or a food for specified health use as a food with a health function.
得られた食品製剤をイヌやネコなどのペットや家畜動物に利用する場合、筋肉の遺伝子の障害の回復、老化の抑制と運動能力の向上を目的とした飼料やペット用サプリメントとして利用される。 When the obtained food preparation is used for pets such as dogs and cats and domestic animals, it is used as a feed or supplement for pets for the purpose of recovering muscle gene disorders, suppressing aging and improving athletic ability.
化粧料として常法に従って界面活性化剤、溶剤、増粘剤、賦形剤等とともに用いることができる。例えば、クリーム、毛髪用ジェル、洗顔剤、美容液、化粧水等の形態とすることができる。 As a cosmetic, it can be used together with a surfactant, a solvent, a thickener, an excipient and the like according to a conventional method. For example, it can be in the form of a cream, a gel for hair, a face wash, a beauty essence, a lotion, or the like.
化粧料の形態は任意であり、溶液状、クリーム状、ペースト状、ゲル状、ジェル状、固形状または粉末状として用いることができる。この誘導体は水溶性と油溶性の両方の溶媒に溶解することから幅広い化粧料に利用できる。すなわち、水溶液とオイルに溶解することができる。 The form of the cosmetic is arbitrary and can be used as a solution, cream, paste, gel, gel, solid or powder. Since this derivative is soluble in both water-soluble and oil-soluble solvents, it can be used in a wide range of cosmetics. That is, it can be dissolved in an aqueous solution and an oil.
ここで製造された化粧料は皮膚の障害された遺伝子の修復やコラーゲンやエラスチンなどの増加及び皮膚の健康維持の目的で利用される。 The cosmetics produced here are used for the purpose of repairing damaged genes in the skin, increasing collagen and elastin, and maintaining the health of the skin.
また、このフマル酸誘導体は遺伝子が障害された歯肉細胞の機能の維持を目的とした歯磨き剤、洗口液や歯磨きペーストなどに利用できる。 In addition, this fumaric acid derivative can be used in dentifrices, mouthwashes, dentifrice pastes, etc. for the purpose of maintaining the function of genetically impaired gingival cells.
また、植物に対しては遺伝子の障害を回復させることにより葉の結実と収穫量の増加を目的とした植物活性化剤として利用することができる。 In addition, for plants, it can be used as a plant activator for the purpose of leaf fruiting and increasing yield by recovering from genetic disorders.
この植物活性化剤は高級で希少な蘭、胡蝶蘭、薔薇やマツバランなどの花の栽培促進の目的で利用でき、葉や野菜、穀類の栽培を安定化させる。植物工場における野菜や葉の栽培にも利用でき、栽培効率を上げることができる。 This plant activator can be used for the purpose of promoting the cultivation of flowers such as high-grade and rare orchids, phalaenopsis orchids, roses and whisk ferns, and stabilizes the cultivation of leaves, vegetables and grains. It can also be used for growing vegetables and leaves in plant factories, and can improve cultivation efficiency.
以下に、ナズナの葉を発酵する工程を特徴とし、ナズナの葉、米糠粉末と納豆本舗製の納豆菌を添加して発酵させた発酵液を紅麹本舗製のベニコウジ菌で発酵する工程からなる遺伝子修復作用を呈する前記の式（１）で示されるフマル酸誘導体の製造方法について説明する。 The following is characterized by the process of fermenting the leaves of Nazuna, which consists of the process of fermenting the fermented liquid by adding the leaves of Nazuna, rice bran powder and Bacillus natto made by Natto Honpo with Monascus purpureus made by Benikoji Honpo. A method for producing a fumaric acid derivative represented by the above formula (1), which exhibits a gene repair action, will be described.
ここでいうフマル酸誘導体は前記の式（１）で示され、フマル酸の２分子、イソラムネチン１分子及びケイ素の１分子から構成されている。これらの結合はすべて天然型であり、物質の間は共有結合を介して結合している。 The fumaric acid derivative referred to here is represented by the above formula (1) and is composed of two molecules of fumaric acid, one molecule of isorhamnetin and one molecule of silicon. All of these bonds are natural and the substances are linked via covalent bonds.
このフマル酸誘導体のフマル酸、イソラムネチン及びケイ素は天然に存在し、食経験も豊富であり、安全性が認められていることから好ましい。 The fumaric acid derivatives fumaric acid, isorhamnetin, and silicon are preferable because they are naturally occurring, have abundant eating experience, and have been confirmed to be safe.
この誘導体は皮膚、神経、骨、筋肉、肝臓や腎臓などにも働き、障害された遺伝子を修復させることにより、組織及び身体機能を回復させる。 This derivative also acts on skin, nerves, bones, muscles, liver, kidneys, etc., and restores tissue and physical function by repairing damaged genes.
この製造方法とはナズナの葉、米糠粉末と納豆本舗製の納豆菌を添加して発酵させた発酵液を紅麹本舗製のベニコウジ菌で発酵する工程からなる。 This production method consists of a step of fermenting a fermented liquid obtained by adding Nazuna leaf, rice bran powder and Bacillus natto made by Natto Honpo with Monascus purpureus made by Benikoji Honpo.
原料となる物質はナズナの葉、米糠粉末、納豆本舗製の納豆菌及び紅麹本舗製のベニコウジ菌である。ナズナの代わりに、ワカメなどの海藻類、フランスカイガンショウ、ヒメマツタケなどの植物やキノコ類、オキアミやプラセンタなどを利用することもできる。 The raw material substances are shepherd's purse leaf, rice bran powder, natto bacterium manufactured by Natto Honpo, and Monascus purpureus manufactured by Benikoji Honpo. Instead of shepherd's purse, seaweeds such as wakame seaweed, plants and mushrooms such as Pinus pinaster and Himematsutake, krill and placenta can also be used.
ここでいうナズナとは学名Ｃａｐｓｅｌｌａ Ｂｕｒｓａ−Ｐａｓｔｏｒｉｓ、アブラナ科ナズナ属の越年草の植物で、別名はペンペングサやシャミセングサといわれる。日本各地、アジア各地に自生しており、その葉や花は食用として利用され、春の七草の一つでもある。また、民間薬として肝臓病、解熱、下痢、高血圧、便秘に利用されている。 Shepherd's purse here is a perennial plant of the scientific name Capsella Bursa-Pastorias, Brassicaceae Shepherd's Purse, and is also known as Penpengusa or Shamisengusa. It grows naturally in various parts of Japan and Asia, and its leaves and flowers are used for food and are one of the seven spring herbs. It is also used as a folk medicine for liver disease, fever, diarrhea, hypertension, and constipation.
使用するのはナズナの葉であり、茎を含有していても良い。 It is the leaves of shepherd's purse that may contain stems.
ナズナの葉は日本、アメリカ、アジア、その他の国で採取されたいずれのものでも良いが、品質が高く、価格の点から、日本産は品質が良いことから好ましい。たとえば、岩手県のきのこ店きのこやおいよでは減農薬で栽培されたナズナの葉を供給しており、品質が良いことから好ましい。 The leaves of shepherd's purse may be collected from Japan, the United States, Asia, or any other country, but from the viewpoint of high quality and price, Japanese products are preferable because of their high quality. For example, the mushroom shop in Iwate Prefecture, Mushrooms and Oiyo, supplies shepherd's purse leaves cultivated with reduced pesticides, which is preferable because of its good quality.
ナズナの葉は乾燥され、粉末化されることが好ましく、発酵の前にオートクレーブ滅菌されることは発酵をスムーズに行うることから好ましい。 The leaves of shepherd's purse are preferably dried and pulverized, and autoclaved before fermentation is preferable because the fermentation proceeds smoothly.
３マイクロメーター以下の粒子サイズの粉末が発酵の工程を実施しやすくすることから好ましい。 A powder having a particle size of 3 micrometers or less is preferable because it facilitates the fermentation process.
原料となる米糠粉末は、日本産、中国産、アメリカ産、ロシア産などいずれの産地の米（学名Ｏｒｙｚａ Ｓａｔｉｖａ）から採取された米糠でも利用できる。トレーサビリティーが確実であり、農薬の利用状況が把握でき、生産者が明確である日本産が好ましい。 The rice bran powder used as a raw material can be used for rice bran collected from rice (scientific name: Oryza Sativa) from any of the production areas such as Japan, China, the United States, and Russia. It is preferable to use Japanese products that have reliable traceability, can grasp the usage status of pesticides, and have a clear producer.
このうち、有機栽培や無農薬で栽培された米糠は有害な農薬や金属を含有しないことから、さらに好ましい。 Of these, rice bran cultivated organically or without pesticides is more preferable because it does not contain harmful pesticides or metals.
米糠は使用に際して、株式会社奈良機械製作所製の自由ミル、スーパー自由ミル、サンプルミル、ゴブリン、スーパークリーンミル、マイクロス、減圧乾燥機として東洋理工製の小型減圧乾燥機、株式会社マツイ製の小型減圧伝熱式乾燥機ＤＰＴＨ−４０、エーキューエム九州テクノス株式会社製のクリーンドライＶＤ−７、ＶＤ−２０、中山技術研究所製ＤＭ−６などの粉砕機で粉砕される。これにより発酵の工程が効率的に進行されやすい。 When using rice bran, Nara Kikai Seisakusho Co., Ltd.'s free mill, super free mill, sample mill, goblin, super clean mill, micros, Toyo Riko's small decompression dryer as a decompression dryer, Matsui's small decompression dryer It is crushed by a crusher such as a vacuum heat transfer dryer DPTH-40, a clean dry VD-7 and VD-20 manufactured by AKUM Kyushu Technos Co., Ltd., and a DM-6 manufactured by Nakayama Institute of Technology. As a result, the fermentation process is likely to proceed efficiently.
さらに、ナズナの葉と米糠は粉砕後、オートクレーブなどにより滅菌されることは雑菌の繁殖を防御できることから好ましい。 Furthermore, it is preferable that the leaves of shepherd's purse and rice bran are crushed and then sterilized by an autoclave or the like because they can prevent the growth of various germs.
用いる納豆本舗製の納豆菌は学名バチルス サブチリスで日本では納豆の製造に汎用され、食経験が豊富で有用な食用菌である。沖縄や鹿児島などの日本産、中国や台湾の東南アジア原産の菌種が用いられる。用いる納豆菌は納豆本舗製であり、高い発酵性を呈する。 The natto bacterium manufactured by Natto Honpo is a edible bacterium with abundant eating experience and is widely used in the production of natto in Japan under the scientific name Bacillus subtilis. Bacterial species native to Japan such as Okinawa and Kagoshima, and Southeast Asia of China and Taiwan are used. The natto bacterium used is made by Natto Honpo and exhibits high fermentability.
この納豆菌はナズナの葉と米糠からなるフマル酸とケイ素の結合反応を促進する。 This natto bacterium promotes the binding reaction between fumaric acid and silicon, which consists of shepherd's purse leaves and rice bran.
前記の発酵に関するそれぞれの添加量はナズナの葉の乾燥粉末１重量に対し、米糠粉末は０．０１〜５重量及び納豆本舗製の納豆菌は０．００１〜０．０５重量が好ましい。納豆菌は発酵される前に、前培養することは、発酵の初発時間を短縮し、発酵時間が短縮されることから好ましい。 The amount of each of the above-mentioned fermentation additions is preferably 0.01 to 5% by weight for rice bran powder and 0.001 to 0.05 weight for Bacillus natto manufactured by Natto Honpo with respect to 1 weight of dry powder of shepherd's purse leaves. Pre-culturing Bacillus natto before fermentation is preferable because it shortens the initial fermentation time and shortens the fermentation time.
前記の発酵は清浄な培養用タンクで実施され、滅菌された水道水により前記の材料を混合することは好ましい。 The fermentation is carried out in a clean culture tank and it is preferred to mix the ingredients with sterilized tap water.
また、この発酵は４１〜４４℃に加温され、発酵は２日間から１０日間行われる。目的とするフマル酸誘導体をＨＰＬＣやＴＬＣにより定量することならびに菌体の増殖性を確認することにより、発酵の工程管理を実施することは産生量が調整されることから好ましい。 In addition, this fermentation is heated to 41 to 44 ° C., and the fermentation is carried out for 2 to 10 days. It is preferable to control the fermentation process by quantifying the desired fumaric acid derivative by HPLC or TLC and confirming the proliferative property of the cells because the production amount is adjusted.
前記の発酵は清浄な培養用タンクで実施され、滅菌された水道水により前記の材料を混合することは好ましい。 The fermentation is carried out in a clean culture tank and it is preferred to mix the ingredients with sterilized tap water.
この発酵の工程によって生成されるフマル酸誘導体はその結合が不安定であり、分解されやすいことから次の紅麹本舗製のベニコウジ菌による発酵を行い、目的とするフマル酸誘導体の結合を安定化させる。 The fumaric acid derivative produced by this fermentation process has an unstable bond and is easily decomposed. Therefore, the next fermentation by Monascus purpureus manufactured by Benikoji Honpo is performed to stabilize the bond of the target fumaric acid derivative. Let me.
用いる紅麹本舗製のベニコウジ菌は学名Ｍｏｎａｓｃｕｓ ｐｕｒｐｕｒｅｕｓの糸状菌であり、古くから日本、中国や台湾において紅酒や豆腐ようなどの発酵食品に利用されている。また、沖縄や鹿児島などの日本産、中国や台湾の東南アジア原産の菌種が用いられる。紅麹本舗製のベニコウジ菌は発酵効率に優れており、また、安全性も高い。 The Monascus purpureus produced by Benikoji Honpo is a filamentous fungus with the scientific name Monascus purpureus, and has long been used in fermented foods such as red wine and tofu in Japan, China and Taiwan. In addition, bacterial species native to Japan such as Okinawa and Kagoshima, and Southeast Asia of China and Taiwan are used. Monascus purpureus manufactured by Benikoji Honpo has excellent fermentation efficiency and is also highly safe.
前記の発酵に関するそれぞれの添加量は前記の発酵物１重量に対してベニコウジ菌は０．０００５〜０．０５重量が好ましい。紅麹本舗製のベニコウジ菌は発酵される前に、前培養することは、発酵の初発時間を短縮し、発酵時間が短縮されることから好ましい。 The amount of each addition for the fermentation is preferably 0.0005 to 0.05 weight by weight of Monascus purpureus with respect to 1 weight of the fermented product. It is preferable to pre-culture the Monascus purpureus manufactured by Benikoji Honpo before it is fermented because it shortens the initial fermentation time and shortens the fermentation time.
前記の発酵は清浄な培養用タンクで実施され、滅菌された水道水により前記の材料を混合することは好ましい。 The fermentation is carried out in a clean culture tank and it is preferred to mix the ingredients with sterilized tap water.
また、この発酵は４０〜４３℃に加温され、発酵は２日間から２０日間行われる。この発酵の工程によってベニコウジ菌の酸化及び還元作用によりこのフマル酸誘導体の構造が安定化される。 In addition, this fermentation is heated to 40 to 43 ° C., and the fermentation is carried out for 2 to 20 days. This fermentation process stabilizes the structure of this fumaric acid derivative by the oxidizing and reducing action of Monascus purpureus.
前記の発酵物は含水エタノールで抽出されることは、生成物を効率良く回収し、菌を滅菌でき、次の工程が実施しやすいことから、好ましい。また、得られた発酵物を超音波処理することは、生成物が分離しやすいことから、好ましい。また、凍結乾燥などにより、濃縮することは、以下の工程が短時間に実施できることから好ましい。 It is preferable that the fermented product is extracted with hydrous ethanol because the product can be efficiently recovered, the bacteria can be sterilized, and the next step can be easily carried out. Further, it is preferable to ultrasonically treat the obtained fermented product because the product can be easily separated. Further, it is preferable to concentrate by freeze-drying or the like because the following steps can be carried out in a short time.
前記の還元反応物から、目的とするフマル酸誘導体を分離し、精製することは純度の高い物質として摂取量を減少させることができる点から好ましい。この精製の方法としては、分離用の樹脂などの精製操作を利用することが好ましい。この精製は繰り返して実施され、または、組み合わせて実施することにより、純度の高い精製物が得られる。 It is preferable to separate and purify the desired fumaric acid derivative from the reduction reaction product from the viewpoint that the intake amount can be reduced as a highly pure substance. As this purification method, it is preferable to use a purification operation such as a resin for separation. This purification is carried out repeatedly or in combination to obtain a highly pure purified product.
例えば、分離用担体または樹脂により分離され、分取されることにより目的とするフマル酸誘導体が得られる。分離用担体または樹脂としては、表面が後述のようにコーティングされた、多孔性の多糖類、酸化珪素化合物、ポリアクリルアミド、ポリスチレン、ポリプロピレン、スチレン−ビニルベンゼン共重合体等が用いられる。０．１〜３００μｍの粒度を有するものが好ましく、粒度が細かい程、精度の高い分離が行なわれるが、分離時間が長い欠点がある。 For example, the desired fumaric acid derivative can be obtained by being separated by a separation carrier or a resin and separated. As the separation carrier or resin, a porous polysaccharide, a silicon oxide compound, polyacrylamide, polystyrene, polypropylene, a styrene-vinylbenzene copolymer or the like whose surface is coated as described later is used. Those having a particle size of 0.1 to 300 μm are preferable, and the finer the particle size, the higher the accuracy of separation, but there is a drawback that the separation time is long.
例えば、逆相担体または樹脂として表面が疎水性化合物でコーティングされたものは、疎水性の高い物質の分離に利用される。陽イオン物質でコーティングされたものは陰イオン性に荷電した物質の分離に適している。また、陰イオン物質でコーティングされたものは陽イオン性に荷電した物質の分離に適している。特異的な抗体をコーティングした場合には、特異的な物質のみを分離するアフィニティ担体または樹脂として利用される。 For example, a reversed-phase carrier or a resin whose surface is coated with a hydrophobic compound is used for separating highly hydrophobic substances. Those coated with a cationic substance are suitable for separating anionically charged substances. Further, those coated with an anionic substance are suitable for separating a cationically charged substance. When coated with a specific antibody, it is used as an affinity carrier or resin that separates only specific substances.
アフィニティ担体または樹脂は、抗原抗体反応を利用して抗原の特異的な調製に利用される。分配性担体または樹脂は、シリカゲル（メルク社製）等のように、物質と分離用溶媒の間の分配係数に差異がある場合、それらの物質の単離に利用される。 The affinity carrier or resin is utilized for the specific preparation of the antigen by utilizing the antigen-antibody reaction. Distributable carriers or resins, such as silica gel (manufactured by Merck & Co., Inc.), are used for isolation of substances when there is a difference in partition coefficient between the substance and the solvent for separation.
これらのうち、製造コストを低減することができる点から、吸着性担体または樹脂、分配性担体または樹脂、分子篩用担体または樹脂及びイオン交換担体または樹脂が好ましい。さらに、分離用溶媒に対して分配係数の差異が大きい点から、逆相担体または樹脂及び分配性担体または樹脂はより好ましい。 Of these, an adsorptive carrier or resin, a distributable carrier or resin, a carrier or resin for molecular sieving, and an ion exchange carrier or resin are preferable from the viewpoint of reducing the production cost. Further, a reversed-phase carrier or resin and a distributable carrier or resin are more preferable because the difference in partition coefficient with respect to the separation solvent is large.
分離用溶媒として有機溶媒を用いる場合には、有機溶媒に耐性を有する担体または樹脂が用いられる。また、医薬品製造または食品製造に利用される担体または樹脂は好ましい。 When an organic solvent is used as the separation solvent, a carrier or resin having resistance to the organic solvent is used. In addition, carriers or resins used for pharmaceutical production or food production are preferable.
これらの点から吸着性担体としてダイヤイオン（三菱化学（株）社製）及びＸＡＤ−２またはＸＡＤ−４（ロームアンドハース社製）、分子篩用担体としてセファデックスＬＨ−２０（アマシャムファルマシア社製）、分配用担体としてシリカゲル、イオン交換担体としてＩＲＡ−４１０（ロームアンドハース社製）、逆相担体としてＤＭ１０２０Ｔ（富士シリシア社製）がより好ましい。 From these points, Diaion (manufactured by Mitsubishi Chemical Corporation) and XAD-2 or XAD-4 (manufactured by Roam & Haas) as the adsorptive carrier, and Sephadex LH-20 (manufactured by Amasham Pharmacia) as the carrier for molecular sieves. , Silica gel as a distribution carrier, IRA-410 (manufactured by Roam & Haas) as an ion exchange carrier, and DM1020T (manufactured by Fuji Silicia) as a reverse phase carrier are more preferable.
これらのうち、ダイヤイオンＡＭＰ０３、セファデックスＬＨ−２０及びＤＭ１０２０Ｔはさらに好ましい。 Of these, Diaion AMP03, Sephadex LH-20 and DM1020T are even more preferred.
得られた抽出物は、分離前に分離用担体または樹脂を膨潤化させるための溶媒に溶解される。その量は、分離効率の点から抽出物の重量に対して２〜４２倍量が好ましく、４〜２２倍量がより好ましい。分離の温度としては物質の安定性の点から１０〜３１℃が好ましく、１２〜２４℃がより好ましい。 The resulting extract is dissolved in a separation carrier or solvent for swelling the resin prior to separation. The amount thereof is preferably 2 to 42 times, more preferably 4 to 22 times, the weight of the extract from the viewpoint of separation efficiency. The separation temperature is preferably 10 to 31 ° C., more preferably 12 to 24 ° C. from the viewpoint of substance stability.
分離用溶媒には、水、または、水を含有する低級アルコール、親水性溶媒、親油性溶媒が用いられる。低級アルコールとしては、メタノール、エタノール、プロパノール、ブタノールが用いられるが、食用として利用されているエタノールが好ましい。また、水溶性の溶媒に加えて油溶性の溶媒である植物油、魚油、ラードなどの動物性油脂に溶解できる。 As the separation solvent, water, a lower alcohol containing water, a hydrophilic solvent, or a lipophilic solvent is used. As the lower alcohol, methanol, ethanol, propanol and butanol are used, but ethanol used for food is preferable. In addition to the water-soluble solvent, it can be dissolved in animal fats and oils such as vegetable oil, fish oil, and lard, which are oil-soluble solvents.
セファデックスＬＨ−２０を用いる場合、分離用溶媒には低級アルコールが好ましい。シリカゲルを用いる場合、分離用溶媒にはクロロホルム、メタノール、酢酸またはそれらの混合液が好ましい。 When Sephadex LH-20 is used, a lower alcohol is preferable as the separation solvent. When silica gel is used, chloroform, methanol, acetic acid or a mixture thereof is preferable as the separation solvent.
ダイヤイオン及びＤＭ１０２０Ｔを用いる場合、分離用溶媒はメタノール、エタノール等の低級アルコールまたは低級アルコールと水の混合液が好ましい。 When Diaion and DM1020T are used, the separation solvent is preferably a lower alcohol such as methanol or ethanol or a mixed solution of a lower alcohol and water.
フマル酸誘導体を含む画分を採取して乾燥または真空乾燥により溶媒を除去し、目的とするフマル酸誘導体を粉末または濃縮液として得ることは溶媒による影響を除外できることから好ましい。 It is preferable to collect a fraction containing the fumaric acid derivative and remove the solvent by drying or vacuum drying to obtain the desired fumaric acid derivative as a powder or a concentrated solution because the influence of the solvent can be excluded.
また、最終抽出を食用油や化粧料に用いる油脂で実施することは、得られるフマル酸誘導体が安定に維持されることから好ましい。例えば、米糠油、米ぬか油、グレープシード油、オリーブ油、ホホバ油で抽出することは好ましい。 Further, it is preferable to carry out the final extraction with oils and fats used for cooking oils and cosmetics because the obtained fumaric acid derivative is stably maintained. For example, it is preferable to extract with rice bran oil, rice bran oil, grape seed oil, olive oil, and jojoba oil.
また、このフマル酸誘導体を粉末化することは防腐の目的から好ましい。 Further, it is preferable to powder the fumaric acid derivative for the purpose of antiseptic.
以下、前記実施形態を実施例及び試験例を用いて具体的に説明する。なお、これらは一例であり、素材、原料や検体の違いに応じて常識の範囲内で条件を変更させることが可能である。 Hereinafter, the embodiment will be specifically described with reference to Examples and Test Examples. These are just examples, and it is possible to change the conditions within the range of common sense according to the difference in the material, raw material, and sample.
岩手県で栽培されたナズナ（学名Ｃａｐｓｅｌｌａ Ｂｕｒｓａ−Ｐａｓｔｏｒｉｓ）のの葉をきのこ店きのこやおいよから購入して用いた。この葉を水道水で水洗後、天日で乾燥させ、粉砕機（株式会社奈良機械製作所製のスーパー自由ミル）にて粉砕し、ナズナの葉の乾燥粉砕物を１．０ｋｇ得た。なお、葉の他に一部、茎も含有していた。 The leaves of shepherd's purse (scientific name Capsella Bursa-Pastorias) cultivated in Iwate Prefecture were purchased from a mushroom shop, Mushroom Yaoiyo, and used. The leaves were washed with tap water, dried in the sun, and crushed with a crusher (Super Free Mill manufactured by Nara Kikai Seisakusho Co., Ltd.) to obtain 1.0 kg of dried crushed shepherd's purse leaves. In addition to the leaves, some stems were also contained.
また、北海道産の米糠（学名Ｏｒｙｚａ Ｓａｔｉｖａ）をミキサー（クイジナート製）に供し、米糠の粉砕物１．０ｋｇを得た。前記のナズナの葉と米糠の粉砕物をオートクレーブ（ＳＤＬ−３２０、トミー製）に供し、１２１℃、２０分間、滅菌した。 Further, rice bran (scientific name: Oryza Sativa) produced in Hokkaido was used in a mixer (manufactured by Cuisinart) to obtain 1.0 kg of crushed rice bran. The crushed shepherd's purse leaves and rice bran were placed in an autoclave (SDL-320, manufactured by Tommy) and sterilized at 121 ° C. for 20 minutes.
これらを清浄な発酵タンク（滅菌された発酵用丸形４０リットルタンク、遠藤科学製）に入れ、滅菌された水道水５ｋｇを添加し、攪拌した。 These were placed in a clean fermentation tank (sterilized round 40 liter tank for fermentation, manufactured by Endo Kagaku), 5 kg of sterilized tap water was added, and the mixture was stirred.
これとは別に、納豆本舗製の粉末納豆菌（学名Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）の１１ｇを上記の発酵タンクに供し、滅菌した米糠粉末と前培養させた発酵準備液を用意した。 Separately, 11 g of powdered Bacillus subtilis (scientific name: Bacillus subtilis) manufactured by Natto Honpo was applied to the above fermentation tank, and sterilized rice bran powder and pre-cultured fermentation preparation liquid were prepared.
前記の前培養した納豆菌の発酵準備液とナズナの葉の乾燥粉末と米糠とを入れた発酵タンクに添加し、攪拌後、４１〜４２℃の温度範囲で加温し、発酵させた。 It was added to a fermentation tank containing the fermentation preparation solution of the pre-cultured Bacillus natto, dried powder of shepherd's purse leaves, and rice bran, and after stirring, the mixture was heated in a temperature range of 41 to 42 ° C. and fermented.
発酵過程では通気によりバブリングと攪拌を行いつつ、発酵液のサンプリングを行い、７日間発酵させた。発酵終了後、発酵タンクより発酵物を取り出し、煮沸滅菌した。この発酵物を濾過布により濾過して、納豆菌による発酵液１．３ｋｇを得た。この発酵液１ｋｇに対して紅麹本舗製のベニコウジ菌（学名Ｍｏｎａｓｃｕｓ ｐｕｒｐｕｒｅｕｓ）の１１ｇを添加して４２〜４３℃で７日間発酵させた。 In the fermentation process, the fermentation broth was sampled while bubbling and stirring by aeration, and the fermentation was carried out for 7 days. After the fermentation was completed, the fermented product was taken out from the fermentation tank and sterilized by boiling. This fermented product was filtered through a filter cloth to obtain 1.3 kg of a fermented liquid prepared by Bacillus natto. To 1 kg of this fermented liquid, 11 g of Monascus purpureus (scientific name: Monascus purpureus) manufactured by Benikoji Honpo was added and fermented at 42 to 43 ° C. for 7 days.
この発酵物にエタノールを添加して発酵を停止した。さらに、煮沸滅菌した。これを濾過し、濾過液を目的とするフマル酸誘導体とした。これを検体１とした。 Ethanol was added to this fermented product to stop the fermentation. Furthermore, it was sterilized by boiling. This was filtered to obtain a fumaric acid derivative for the purpose of the filtrate. This was designated as Specimen 1.
さらに、構造解析及び機能性の実験の目的で精製物を得た。つまり、前述の検体１のフマル酸誘導体の１００ｇに６％エタノール含有精製水の２Ｌを添加し、これをダイヤイオン（ＡＭＰ０３型、三菱化学製）５００ｇを６％エタノール液に懸濁して充填したガラス製カラム（遠藤科学製）に供した。 In addition, purified products were obtained for structural analysis and functional experiments. That is, a glass in which 2 L of purified water containing 6% ethanol was added to 100 g of the fumaric acid derivative of Sample 1 described above, and 500 g of Diaion (AMP03 type, manufactured by Mitsubishi Chemical Corporation) was suspended in a 6% ethanol solution and filled. It was used for a column made by Endo Kagaku.
これに１０Ｌの６％エタノール液を添加して清浄し、さらに、２５％エタノール液を１Ｌ添加して洗浄した。また、６０％エタノール液を１Ｌ添加して目的とするフマル酸誘導体を溶出させ、この溶出液を濃縮して精製した。この精製工程を３回実施し、最後に得られた精製されたフマル酸誘導体を減圧蒸留により、エタノール部分を除去し、水溶液とした。これを真空乾燥させ、フマル酸誘導体の精製物４４ｇを得てこれを検体２とした。収率は約４．４％であり、天然物から製造するには十分な収量であり、この製造方法が優れた製法であることが確認された。 To this was cleaned by adding 10 L of 6% ethanol solution, and further, 1 L of 25% ethanol solution was added for washing. Further, 1 L of a 60% ethanol solution was added to elute the desired fumaric acid derivative, and this eluate was concentrated and purified. This purification step was carried out three times, and the ethanol moiety was removed from the finally obtained purified fumaric acid derivative by vacuum distillation to obtain an aqueous solution. This was vacuum dried to obtain 44 g of a purified fumaric acid derivative, which was used as sample 2. The yield was about 4.4%, which was sufficient for production from natural products, confirming that this production method was an excellent production method.
以下に、フマル酸誘導体の構造解析に関する試験方法及び結果について説明する。
（試験例１）
The test methods and results for structural analysis of fumaric acid derivatives will be described below.
(Test Example 1)
上記のように得られた検体２を重水素化ジメチルスルホキシド（シグマアルドリッチ製）に溶解し、質量分析器付き高速液体クロマトグラフィ（ＨＰＬＣ、島津製作所）で分析した。また、ケイ素については原子吸光装置（日立ハイテクサイエンス製）により定量した。検体２のフマル酸誘導体の純堂は、９８．２％であり、検体１の純度は８０．３％であった。 The sample 2 obtained as described above was dissolved in deuterated dimethyl sulfoxide (manufactured by Sigma-Aldrich) and analyzed by high performance liquid chromatography (HPLC, Shimadzu Corporation) with a mass spectrometer. Silicon was quantified by an atomic absorption spectrophotometer (manufactured by Hitachi High-Tech Science). The pure temple of the fumaric acid derivative of Specimen 2 was 98.2%, and the purity of Specimen 1 was 80.3%.
さらに、これを核磁気共鳴装置（２００ＭＨｚ、ＮＭＲ、ブルカー製）で解析した結果、検体１と検体２からフマル酸の２分子とケイ素の１分子からなるフマル酸誘導体が検出された。 Further, as a result of analyzing this with a nuclear magnetic resonance apparatus (200 MHz, NMR, manufactured by Bruker), a fumaric acid derivative composed of two molecules of fumaric acid and one molecule of silicon was detected from the sample 1 and the sample 2.
すなわち、検体２の重水素化ジメチルスルホキシド中のＨ−ＮＭＲ測定の結果、ピークの位置は６．１７、６．２４、６．２５、６．３２、６．４８、６．６２、９．７２、９．８６、９．８７、９．９４、１０．１５及び１０．３８ｐｐｍに認められた。 That is, as a result of 1 H-NMR measurement in deuterated dimethyl sulfoxide of Specimen 2, the peak positions were 6.17, 6.24, 6.25, 6.32, 6.48, 6.62, 9.72. , 9.86, 9.87, 9.94, 10.15 and 10.38 ppm.
また、検体２のＣ−ＮＭＲ測定の結果、ピークの位置は９４．５、９４．７、９５．９、９７．３、９９．３、９９．４、１２３．５、１２３．９、１２４．６、１２５．４、１３８．６、１４３．５、１４４．０、１４７．５、１４７．７、１５２．９、１５６．２、１５６．５、１６０．５及び１６２．１ｐｐｍに認められた。 As a result of C-NMR measurement of Specimen 2, the peak positions were 94.5, 94.7, 95.9, 97.3, 99.3, 99.4, 123.5, 123.9, 124. It was observed at 6, 125.4, 138.6, 143.5, 144.0, 147.5, 147.7, 152.9, 156.2, 156.5, 160.5 and 162.1 ppm.
以下に、Ｃ−ＮＭＲの解析結果のチャートを示した。（横軸単位はｐｐｍ、縦軸単位はピーク強度を示す。）
上記の解析結果から化学的に合成した標準品と同一構造を呈することが判明した。すなわち、検体２からフマル酸２分子、イソラムネチン１分子とケイ素の１分子が結合した目的とするフマル酸誘導体であると確認できた。また、検体２を粉末化した場合、水溶液に溶解した結果、水素ガスの発生がガスクロマトグラフィー（島津製作所製）により確認された。この場合の水素ガスの発生量は１．６ｐｐｍであった。 From the above analysis results, it was found that the product has the same structure as the chemically synthesized standard product. That is, it was confirmed from the sample 2 that the desired fumaric acid derivative was obtained by binding two molecules of fumaric acid, one molecule of isorhamnetin and one molecule of silicon. Further, when the sample 2 was pulverized, as a result of dissolving it in an aqueous solution, the generation of hydrogen gas was confirmed by gas chromatography (manufactured by Shimadzu Corporation). The amount of hydrogen gas generated in this case was 1.6 ppm.
以下にヒト皮膚表皮細胞を用いた皮膚作用試験について述べる。なお、この試験方法は生化学的に成分の効果を検証できる再現性のある常法である。
（試験例２）
The skin action test using human skin epidermal cells will be described below. This test method is a reproducible conventional method that can biochemically verify the effects of the components.
(Test Example 2)
クラボウより購入したヒト由来表皮細胞（表皮由来、エピーダーセル）を用いた。培養液として５％牛胎児血清含有ＭＥＭ培地（Ｓｉｇｍａ製）を用いて培養した１０００個の細胞を３５ｍｍ培養シャーレ（ＦＡＬＣＯＮ製）に播種し、５％炭酸ガス下、３７℃で培養した。ここに紫外線照射装置（クオークテクノロジー製）により２８０ｎｍの紫外線を１時間照射した。ここに、前記の検体１、検体２及び陽性対照としてＥＧＦ（フナコシ製、表皮成長因子）をいずれも１０ｍｇ／ｍｌの最終濃度で添加した。これを４８時間培養して試験した。培養液を採取後、表皮細胞の生存率をトリパンブルー法により計数した。その後、表皮細胞の懸濁液を調製した。ここからｍＲＮＡを核酸抽出キット（フナコシ製）により抽出した。常法に従い、ＲＴ−ＰＣＲ法によりＤＮＡポリメラーゼ及び８−オキソグアニンＤＮＡグリコシラーゼ（ＯＧＧ１と略す）のｍＲＮＡを定量した。同時に、細胞懸濁液中の８−ＯＨｄＧ量をキット（日本老化制御研究所製）にて定量した。８−ＯＨｄＧに特異的なモノクローナル抗体を使用したＥＬＩＳＡキットである。なお、シャーレは５枚を用いてその平均値を算出した。溶媒を添加した溶媒対照群と比較した。 Human-derived epidermal cells (epidermal-derived, epidermis cells) purchased from Kurabo Industries were used. 1000 cells cultured using MEM medium containing 5% fetal bovine serum (manufactured by Sigma) as a culture solution were seeded in a 35 mm culture dish (manufactured by FALCON) and cultured at 37 ° C. under 5% carbon dioxide gas. This was irradiated with ultraviolet rays of 280 nm for 1 hour by an ultraviolet irradiation device (manufactured by Quark Technology). To this, EGF (manufactured by Funakoshi, epidermal growth factor) was added to each of the above-mentioned Specimen 1, Specimen 2 and as a positive control at a final concentration of 10 mg / ml. This was cultured for 48 hours and tested. After collecting the culture medium, the survival rate of epidermal cells was counted by the trypan blue method. Then, a suspension of epidermal cells was prepared. From this, mRNA was extracted with a nucleic acid extraction kit (manufactured by Funakoshi). The mRNAs of DNA polymerase and 8-oxoguanine DNA glycosylase (abbreviated as OGG1) were quantified by the RT-PCR method according to a conventional method. At the same time, the amount of 8-OHdG in the cell suspension was quantified with a kit (manufactured by Japan Aging Control Laboratory). An ELISA kit using a monoclonal antibody specific for 8-OHdG. The average value of the petri dish was calculated using five petri dishes. It was compared with the solvent control group to which the solvent was added.
その結果、検体１の１０ｍｇ／ｍｌの添加によりヒト由来表皮細胞数は溶媒対照群に比して平均値として１７０％に増加した。また、検体２では２２２％に増加した。一方、ＥＧＦでは１５５％となった。この結果、検体１及び検体２の方がＥＧＦよりも優れた細胞活性化作用を呈した。また、検体１とＥＧＦを同時に添加した場合、細胞数は３３０％となり、検体１とＥＧＦの相乗的な作用が確認された。 As a result, the number of human-derived epidermal cells increased to 170% on average as compared with the solvent control group by adding 10 mg / ml of Specimen 1. In sample 2, it increased to 222%. On the other hand, it was 155% in EGF. As a result, Specimen 1 and Specimen 2 exhibited a better cell activating effect than EGF. Moreover, when Specimen 1 and EGF were added at the same time, the number of cells became 330%, and the synergistic action of Specimen 1 and EGF was confirmed.
上記の細胞中のＤＮＡポリメラーゼのｍＲＮＡ発現量（コピー数）は溶媒対照群では１２コピー、検体１処理群では１６６コピー、検体２処理群では４７７コピ−、ＥＧＦ処理群では９８コピーであった。 The mRNA expression level (copy number) of the DNA polymerase in the above cells was 12 copies in the solvent control group, 166 copies in the sample 1 treatment group, 477 copies in the sample 2 treatment group, and 98 copies in the EGF treatment group.
ＤＮＡポリメラーゼのｍＲＮＡ発現量は検体１及び検体２で著しく、ＥＧＦより優っていた。これは検体１及び検体２による遺伝子修復酵素の活性化作用を示していた。なお、検体１及び検体２を添加した培養液では添加後１時間に１．６ｐｐｍの水素ガスの発生が確認された。 The mRNA expression level of DNA polymerase was remarkable in Specimen 1 and Specimen 2, and was superior to EGF. This showed the activating action of the gene repair enzyme by Specimen 1 and Specimen 2. In the culture solution to which Specimen 1 and Specimen 2 were added, it was confirmed that 1.6 ppm of hydrogen gas was generated 1 hour after the addition.
上記の細胞中のＯＧＧ１のｍＲＮＡ発現量（コピー数）は溶媒対照群では１８コピー、検体１処理群では１１７コピー、検体２処理群では６９０コピ−、ＥＧＦ処理群では７７コピーであった。 The mRNA expression level (number of copies) of OGG1 in the above cells was 18 copies in the solvent control group, 117 copies in the sample 1 treatment group, 690 copies in the sample 2 treatment group, and 77 copies in the EGF treatment group.
ＯＧＧ１のｍＲＮＡ発現量は検体１及び検体２で高く、ＥＧＦより優っていた。これは検体１及び検体２が遺伝子修復作用を有することを示していた。 The mRNA expression level of OGG1 was higher in Specimen 1 and Specimen 2 and was superior to that of EGF. This indicated that Specimen 1 and Specimen 2 had a gene repair effect.
上記の細胞中の８ＯＨｄＧ量は溶媒対照群では５９９ｎｇ、検体１処理群では７９ｎｇ、検体２処理群では３９ｎｇ、ＥＧＦ処理群では４８２ｎｇであった。 The amount of 8 OHdG in the cells was 599 ng in the solvent control group, 79 ng in the sample 1 treated group, 39 ng in the sample 2 treated group, and 482 ng in the EGF treated group.
８ＯＨｄＧは遺伝子が活性酸素により修飾された変異した状態であり、遺伝子の障害をあらわしている。検体１及び検体２でこの値が低く、ＥＧＦの働きより優っていた。これは検体１及び検体２による遺伝子修復作用を示していた。 8OHdG is a mutated state in which a gene is modified by active oxygen, and represents a gene disorder. This value was low in Specimen 1 and Specimen 2, which was superior to the function of EGF. This showed the gene repair effect of Specimen 1 and Specimen 2.
一方、安全性試験の一環として人工皮膚であるＥｐｉＳｋｉｎ（ＳｋｉｎＥｔｈｉｃ社製）を用いた皮膚刺激性実験では、検体１及び検体２の添加により刺激性は認められず、安全性が確認された。なお、この方法は細胞を用いる皮膚刺激性試験評価法として動物を使用しない方法として確立されている。 On the other hand, in a skin irritation experiment using EpiSkin (manufactured by SkinEthic), which is an artificial skin, as part of the safety test, no irritation was observed due to the addition of Specimen 1 and Specimen 2, and the safety was confirmed. This method has been established as a method that does not use animals as a skin irritation test evaluation method that uses cells.
以下にヒト神経細胞の障害モデルを用いた障害に対する試験について述べる。なお、この試験方法は生化学的に成分の働きを検証できる再現性のある常法である。
（試験例３）
The test for the disorder using the disorder model of human nerve cells is described below. This test method is a reproducible conventional method that can biochemically verify the function of the components.
(Test Example 3)
コスモバイオから購入したヒト神経細胞（Ｈｕｍａｎ Ｎｅｕｒｏｎｓ（ＨＮ））を用いた。培養液として専用の培養液（神経細胞増殖培地）を用いて培養した１０００個の細胞を３５ｍｍ培養シャーレに播種し、５％炭酸ガス下、３７℃で培養した。これに１％の神経毒であるアクリルアミド水溶液を添加して神経細胞を刺激した。 Human neurons (Human Neurons (HN)) purchased from Cosmo Bio were used. 1000 cells cultured using a special culture medium (nerve cell growth medium) as a culture medium were seeded in a 35 mm culture dish and cultured at 37 ° C. under 5% carbon dioxide gas. A 1% aqueous solution of acrylamide, which is a neurotoxin, was added to stimulate nerve cells.
ここに、前記の実施例１で得られた検体１及び検体２、陽性対照としてＮＧＦ（フナコシ(株)、ヒトタイプ）をいずれも１０ｍｇ／ｍｌの最終濃度で添加した。これを４８時間培養した。 To this, Specimens 1 and 2 obtained in Example 1 above, and NGF (Funakoshi Co., Ltd., human type) as a positive control were all added at a final concentration of 10 mg / ml. This was cultured for 48 hours.
培養終了後、細胞数を顕微鏡的に計数した。さらに、上記と同様の方法により、遺伝子修復の働きを検証した。すなわち、細胞の懸濁液を調製し、ここからｍＲＮＡを核酸抽出キット（フナコシ製）により抽出した。常法に従い、ＲＴ−ＰＣＲ法によりＤＮＡポリメラーゼ及び８−オキソグアニンＤＮＡグリコシラーゼ（ＯＧＧ１と略す）のｍＲＮＡを定量した。同時に、細胞懸濁液中の８−ＯＨｄＧ量をキット（日本老化制御研究所製）にて定量した。８−ＯＨｄＧに特異的なモノクローナル抗体を使用したＥＬＩＳＡキットである。 After completion of the culture, the number of cells was counted microscopically. Furthermore, the function of gene repair was verified by the same method as above. That is, a suspension of cells was prepared, and mRNA was extracted from this with a nucleic acid extraction kit (manufactured by Funakoshi). The mRNAs of DNA polymerase and 8-oxoguanine DNA glycosylase (abbreviated as OGG1) were quantified by the RT-PCR method according to a conventional method. At the same time, the amount of 8-OHdG in the cell suspension was quantified with a kit (manufactured by Japan Aging Control Laboratory). An ELISA kit using a monoclonal antibody specific for 8-OHdG.
その結果、検体１の１０ｍｇ／ｍｌの添加により神経細胞数は溶媒対照群に比して平均値として１４４％に増加した。また、検体２では１８９％に増加した。一方、ＮＧＦでは１３７％となった。この結果、検体１及び検体２の方がＮＧＦよりも優れた細胞活性化作用を呈した。また、検体１とＮＧＦの併用により細胞数は３３０％となり、検体１とＮＧＦの相乗作用が確認された。 As a result, the addition of 10 mg / ml of Specimen 1 increased the number of nerve cells to 144% on average as compared with the solvent control group. In sample 2, it increased to 189%. On the other hand, it was 137% in NGF. As a result, Specimen 1 and Specimen 2 exhibited a better cell activating effect than NGF. In addition, the combined use of Specimen 1 and NGF resulted in a cell number of 330%, confirming the synergistic effect of Specimen 1 and NGF.
上記の細胞中のＤＮＡポリメラーゼのｍＲＮＡ発現量（コピー数）は溶媒対照群では１９コピー、検体１処理群では１８３コピー、検体２処理群では７４０コピ−、ＮＧＦ処理群では９０コピーであった。 The mRNA expression level (copy number) of the DNA polymerase in the above cells was 19 copies in the solvent control group, 183 copies in the sample 1 treatment group, 740 copies in the sample 2 treatment group, and 90 copies in the NGF treatment group.
ＤＮＡポリメラーゼのｍＲＮＡ発現量は検体１及び検体２で高く、ＮＧＦより優っていた。これは検体１及び検体２による遺伝子修復酵素の活性化作用を示していた。 The mRNA expression level of DNA polymerase was high in Specimen 1 and Specimen 2, which was superior to that of NGF. This showed the activating action of the gene repair enzyme by Specimen 1 and Specimen 2.
上記の細胞中のＯＧＧ１のｍＲＮＡ発現量（コピー数）は溶媒対照群では２２コピー、検体１処理群では１０６コピー、検体２処理群では３７７コピ−、ＮＧＦ処理群では１６７コピーであった。 The mRNA expression level (number of copies) of OGG1 in the above cells was 22 copies in the solvent control group, 106 copies in the sample 1 treatment group, 377 copies in the sample 2 treatment group, and 167 copies in the NGF treatment group.
ＯＧＧ１のｍＲＮＡ発現量は検体１及び検体２で高く、ＮＧＦより優っていた。これは検体１及び検体２による遺伝子修復酵素の活性化作用を示していた。 The mRNA expression level of OGG1 was higher in Specimen 1 and Specimen 2 and was superior to that of NGF. This showed the activating action of the gene repair enzyme by Specimen 1 and Specimen 2.
上記の細胞中の８ＯＨｄＧ量は溶媒対照群では４２０ｎｇ、検体１処理群では９８ｎｇ、検体２処理群では２４ｎｇ、ＮＧＦ処理群では１６６ｎｇであった。 The amount of 8 OHdG in the cells was 420 ng in the solvent control group, 98 ng in the sample 1 treatment group, 24 ng in the sample 2 treatment group, and 166 ng in the NGF treatment group.
８ＯＨｄＧは遺伝子が活性酸素により修飾された変異した状態であり、遺伝子の障害をあらわしている。検体１及び検体２でこの値が低く、ＮＧＦの働きに比して優れていた。これは検体１及び検体２による遺伝子修復作用を示していた。なお、検体１及び検体２を添加した培養液では添加後１時間に１．６ｐｐｍの水素ガスの発生が確認された。 8OHdG is a mutated state in which a gene is modified by active oxygen, and represents a gene disorder. This value was low in Specimen 1 and Specimen 2, and was superior to the action of NGF. This showed the gene repair effect of Specimen 1 and Specimen 2. In the culture solution to which Specimen 1 and Specimen 2 were added, it was confirmed that 1.6 ppm of hydrogen gas was generated 1 hour after the addition.
本発明で得られるフマル酸誘導体は遺伝子修復作用を呈し、皮膚細胞や神経細胞などの細胞機能を増進させる。これにより国民のＱＯＬを改善し、健康な労働人口を増加させ、かつ、医療費を削減できる。 The fumaric acid derivative obtained in the present invention exhibits a gene repair action and enhances cell functions such as skin cells and nerve cells. As a result, the QOL of the people can be improved, the healthy working population can be increased, and medical expenses can be reduced.
本発明で得られるフマル酸誘導体は遺伝子修復作用を呈し、これにより皮膚の細胞を増加させ、化粧料としてシワやタルミなどの皮膚トラブルに悩む方の皮膚の改善に貢献して化粧品業界の発展に寄与する。 The fumaric acid derivative obtained by the present invention exhibits a gene repairing action, thereby increasing the number of skin cells, contributing to the improvement of the skin of those suffering from skin troubles such as wrinkles and tarmi as cosmetics, and for the development of the cosmetics industry. Contribute.
本発明で得られるフマル酸誘導体は発酵法により製造されることから機能性を有する食品として利用でき、食品産業や発酵業界の発展に寄与する。 Since the fumaric acid derivative obtained by the present invention is produced by a fermentation method, it can be used as a functional food and contributes to the development of the food industry and the fermentation industry.
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