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JP7570615B2 - Antioxidant, method for producing antioxidant, and method for inhibiting the progress of oxidation reaction - Google Patents

Antioxidant, method for producing antioxidant, and method for inhibiting the progress of oxidation reaction Download PDF

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JP7570615B2
JP7570615B2 JP2020156594A JP2020156594A JP7570615B2 JP 7570615 B2 JP7570615 B2 JP 7570615B2 JP 2020156594 A JP2020156594 A JP 2020156594A JP 2020156594 A JP2020156594 A JP 2020156594A JP 7570615 B2 JP7570615 B2 JP 7570615B2
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quercetin
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glucoside
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未沙 藤浪
豊 小谷野
嘉純 佐藤
令以子 小松
航 門脇
沙理 本田
茂則 熊澤
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Shizuoka University NUC
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Description

本発明は、抗酸化剤およびケルセチン配糖体の製造方法に関する。より詳細には、ケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシドまたはその塩を有効成分とする抗酸化剤、ケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシドまたはその塩の製造方法、および、ケルセチン3-O-(6″-O-マロニル)-β-D-グルコシドまたはその塩の製造方法に関する。 The present invention relates to an antioxidant and a method for producing quercetin glycoside. More specifically, the present invention relates to an antioxidant containing quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside or its salt as an active ingredient, a method for producing quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside or its salt, and a method for producing quercetin 3-O-(6"-O-malonyl)-β-D-glucoside or its salt.

生体内では、好気呼吸により酸素を利用する過程において、あるいは紫外線や大気汚染物質、放射線、ある種の薬剤、タバコなどの刺激により、種々の活性酸素やフリーラジカルが発生している。活性酸素やフリーラジカルは高い酸化力を持ち、細胞伝達物質として働いたり、殺菌や抗腫瘍など免疫機能を果たす。しかしその一方で、過剰に産生されたものは細胞を傷害し、動脈硬化症、脳梗塞、心疾患、癌、糖尿病、高脂血症、睡眠障害、認知症などの様々な疾患をもたらしたり、老化を亢進する要因となることが報告されている(非特許文献1)。 In the body, various types of active oxygen and free radicals are generated during the process of utilizing oxygen through aerobic respiration, or due to stimuli such as ultraviolet light, air pollutants, radiation, certain drugs, and tobacco. Active oxygen and free radicals have high oxidizing power, and act as cell transmitters and perform immune functions such as bactericidal and antitumor effects. However, on the other hand, it has been reported that excessive production of these substances can damage cells and cause various diseases such as arteriosclerosis, cerebral infarction, heart disease, cancer, diabetes, hyperlipidemia, sleep disorders, and dementia, and can also be a factor in accelerating aging (Non-Patent Document 1).

また、生体外においても、例えば、食品や飼料、化粧品、合成樹脂、洗浄剤などでは、空気中の酸素によって含有成分が酸化され、褐変や退色、においや風味の変化、機能低下、栄養価の低下など、品質が劣化することが課題になっている。 Even outside the body, for example in food, feed, cosmetics, synthetic resins, and cleaning agents, oxygen in the air can oxidize the components contained in these products, causing problems such as browning, discoloration, changes in odor and flavor, reduced functionality, and reduced nutritional value, resulting in deterioration in quality.

そこで、酸化力の高い物質を消去したり、対象物における酸化反応の進行を抑制したりする活性(抗酸化活性)を有する物質が求められている。例えば、特許文献1には、ネギ属抽出物やタマネギ抽出物に含まれるS-1-プロペニルシステインを有効成分とする抗酸化剤が開示されている。 Therefore, there is a demand for substances that have the activity (antioxidant activity) to eliminate highly oxidizing substances and inhibit the progress of oxidation reactions in the target object. For example, Patent Document 1 discloses an antioxidant whose active ingredient is S-1-propenylcysteine, which is contained in Allium extract and onion extract.

特開第2020-029529号公報JP 2020-029529 A

吉川敏一、フリーラジカルの医学、京府医大誌、第120号、第6巻、第381~391頁、2011年Toshikazu Yoshikawa, Medicine of Free Radicals, Journal of Kyoto Prefectural University of Medicine, Vol. 120, 6, pp. 381-391, 2011

しかしながら、未だ、酸化反応の進行を効果的に抑制する物質は十分に供給されている状況とはいえない。本発明は、係る課題を解決するためになされたものであって、酸化反応の進行を効果的に抑制する抗酸化剤を提供することを目的とする。また、高い抗酸化活性を有するケルセチン配糖体を製造する方法を提供することを目的とする。 However, there is still not a sufficient supply of substances that effectively inhibit the progression of oxidation reactions. The present invention has been made to solve this problem, and aims to provide an antioxidant that effectively inhibits the progression of oxidation reactions. It also aims to provide a method for producing quercetin glycosides that have high antioxidant activity.

本発明者らは、鋭意研究の結果、キンセンカ属(Calendula)に、ケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシド、および、ケルセチン3-O-(6″-O-マロニル)-β-D-グルコシドが多く含まれることを見出した。また、これらのケルセチン配糖体が高い抗酸化活性を有することを見出した。そこで、これらの知見に基づいて下記の各発明を完成した。 As a result of intensive research, the inventors have discovered that Calendula contains large amounts of quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside and quercetin 3-O-(6"-O-malonyl)-β-D-glucoside. They have also discovered that these quercetin glycosides have high antioxidant activity. Based on these findings, the inventors have completed the following inventions.

(1)本発明に係る抗酸化剤は、ケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシドまたはその塩を有効成分とする。 (1) The antioxidant of the present invention contains quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside or its salt as an active ingredient.

(2)本発明において、ケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシドまたはその塩は、キンセンカ属から抽出されたものであってもよい。 (2) In the present invention, quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside or its salt may be extracted from Calendula genus.

(3)本発明に係るケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシドまたはその塩の製造方法は、キンセンカ属からケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシドまたはその塩を抽出する工程を有する。 (3) The method for producing quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside or its salt according to the present invention comprises the step of extracting quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside or its salt from Calendula officinalis.

(4)本発明に係るケルセチン3-O-(6″-O-マロニル)-β-D-グルコシドまたはその塩の製造方法は、キンセンカ属からケルセチン3-O-(6″-O-マロニル)-β-D-グルコシドまたはその塩を抽出する工程を有する。 (4) The method for producing quercetin 3-O-(6"-O-malonyl)-β-D-glucoside or its salt according to the present invention includes a step of extracting quercetin 3-O-(6"-O-malonyl)-β-D-glucoside or its salt from Calendula officinalis.

(5)本発明において、キンセンカ属の植物は、トウキンセンカ(Calendula officinalis)であってもよい。 (5) In the present invention, the plant of the genus Calendula may be Calendula officinalis.

本発明の抗酸化剤は、還元能ないしラジカル消去能力に優れ、高い抗酸化作用を発揮する。よって、酸化ストレスによる老化の進行の抑制、酸化ストレスによる疾病の発症や不健康状態の予防ないし改善、あるいは、酸化による製品の品質劣化の抑制に寄与することができる。また、本発明によれば、有用な化合物であるケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシドやケルセチン3-O-(6″-O-マロニル)-β-D-グルコシドまたはそれらの塩を製造することができる。 The antioxidant of the present invention has excellent reducing and radical scavenging abilities and exerts a strong antioxidant effect. Therefore, it can contribute to inhibiting the progression of aging caused by oxidative stress, preventing or improving the onset of diseases and ill health caused by oxidative stress, and inhibiting the deterioration of product quality caused by oxidation. Furthermore, according to the present invention, it is possible to produce the useful compounds quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside and quercetin 3-O-(6"-O-malonyl)-β-D-glucoside, or salts thereof.

トウキンセンカ葉エタノール抽出物のHPLCクロマトグラムを示す図である。FIG. 1 shows an HPLC chromatogram of an ethanol extract of Calendula officinalis leaves. FRAP法により測定した、クロロゲン酸(化1)、ケルセチン3-O-ネオヘスペリジン(化2)、ケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシド(化3)およびケルセチン3-O-(6″-O-マロニル)-β-D-グルコシド(化4)のトロロックス相当量(mol TE/mol)を示す棒グラフである。1 is a bar graph showing the Trolox equivalents (mol TE/mol) of chlorogenic acid (chemical formula 1), quercetin 3-O-neohesperidin (chemical formula 2), quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside (chemical formula 3), and quercetin 3-O-(6"-O-malonyl)-β-D-glucoside (chemical formula 4) as measured by the FRAP method. ABTS法により測定した、クロロゲン酸(化1)およびケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシド(化3)のトロロックス相当量(mol TE/mol)を示す棒グラフである。1 is a bar graph showing the trolox equivalent (mol TE/mol) of chlorogenic acid (Chemical formula 1) and quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside (Chemical formula 3) measured by the ABTS method. DPPH法により測定した、クロロゲン酸(化1)およびケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシド(化3)のトロロックス相当量(mol TE/mol)を示す棒グラフである。1 is a bar graph showing the trolox equivalent (mol TE/mol) of chlorogenic acid (Chemical formula 1) and quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside (Chemical formula 3) measured by the DPPH method.

以下、本発明について詳細に説明する。 The present invention will be described in detail below.

本発明において、「抗酸化活性」とは、活性酸素やフリーラジカルといった酸化力の強い物質(酸化物質)を消去する活性、もしくは、酸化物質の酸化力を弱める活性、または、対象物における酸化反応の進行を抑制する活性をいう。また、「抗酸化剤」とは、抗酸化活性を有する物質をいう。 In the present invention, "antioxidant activity" refers to the activity of eliminating highly oxidizing substances (oxidizing substances) such as active oxygen and free radicals, or the activity of weakening the oxidizing power of oxidizing substances, or the activity of inhibiting the progress of oxidation reactions in a target object. In addition, "antioxidant" refers to a substance that has antioxidant activity.

キンセンカ属は、キク科キンセンカ属に属する植物をいう。キンセンカ属としては、例えば、トウキンセンカ(Calendula officinalis、ポットマリーゴールド、カレンデュラ)や、ヒメキンセンカ(Calendula arvensis、ホンキンセンカ、フユシラズ、フユザキキンセンカ)などを例示することができる。 The genus Calendula refers to plants belonging to the genus Calendula in the family Asteraceae. Examples of Calendula include Calendula officinalis (pot marigold, calendula) and Calendula arvensis (Japanese calendula, winter marigold, winter calendula).

ケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシド(quercetin 3-O-(2"-O-α-rhamnosyl-6''-O-malonyl)-β-D-glucoside)は、下記の化3に示す構造の化合物である。
Quercetin 3-O-(2"-O-α-rhamnosyl-6''-O-malonyl)-β-D-glucoside is a compound having the structure shown in Chemical Formula 3 below.

ケルセチン3-O-(6″-O-マロニル)-β-D-グルコシド(quercetin 3-O-(6''-O-malonyl)-β-D-glucoside)は、下記の化4に示す構造の化合物である。
Quercetin 3-O-(6″-O-malonyl)-β-D-glucoside is a compound having the structure shown in Chemical Formula 4 below.

ケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシド(化3)およびその塩、ならびにケルセチン3-O-(6″-O-マロニル)-β-D-グルコシド(化4)およびその塩(以下、化3および化4の化合物ならびにそれらの塩をまとめて、またはいずれかを指して、「本化合物」という場合がある。)は、後述する実施例で示すように高い抗酸化活性を有する。よって、少なくともこの点で有用な物質であり、本化合物は抗酸化剤として用いることができる。 Quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside (chemical formula 3) and its salts, and quercetin 3-O-(6"-O-malonyl)-β-D-glucoside (chemical formula 4) and its salts (hereinafter, the compounds of chemical formulas 3 and 4 and their salts may be collectively referred to as "the present compounds" or either of them may be referred to as "the present compounds") have high antioxidant activity, as shown in the examples described later. Therefore, they are useful substances at least in this respect, and the present compounds can be used as antioxidants.

化3および化4の化合物はそのマロニル基で塩を形成しうるが、本発明においては、これらの塩を用いてもよい。ここで、塩は、「薬理学的に許容される塩」を含み、広義に解釈される。例えば、金属塩、アンモニウム塩、有機アミン付加塩、アミノ酸付加塩等、各種の塩であってよい。金属塩の例としてはナトリウム塩、カリウム塩、リチウム塩などのアルカリ金属塩、マグネシウム塩、カルシウム塩などのアルカリ土類金属塩、アルミニウム塩、亜鉛塩が挙げられる。アンモニウム塩の例としてはアンモニウム、テトラメチルアンモニウムなどの塩が挙げられる。有機アミン付加塩の例としてはモルホリン付加塩、ピペリジン付加塩が挙げられる。アミノ酸付加塩の例としてはグリシン付加塩、フェニルアラニン付加塩、リジン付加塩、アスパラギン酸付加塩、グルタミン酸付加塩が挙げられる。 The compounds of formula 3 and formula 4 can form salts with the malonyl group, and these salts may be used in the present invention. Here, the term "salt" includes "pharmacologically acceptable salts" and is interpreted in a broad sense. For example, various salts such as metal salts, ammonium salts, organic amine addition salts, and amino acid addition salts may be used. Examples of metal salts include alkali metal salts such as sodium salts, potassium salts, and lithium salts, alkaline earth metal salts such as magnesium salts and calcium salts, aluminum salts, and zinc salts. Examples of ammonium salts include ammonium and tetramethylammonium salts. Examples of organic amine addition salts include morpholine addition salts and piperidine addition salts. Examples of amino acid addition salts include glycine addition salts, phenylalanine addition salts, lysine addition salts, aspartic acid addition salts, and glutamic acid addition salts.

本化合物は、合成品を用いてもよく、市販の試薬を用いてもよい。または、本化合物を含む動植物等の天然物から抽出、精製等して用いることもでき、本化合物を化学的に合成して用いることもできる。 The compound may be a synthetic product or a commercially available reagent. Alternatively, the compound may be extracted and purified from natural products such as plants and animals that contain the compound, or the compound may be chemically synthesized and used.

本化合物は、キンセンカ属に多く含まれる。よって、キンセンカ属から抽出することにより、本化合物を効率的に得ることができる。キンセンカ属からの本化合物の抽出は、例えば、抽出溶媒にキンセンカ属の植物体を浸漬することにより行うことができる。具体的には、以下の方法を例示することができる。
≪キンセンカ属の植物体≫
キンセンカ属の植物体は、葉、茎、花、根あるいはこれらを含む全部(全草)など、いずれの部位を用いてもよいが、葉部を含むことが好ましい。また、生育地から採集したものをそのまま用いてもよく、乾燥させてから用いてもよい。また、葉、茎、花、根などの形態のものをそのまま用いてもよく、破片状や粉末状に砕いてから用いてもよい。
≪抽出溶媒≫
キンセンカ属から本化合物を抽出することができれば特に制限はなく、製品の最終用途に応じて適宜設定できる。例えば、溶媒は、水や低級アルコール類(エタノール、プロパノールなど)、グリコール類(グリセリン、1,3-ブチレングリコール、プロピレングリコール、1,3-プロパンジオールなど)、これらの混液などの極性溶媒を例示することができる。
≪抽出条件≫
抽出溶媒に植物体を浸漬し、温度1~30℃あるいは室温で、2~24時間、静置または攪拌しながら置いておく。
The present compound is contained in large amounts in Calendula genus. Therefore, the present compound can be efficiently obtained by extracting it from Calendula genus. The present compound can be extracted from Calendula genus by, for example, immersing the plant body of Calendula genus in an extraction solvent. Specifically, the following method can be exemplified.
<Calendula plant>
The plant body of the Calendula genus may be used in any part, such as leaves, stems, flowers, roots, or the whole plant including these (whole plant), but preferably includes leaves. Also, the plant collected from the growing area may be used as it is, or may be used after drying. Also, the plant body in the form of leaves, stems, flowers, roots, etc. may be used as it is, or may be used after crushing into pieces or powder.
<Extraction solvent>
As long as the present compound can be extracted from the genus Calendula, there is no particular limitation and the solvent can be appropriately selected depending on the final use of the product. For example, the solvent can be water, lower alcohols (ethanol, propanol, etc.), glycols (glycerin, 1,3-butylene glycol, propylene glycol, 1,3-propanediol, etc.), and polar solvents such as mixtures thereof.
<Extraction conditions>
The plant body is immersed in the extraction solvent and allowed to stand or be stirred at a temperature of 1 to 30° C. or at room temperature for 2 to 24 hours.

上記のように抽出操作を行った後の溶媒には、本化合物が含まれるため、これをそのまま用いてもよく、必要に応じて精製や濃縮あるいは希釈、殺菌などを行ってから用いてもよい。精製は、濾過や遠心分離による植物体残渣の除去や、液体クロマトグラフィーによる分画精製などを行うことができる。また、スプレードライや凍結乾燥などの方法により固体化してから用いてもよい。 The solvent after the extraction procedure described above contains the present compound, and may be used as is, or may be purified, concentrated, diluted, sterilized, or the like as necessary before use. Purification may be performed by removing plant residues through filtration or centrifugation, or by fractional purification through liquid chromatography. The compound may also be solidified by methods such as spray drying or freeze drying before use.

また、化学合成による場合、例えば、化4は、非特許文献2(Riva, S., Danieli, B., Luisetti, M., A two step efficient chemoenzymic synthesis of flavonoid glycoside malonates, J. Nat. Prod, 59, 618-621 (1996))に記載の方法で得ることができる。すなわち、当該方法によれば、まず、Candida antarctica由来のリパーゼの触媒下でイソクェルシトリンとマロン酸ジベンジルとを反応させて、イソクェルシトリンのグルコース残基をアシル化する。次に、パラジウム炭素触媒を用いた水素化還元反応を行ってベンジル基を外す。続いて、濾過により触媒を除去し、溶媒を減圧留去すれば、化4を得ることができる。 In addition, in the case of chemical synthesis, for example, Chemical formula 4 can be obtained by the method described in Non-Patent Document 2 (Riva, S., Danieli, B., Luisetti, M., A two step efficient chemoenzymic synthesis of flavonoid glycoside malonates, J. Nat. Prod, 59, 618-621 (1996)). That is, according to this method, isoquercitrin is first reacted with dibenzyl malonate under the catalyst of lipase derived from Candida antarctica to acylate the glucose residue of isoquercitrin. Next, a hydrogenation reduction reaction using a palladium carbon catalyst is performed to remove the benzyl group. The catalyst is then removed by filtration, and the solvent is distilled off under reduced pressure to obtain Chemical formula 4.

また、非特許文献3(Danieli, B., Bertario, A., Carrea, G., Redigolo, B., Secundo, F., Riva, S., Helv. Chim. Acta, 1993, 73, 2981-2991)に記載の方法により化4を得ることもできる。当該方法によれば、まず、タンパク分解酵素サブチリシンの触媒下でイソクェルシトリンと2-クロロエチルメチルマロン酸とを反応させて、イソクェルシトリンのグルコース残基の最初のヒドロキシ基にメチルマロン酸残基を導入する。次に、エステラーゼ触媒による酵素反応を行ってメトキシカルボニル基を選択的に加水分解する。続いて、逆相HPLCにより精製すれば、高純度の化4を得ることができる。 Chemical formula 4 can also be obtained by the method described in Non-Patent Document 3 (Danieli, B., Bertario, A., Carrea, G., Redigolo, B., Secundo, F., Riva, S., Helv. Chim. Acta, 1993, 73, 2981-2991). According to this method, isoquercitrin is first reacted with 2-chloroethylmethylmalonic acid under the catalysis of the proteolytic enzyme subtilisin to introduce a methylmalonic acid residue into the first hydroxyl group of the glucose residue of isoquercitrin. Next, an enzymatic reaction is carried out with the esterase catalyst to selectively hydrolyze the methoxycarbonyl group. Then, by purifying the product by reversed-phase HPLC, a highly pure product 4 can be obtained.

本発明において、抗酸化剤は、任意の形態ないし任意の用途で用いることができる。例えば、生体内で抗酸化活性を発揮させる目的で用いることができ、そのような場合には、経口摂取で用いられる医薬品や医薬部外品、健康食品、食品、飲料、飼料の形態、またはこれらに配合して使用される原料ないし添加物とすることができる。また、例えば、皮膚において抗酸化活性を発揮させる目的で用いることができ、そのような場合には、経皮的に用いられる医薬品や医薬部外品(湿布や軟膏など)、化粧品(パックや化粧水、乳液、ジェル、クリーム、リップクリームなど)の形態、またはこれらに配合して使用される原料ないし添加物とすることができる。また、例えば、製品(医薬品や医薬部外品、健康食品、食品、飲料、飼料、化粧品、合成樹脂、洗浄剤など)内において抗酸化活性を発揮させ、当該製品の品質劣化を抑制する目的で用いることができ、そのような場合には、当該製品に配合される原料ないし添加物の形態とすることができる。いずれの形態であっても、本発明の有効成分を配合した上で、常法により製造することができる。製品における有効成分の配合量または生体への投与量も、当該製品の用途や安全性、他の原材料などに応じて適宜設定することができる。 In the present invention, the antioxidant can be used in any form or for any purpose. For example, it can be used for the purpose of exerting antioxidant activity in the living body, and in such a case, it can be in the form of a medicine or quasi-drug, health food, food, beverage, or feed used by oral ingestion, or as a raw material or additive used by blending with these. It can also be used for the purpose of exerting antioxidant activity on the skin, and in such a case, it can be in the form of a medicine or quasi-drug (such as a compress or ointment) or a cosmetic (such as a pack, lotion, milky lotion, gel, cream, lip balm, etc.) used percutaneously, or as a raw material or additive used by blending with these. It can also be used for the purpose of exerting antioxidant activity in a product (such as a medicine or quasi-drug, health food, food, beverage, feed, cosmetic, synthetic resin, detergent, etc.) and suppressing deterioration of the quality of the product, and in such a case, it can be in the form of a raw material or additive used by blending with the product. In any form, it can be manufactured by a conventional method after blending the active ingredient of the present invention. The amount of active ingredient in the product or the amount administered to the living body can be set appropriately depending on the product's intended use, safety, other raw materials, etc.

以下、本発明について、各実施例に基づいて説明するが、本発明の技術的範囲は、これらの実施例によって示される特徴に限定されない。本実施例において、単位に用いられるMはmol/Lを表す。 The present invention will be described below based on each example, but the technical scope of the present invention is not limited to the characteristics shown in these examples. In the examples, the unit M represents mol/L.

<実施例1>化合物の同定
(1)トウキンセンカ葉エタノール抽出物の調製
北海道白老町にて採集したトウキンセンカの全草を風通しのよい室内に72時間置くことにより、または、45℃の乾燥機に28~32時間置くことにより、乾燥させた後に保存した。この乾燥物から葉部を分別回収し、ミキサーを用いて粉末状にした。得られた葉の乾燥粉末20gに70%(v/v)エタノール200mLを加え、スターラーを用いて500回転/分(rpm)、室温にて24時間攪拌し、抽出液を得た。抽出液を吸引濾過して濾液を回収し、エバポレーターを用いて減圧濃縮し、濃縮液を得た。続いて、濃縮液を24時間凍結乾燥し、固体状のトウキンセンカ葉エタノール抽出物5.2gを得た。
Example 1: Identification of Compounds (1) Preparation of Calendula officinalis Leaf Ethanol Extract The whole plant of Calendula officinalis collected in Shiraoi-cho, Hokkaido was dried and stored by placing it in a well-ventilated room for 72 hours or by placing it in a dryer at 45°C for 28 to 32 hours. The leaves were separated and collected from the dried matter and powdered using a mixer. 200 mL of 70% (v/v) ethanol was added to 20 g of the obtained dried leaf powder, and the mixture was stirred at 500 revolutions per minute (rpm) at room temperature for 24 hours using a stirrer to obtain an extract. The extract was filtered by suction to collect the filtrate, which was then concentrated under reduced pressure using an evaporator to obtain a concentrate. The concentrate was then freeze-dried for 24 hours to obtain 5.2 g of a solid Calendula officinalis leaf ethanol extract.

(2)化合物の単離および同定
トウキンセンカ葉エタノール抽出物5.2gを超純水(MilliQ水)に溶解させて全量を150mLとした。酢酸エチル325mLを添加し、水層と酢酸エチル層とに分画した。水層2.16gを下記条件の中圧分取液体クロマトグラフに供した。保持時間60分の第1画分、同88分の第2画分、同95分の第3画分および同106分の第4画分を分取して、乾燥させ、重量を測定した。
《中圧分取液体クロマトグラフの条件》
カラム:YAMAZEN ULTRAPAK ODS-SM-50D (50μm,φ50×300mm)
溶媒:(A)H2O(0.1%(v/v) TFA) (B)アセトニトリル(MeCN)(0.1%(v/v) TFA)
勾配:(B)10%(v/v)(15分)→(B)40%(v/v)(150分)→(B)100%(155分)
流速:45mL/分
検出:吸光光度検出器(280nm)
(2) Isolation and Identification of Compounds 5.2 g of ethanol extract of Calendula officinalis leaves was dissolved in ultrapure water (MilliQ water) to a total volume of 150 mL. 325 mL of ethyl acetate was added, and the mixture was fractionated into an aqueous layer and an ethyl acetate layer. 2.16 g of the aqueous layer was subjected to medium pressure preparative liquid chromatography under the following conditions. The first fraction with a retention time of 60 minutes, the second fraction with a retention time of 88 minutes, the third fraction with a retention time of 95 minutes, and the fourth fraction with a retention time of 106 minutes were separated, dried, and weighed.
<Conditions for medium pressure preparative liquid chromatography>
Column: YAMAZEN ULTRAPAK ODS-SM-50D (50 μm, φ50 × 300 mm)
Solvent: (A) H2O (0.1% (v/v) TFA) (B) Acetonitrile (MeCN) (0.1% (v/v) TFA)
Gradient: (B) 10% (v/v) (15 min) → (B) 40% (v/v) (150 min) → (B) 100% (155 min)
Flow rate: 45 mL/min Detection: spectrophotometric detector (280 nm)

[2-1]第1画分の化合物の単離・同定
第1画分(保持時間60分、乾燥重量142.5mg)を下記条件の高速液体クロマトグラフィー(HPLC)に供し、保持時間16分の画分を分取した。続いて、当該画分を乾燥させることにより、化合物30.9mg(乾燥重量)を単離した。
《HPLCの条件》
カラム:SHISEIDO CAPCELLPAK UG120 (5μm,φ20×250mm)
溶媒:H2O : MeCN = 88 : 12 (v:v)(0.1%(v/v) TFA)
流速:9.6 mL/分
検出:吸光光度検出器(280nm)
[2-1] Isolation and identification of the compound in the first fraction The first fraction (retention time 60 minutes, dry weight 142.5 mg) was subjected to high performance liquid chromatography (HPLC) under the following conditions to separate a fraction with a retention time of 16 minutes. The fraction was then dried to isolate 30.9 mg (dry weight) of the compound.
HPLC conditions
Column: SHISEIDO CAPCELLPAK UG120 (5μm, φ20×250mm)
Solvent: H 2 O : MeCN = 88 : 12 (v:v)(0.1%(v/v) TFA)
Flow rate: 9.6 mL/min Detection: spectrophotometric detector (280 nm)

核磁気共鳴(Nuclear Magnetic Resonance:NMR)装置および液体クロマトグラフィー質量分析計(LC-MS)を用いて当該化合物の構造解析を行った結果、化1に示すクロロゲン酸(chrologenic acid)であることが明らかになった。NMRおよびLC-MSのデータを以下に示す。
The structural analysis of the compound was carried out using a nuclear magnetic resonance (NMR) device and a liquid chromatography mass spectrometer (LC-MS), and it was found to be chlorogenic acid shown in Chemical formula 1. The NMR and LC-MS data are shown below.

<化1のNMRおよびLC-MSデータ>
1H NMR: 400 MHz, 13C NMR:100MHz (測定溶媒CD3OD)
1H NMR: δ(ppm) 2.07 [m, 2H, 2.02 - 2.10], 2.20 [m, 2H, 2.16 - 2.26], 3.73 [dd, 1H, J = 8.5, 3.2 Hz], 4.17 [s, 1H], 5.33[m, 1H, 5.31 - 5.36], 6.26 [d, 1H, J = 15.9 Hz], 6.78[d, 1H, J = 8.2 Hz], 6.95 [dd, 1H, J = 8.2, 2.0 Hz], 7.05 [d, 1H, J = 2.0 Hz], 7.56 [d, 1H, J = 15.9 Hz];
13C NMR: δ(ppm) 38.2 [CH2], 38.78 [CH2], 71.30 [CH], 71.95 [CH], 73.49 [CH], 76.14 [C], 115.21 [CH], 115.26 [CH], 116.48 [CH], 122.97 [CH], 127.80 [C], 146.77 [CH], 147.08 [CH], 149.54 [CH], 168.68 [C], 177.00 [C].
HR-ESIMS : m/z = 353.0876([M-H]-)
<NMR and LC-MS data of Chemical Formula 1>
1 H NMR: 400 MHz, 13 C NMR: 100MHz (measurement solvent: CD 3 OD)
1 H NMR: δ(ppm) 2.07 [m, 2H, 2.02 - 2.10], 2.20 [m, 2H, 2.16 - 2.26], 3.73 [dd, 1H, J = 8.5, 3.2 Hz], 4.17 [s, 1H], 5.33[m, 1H, 5.31 - 5. 36], 6.26 [d, 1H, J = 15.9 Hz], 6.78[d, 1H, J = 8.2 Hz], 6.95 [dd, 1H, J = 8.2, 2.0 Hz], 7.05 [d, 1H, J = 2.0 Hz], 7.56 [d, 1H, J = 15.9 Hz];
13 C NMR: δ(ppm) 38.2 [CH 2 ], 38.78 [CH 2 ], 71.30 [CH], 71.95 [CH], 73.49 [CH], 76.14 [C], 115.21 [CH], 115.26 [CH], 116.48 [CH], 122.97 [CH], 12 7.80 [C], 146.77 [CH], 147.08 [CH], 149.54 [CH], 168.68 [C], 177.00 [C].
HR-ESIMS: m/z = 353.0876([MH] - )

[2-2]第2画分の化合物の単離・同定
第2画分(保持時間88分、乾燥重量41.6mg)を下記条件のHPLCに供し、保持時間15.5分の画分を分取した。続いて、当該画分を乾燥させることにより、化合物1.8mg(乾燥重量)を単離した。
《HPLCの条件》
カラム:SHISEIDO CAPCELLPAK UG120 (5μm,φ20×250mm)
溶媒:H2O : MeCN = 82 : 18 (v:v)(0.1%(v/v) TFA)
流速:9.6 mL/分
検出:吸光光度検出器(280nm)
[2-2] Isolation and identification of the compound in the second fraction The second fraction (retention time 88 minutes, dry weight 41.6 mg) was subjected to HPLC under the following conditions to separate a fraction with a retention time of 15.5 minutes. The fraction was then dried to isolate 1.8 mg (dry weight) of the compound.
HPLC conditions
Column: SHISEIDO CAPCELLPAK UG120 (5μm, φ20×250mm)
Solvent: H 2 O : MeCN = 82 : 18 (v:v)(0.1%(v/v) TFA)
Flow rate: 9.6 mL/min Detection: spectrophotometric detector (280 nm)

NMR装置およびLC-MSを用いて当該化合物の構造解析を行った結果、化2に示すケルセチン3-O-ネオヘスペリジン(quercetin 3-O-neohesperidin)であることが明らかになった。NMRおよびLC-MSのデータを以下に示す。
As a result of structural analysis of the compound using an NMR device and LC-MS, it was found to be quercetin 3-O-neohesperidin shown in Chemical formula 2. The NMR and LC-MS data are shown below.

<化2のNMRおよびLC-MSデータ>
1H NMR: 400 MHz, 13C NMR:100MHz (測定溶媒CD3OD)
1H NMR: δ(ppm) 0.97 [d, 3H, J = 6.2 Hz], 3.23 [m, 1H], 3.35 [m, 2H, 3.34 - 3.36], 3.55 [m, 2H, 3.52 - 3.58], 3.66 [dd, 1H, J = 7.6, 1.4 Hz], 3.76[m, 2H, 3.72 - 3.77], 4.02[m, 2H 3.99 - 4.06], 5.23 [d, 1H, J = 1.4 Hz], 5.75 [d, 1H, J = 7.6 Hz], 6.18 [d, 1H, J = 2.2 Hz], 6.37 [d, 1H, J = 2.1 Hz], 6.87[d, 1H, J = 7.8 Hz], 7.61[m, 2H, 7.60 - 7.63];
13C NMR: δ(ppm) 17.46 [CH3], 62.57 [CH2], 69.96 [CH], 71.71 [CH], 72.31 [CH], 72.41 [CH], 74.06 [CH], 78.35 [CH], 78.96 [CH], 80.11 [CH], 94.48 [CH], 99.65 [CH], 100.33[CH], 102.65 [CH], 105.97 [C], 115.98 [CH], 117.19 [CH], 123.22 [C], 123.48 [CH], 134.57 [C], 146.01 [C], 149.56 [C], 158.39 [C], 158.42 [C], 163.22 [C], 165.63 [C], 179.37 [C].
HR-ESIMS: m/z = 609.1462([M-H]-)
<NMR and LC-MS data of Chemical formula 2>
1 H NMR: 400 MHz, 13 C NMR: 100MHz (measurement solvent: CD 3 OD)
1 H NMR: δ(ppm) 0.97 [d, 3H, J = 6.2 Hz], 3.23 [m, 1H], 3.35 [m, 2H, 3.34 - 3.36], 3.55 [m, 2H, 3.52 - 3.58], 3.66 [dd, 1H, J = 7.6, 1.4 Hz] , 3.76[m, 2H, 3.72 - 3.77], 4.02[m, 2H 3.99 - 4.06], 5.23 [d, 1H, J = 1.4 Hz], 5.75 [d, 1H, J = 7.6 Hz], 6.18 [d, 1H, J = 2.2 Hz], 6.37 [d, 1H, J = 2.1 Hz], 6.87[d, 1H, J = 7.8 Hz], 7.61[m, 2H, 7.60 - 7.63];
13 C NMR: δ(ppm) 17.46 [CH 3 ], 62.57 [CH 2 ], 69.96 [CH], 71.71 [CH], 72.31 [CH], 72.41 [CH], 74.06 [CH], 78.35 [CH], 78.96 [CH], 80.11 [CH], 94.48 [CH], 99.65 [CH], 100.33[CH], 102.65 [CH], 105.97 [C], 115.98 [CH], 117.19 [CH], 123.22 [C], 123.48 [CH], 134.57 [C], 146.01 [C], 149.56 [C], 158.39 [C], 158.42 [C], 163.22 [C], 165.63 [C], 179.37 [C].
HR-ESIMS: m/z = 609.1462([MH] - )

[2-3]第3画分の化合物の単離・同定
第3画分(保持時間95分、乾燥重量58.9mg)を下記条件のHPLCに供し、保持時間14.3分の画分を分取した。続いて、当該画分を乾燥させることにより、化合物21.9mg(乾燥重量)を単離した。
《HPLCの条件》
カラム:SHISEIDO CAPCELLPAK UG120 (5μm,φ20×250mm)
溶媒:H2O : MeCN = 80 : 20 (v:v)(0.1%(v/v) TFA)
流速:9.6 mL/分
検出:吸光光度検出器(280nm)
[2-3] Isolation and identification of the compound in the third fraction The third fraction (retention time 95 minutes, dry weight 58.9 mg) was subjected to HPLC under the following conditions to separate a fraction with a retention time of 14.3 minutes. The fraction was then dried to isolate 21.9 mg (dry weight) of the compound.
HPLC conditions
Column: SHISEIDO CAPCELLPAK UG120 (5μm, φ20×250mm)
Solvent: H 2 O : MeCN = 80 : 20 (v:v)(0.1%(v/v) TFA)
Flow rate: 9.6 mL/min Detection: spectrophotometric detector (280 nm)

NMR装置およびLC-MSを用いて当該化合物の構造解析を行った結果、化3に示すケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシドであることが明らかになった。NMRおよびLC-MSのデータを以下に示す。
Structural analysis of the compound using an NMR device and LC-MS revealed that it was quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside, as shown in Chemical formula 3. The NMR and LC-MS data are shown below.

<化3のNMRおよびLC-MSデータ>
1H NMR: 400 MHz, 13C NMR:100MHz (測定溶媒CD3OD)
1H NMR: δ(ppm) 1.02 [d, 3H, J = 6.2 Hz], 3.14 [s, 2H], 3.35 [m, 3H, 3.31 - 3.39], 3.56 [t, 1H, J = 11.1 Hz], 3.66 [dd, 1H, J = 7.7, 1.4 Hz], 3.80[dd, 1H, J = 9.5, 3.5 Hz], 4.01[m, 1H], 4.11 [m, 2H, 4.06 - 4.16], 4.28 [dd, 2H, J = 1.8, 11.8 Hz], 5.23 [d, 1H, J = 1.4 Hz], 5.59 [d, 1H, J = 7.6 Hz], 6.18[d, 1H, J = 2.1 Hz], 6.37 [d, 1H, J = 2.1 Hz], 6.87 [d, 1H, J = 9.0 Hz], 7.57[m, 2H, 7.56 - 7.59];
13C NMR: δ(ppm) 17.51 [CH3], 41.60 [CH2], 64.57 [CH2], 69.97 [CH], 71.36 [CH], 72.29 [CH], 72.38 [CH], 74.04 [CH], 75.27 [CH], 78.66 [CH], 79.95 [CH], 94.61 [CH], 99.71 [CH], 100.38 [CH], 102.66 [CH], 105.86 [C], 115.91 [CH], 117.28 [CH], 123.29 [C], 123.53 [CH], 134.37 [C], 145.92 [C], 149.46 [C], 158.37 [C], 159.02 [C], 163.13 [C], 165.64 [C], 168.31 [C], 170.15 [C], 179.21 [C].
HR-ESIMS: m/z = 695.1469([M-H]-)
<NMR and LC-MS data of Chemical formula 3>
1 H NMR: 400 MHz, 13 C NMR: 100MHz (measurement solvent: CD 3 OD)
1 H NMR: δ(ppm) 1.02 [d, 3H, J = 6.2 Hz], 3.14 [s, 2H], 3.35 [m, 3H, 3.31 - 3.39], 3.56 [t, 1H, J = 11.1 Hz], 3.66 [dd, 1H, J = 7.7, 1.4 Hz], 3.80[dd, 1H, J = 9.5, 3.5 Hz], 4.01[m, 1H], 4.11 [m, 2H, 4.06 - 4.16], 4.28 [dd, 2H, J = 1.8, 11.8 Hz], 5.23 [d, 1H, J = 1.4 Hz], 5.59 [d, 1H, J = 7.6 Hz], 6.18[d, 1H, J = 2.1 Hz], 6.37 [d, 1H, J = 2.1 Hz], 6.87 [d, 1H, J = 9.0 Hz], 7.57[m, 2H, 7.56 - 7.59];
13 C NMR: δ(ppm) 17.51 [CH 3 ], 41.60 [CH 2 ], 64.57 [CH 2 ], 69.97 [CH], 71.36 [CH], 72.29 [CH], 72.38 [CH], 74.04 [CH], 75.27 [CH], 78.66 [CH], 79. 95 [CH], 94.61 [CH], 99.71 [CH], 100.38 [CH], 102.66 [CH], 105.86 [C], 115.91 [CH], 117.28 [CH], 123.29 [C], 123.53 [CH], 134.37 [C], 145.92 [C], 149.46 [C], 158.37 [C], 159.02 [C], 163.13 [C], 165.64 [C], 168.31 [C], 170.15 [C], 179.21 [C].
HR-ESIMS: m/z = 695.1469([MH] - )

[2-4]第4画分の化合物の単離・同定
第4画分(保持時間106分、乾燥重量70.2mg)を下記条件のHPLCに供し、保持時間18.6分の画分を分取した。続いて、当該画分を乾燥させることにより、化合物28.3mg(乾燥重量)を単離した。
《HPLCの条件》
カラム:SHISEIDO CAPCELLPAK UG120 (5μm,φ20×250mm)
溶媒:H2O : MeCN = 80 : 20 (v:v)(0.1%(v/v) TFA)
流速:9.6 mL/分
検出:吸光光度検出器(280nm)
[2-4] Isolation and identification of the compound in the fourth fraction The fourth fraction (retention time 106 minutes, dry weight 70.2 mg) was subjected to HPLC under the following conditions to separate a fraction with a retention time of 18.6 minutes. The fraction was then dried to isolate 28.3 mg (dry weight) of the compound.
HPLC conditions
Column: SHISEIDO CAPCELLPAK UG120 (5μm, φ20×250mm)
Solvent: H 2 O : MeCN = 80 : 20 (v:v)(0.1%(v/v) TFA)
Flow rate: 9.6 mL/min Detection: spectrophotometric detector (280 nm)

NMR装置およびLC-MSを用いて当該化合物の構造解析を行った結果、化4に示すケルセチン3-O-(6″-O-マロニル)-β-D-グルコシドであることが明らかになった。NMRおよびLC-MSのデータを以下に示す。
Structural analysis of the compound was performed using an NMR device and LC-MS, and it was found to be quercetin 3-O-(6"-O-malonyl)-β-D-glucoside shown in Chemical formula 4. The NMR and LC-MS data are shown below.

<化4のNMRおよびLC-MSデータ>
1H NMR: 400 MHz, 13C NMR:100MHz (測定溶媒CD3OD)
1H NMR: δ(ppm) 3.21 [d, 2H, J = 1.0 Hz], 3.45 [m, 3H, 3.37 - 3.52], 4.12 [dd, 1H, J = 5.2, 12.0 Hz], 4.26[dd, 1H, J = 1.9, 12.0 Hz], 5.12 [d, 1H, J = 7.6 Hz], 6.21[d, 1H, J = 2.1 Hz], 6.40 [d, 1H, J = 2.1 Hz], 6.85 [d, 1H, J = 8.5 Hz], 7.58[dd, 1H, J = 2.2, 8.5 Hz], 7.62[d, 1H, J = 2.2 Hz];
13C NMR: δ(ppm) 41.68 [CH2], 64.95 [CH2], 71.11 [CH], 75.46 [CH], 75.57 [CH], 77.88 [CH], 94.82 [CH], 99.94 [CH], 104.53 [CH], 105.62 [C], 115.92 [CH], 117.52 [CH], 123.10 [C], 123.38 [CH], 135.45 [C], 145.86 [C], 149.78 [C], 158.48 [C], 159.53 [C], 163.01 [C], 166.04 [C], 168.42 [C], 170.25 [C], 179.43 [C].
HR-ESIMS: m/z = 549.0889([M-H]-)
<NMR and LC-MS data of Chemical Formula 4>
1 H NMR: 400 MHz, 13 C NMR: 100MHz (measurement solvent: CD 3 OD)
1 H NMR: δ(ppm) 3.21 [d, 2H, J = 1.0 Hz], 3.45 [m, 3H, 3.37 - 3.52], 4.12 [dd, 1H, J = 5.2, 12.0 Hz], 4.26[dd, 1H, J = 1.9, 12.0 Hz], 5.12 [d, 1H, J = 7.6 Hz], 6.21[d, 1H, J = 2.1 Hz], 6.40 [d, 1H, J = 2.1 Hz], 6.85 [d, 1H, J = 8.5 Hz], 7.58[dd, 1H, J = 2.2, 8.5 Hz], 7.62[d, 1H, J = 2.2 Hz];
13 C NMR: δ(ppm) 41.68 [CH 2 ], 64.95 [CH 2 ], 71.11 [CH], 75.46 [CH], 75.57 [CH], 77.88 [CH], 94.82 [CH], 99.94 [CH], 104.53 [CH], 105.62 [C], 115 .92 [CH], 117.52 [CH], 123.10 [C], 123.38 [CH], 135.45 [C], 145.86 [C], 149.78 [C], 158.48 [C], 159.53 [C], 163.01 [C], 166.04 [C], 168.42 [C], 170.25 [C], 179.43 [C].
HR-ESIMS: m/z = 549.0889([MH] - )

<実施例2>化合物の定量
(1)トウキンセンカ葉エタノール抽出物の調製
実施例1(1)のトウキンセンカ葉部乾燥粉末1.0gに70%(v/v)エタノール20mLを加え、攪拌子とスターラーとを用いて900rpm、室温にて2時間攪拌し、抽出液を得た。濾紙と漏斗を用いて抽出液を濾過して濾液を回収し、100mLナスフラスコへ移して、エバポレーターで減圧濃縮し、濃縮物を得た。濃縮物をメタノールに溶解させ、6mLスクリュー管瓶に移してコンビニ・エバポ(バイオクロマト)で再度濃縮し、濃縮物を得た。濃縮物を18時間凍結乾燥し、固体状のトウキンセンカ葉エタノール抽出物を得て、重量を測定した。同様の操作を3回行ったところ、抽出物の重量はそれぞれ195.27mg、189.29mgおよび170.67mgであった。
Example 2: Quantitative determination of compounds (1) Preparation of ethanol extract of Calendula officinalis leaves 20 mL of 70% (v/v) ethanol was added to 1.0 g of dried powder of Calendula officinalis leaves of Example 1 (1), and the mixture was stirred at 900 rpm and room temperature for 2 hours using a stirrer and a stirrer to obtain an extract. The extract was filtered using a filter paper and a funnel to collect the filtrate, which was transferred to a 100 mL eggplant flask and concentrated under reduced pressure using an evaporator to obtain a concentrate. The concentrate was dissolved in methanol, transferred to a 6 mL screw tube bottle, and concentrated again using a convenience evaporator (Biochromate) to obtain a concentrate. The concentrate was freeze-dried for 18 hours to obtain a solid ethanol extract of Calendula officinalis leaves, and its weight was measured. The same operation was performed three times, and the weights of the extracts were 195.27 mg, 189.29 mg, and 170.67 mg, respectively.

(2)検量線の作成
市販の試薬クロロゲン酸(化1)および実施例1(2)[2-2]~[2-4]で得られた化2~4の化合物を1.0mg/mLとなるようメタノールに溶解した。さらにメタノールを用いて、化1は100、250および1000μg/mL、化2は25、50および100μg/mL、化3および化4は100、250および500μg/mLとなるよう希釈して、測定試料を調製した。これらの測定試料を下記条件のHPLCに供してクロマトグラムを得た。当該クロマトグラムにて検出されたピークの面積と試料の濃度とに基づいて、検量線を作成した。
《HPLCの条件》
カラム:SHISEIDO CAPCELL PAK C18 UG120 (5μm,φ4.6×250mm)
溶媒:(A)H2O(0.1%(v:v) TFA) (B)MeCN(0.1%(v:v) TFA)
勾配:(B)0%(0分)→(B)30%(v:v)(40分)
流速:1.0mL/分
検出:吸光光度検出器(280nm)
注入量:10μL
(2) Preparation of calibration curve Commercially available reagent chlorogenic acid (chemical formula 1) and the compounds of chemical formulas 2 to 4 obtained in Example 1 (2) [2-2] to [2-4] were dissolved in methanol to a concentration of 1.0 mg/mL. Further, using methanol, chemical formula 1 was diluted to 100, 250 and 1000 μg/mL, chemical formula 2 was diluted to 25, 50 and 100 μg/mL, and chemical formulas 3 and 4 were diluted to 100, 250 and 500 μg/mL to prepare measurement samples. These measurement samples were subjected to HPLC under the following conditions to obtain chromatograms. A calibration curve was prepared based on the areas of the peaks detected in the chromatograms and the concentrations of the samples.
HPLC conditions
Column: SHISEIDO CAPCELL PAK C18 UG120 (5 μm, φ4.6 × 250 mm)
Solvent: (A) H 2 O (0.1% (v:v) TFA) (B) MeCN (0.1% (v:v) TFA)
Gradient: (B) 0% (0 min) → (B) 30% (v:v) (40 min)
Flow rate: 1.0 mL/min Detection: spectrophotometric detector (280 nm)
Injection volume: 10 μL

(3)化合物の定量
本実施例2(1)のトウキンセンカ葉エタノール抽出物を10mg/mLとなるようメタノールに溶解した後、孔径0.22μmのフィルターで濾過して測定試料とした。この測定試料を、本実施例2(2)に記載の条件のHPLCに供した。当該HPLCにより得られたクロマトグラムを図1に示す。図1に示すクロマトグラムでは、保持時間9.4分、19.6分、22.5分および25.4分のピークが、それぞれ、化1、化2、化3および化4に該当する。
(3) Quantitative Determination of Compounds The ethanol extract of Calendula officinalis leaves of Example 2(1) was dissolved in methanol to a concentration of 10 mg/mL, and then filtered through a filter with a pore size of 0.22 μm to obtain a measurement sample. This measurement sample was subjected to HPLC under the conditions described in Example 2(2). The chromatogram obtained by the HPLC is shown in FIG. 1. In the chromatogram shown in FIG. 1, the peaks at retention times of 9.4 minutes, 19.6 minutes, 22.5 minutes, and 25.4 minutes correspond to Chemical 1, Chemical 2, Chemical 3, and Chemical 4, respectively.

そこで、上記ピークの面積をそれぞれ算出し、本実施例2(2)の検量線を用いて化1~化4の濃度を決定した。決定した濃度に基づいて、トウキンセンカ葉部乾燥物あたり、および、トウキンセンカ葉エタノール抽出物あたりの化1~化4の含有量について、平均値および標準偏差を算出した。その結果を表1に示す。
The areas of the above peaks were calculated, and the concentrations of Chemicals 1 to 4 were determined using the calibration curve of Example 2 (2). Based on the determined concentrations, the average values and standard deviations of the contents of Chemicals 1 to 4 per dried Calendula officinalis leaf material and per ethanol extract of Calendula officinalis leaf were calculated. The results are shown in Table 1.

表1に示すように、トウキンセンカ葉部には、クロロゲン酸(化1)、ケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシド(化3)およびケルセチン3-O-(6″-O-マロニル)-β-D-グルコシド(化4)が高い割合で含まれることが明らかになった。 As shown in Table 1, it was revealed that the leaves of Calendula officinalis contain high proportions of chlorogenic acid (chemical formula 1), quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside (chemical formula 3) and quercetin 3-O-(6"-O-malonyl)-β-D-glucoside (chemical formula 4).

<実施例3>化合物の抗酸化能の評価
実施例1に記載の方法により製造した化1~化4の化合物の抗酸化能を、FRAP法、ABTS法およびDPPH法により評価した。評価にあたっては、表2に示すa~dの4つのサンプルを調製した。標準物質は6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid(トロロックス)を用いた。サンプルa、b、cおよびdの吸光度をそれぞれAa、Ab、AcおよびAdで示す。
Example 3: Evaluation of the antioxidant activity of compounds The antioxidant activity of compounds 1 to 4 produced by the method described in Example 1 was evaluated by the FRAP method, the ABTS method, and the DPPH method. For the evaluation, four samples a to d shown in Table 2 were prepared. 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was used as the standard substance. The absorbances of samples a, b, c, and d are indicated as Aa, Ab, Ac, and Ad, respectively.

(1)FRAP(Ferric Reducing Antioxidant Power)法:鉄イオンに対する還元能
FRAP試薬は、0.3Mの酢酸緩衝液(pH3.6):10mMの2,4,6-Tris(2-pyridyl)-1,3,5-triazine(TPTZ)水溶液:20mMのFeCl水溶液=10:1:1で混合されたものを用いた。
(1) FRAP (Ferric Reducing Antioxidant Power) Method: Reducing Ability for Iron Ions The FRAP reagent used was a mixture of 0.3 M acetate buffer (pH 3.6): 10 mM 2,4,6-Tris(2-pyridyl)-1,3,5-triazine (TPTZ) aqueous solution: 20 mM FeCl3 aqueous solution = 10:1:1.

まず、トロロックスを50%(v/v)エタノールに溶解することにより、濃度12.5、25、50、100および200μMのトロロックス溶液を調製した。96穴プレートにトロロックス溶液(サンプルa、b)または50%(v/v)エタノール(サンプルc、d)を各30μLずつ分注した。続いて、この96穴プレートに、37℃で加温したFRAP試薬(サンプルa、c)または0.3M酢酸緩衝液(pH3.6)(サンプルb、d)を各150μLずつ分注した。室温、暗所で5分間反応させた後、593nmでの吸光度を測定した。続いて、下記の式1によりトロロックス吸光値を算出し、トロロックス溶液の濃度に対してプロットすることにより、検量線を作成した。
式1:トロロックス吸光値= (Aa-Ab) -(Ac-Ad)
First, Trolox solutions with concentrations of 12.5, 25, 50, 100 and 200 μM were prepared by dissolving Trolox in 50% (v/v) ethanol. 30 μL of each of Trolox solutions (samples a, b) or 50% (v/v) ethanol (samples c, d) were dispensed into a 96-well plate. Then, 150 μL of each of FRAP reagent (samples a, c) or 0.3 M acetate buffer (pH 3.6) (samples b, d) warmed at 37° C. was dispensed into the 96-well plate. After reacting for 5 minutes at room temperature in the dark, the absorbance at 593 nm was measured. Then, the Trolox absorbance was calculated according to the following formula 1, and a calibration curve was created by plotting against the concentration of the Trolox solution.
Equation 1: Trolox absorbance value = (Aa-Ab) - (Ac-Ad)

実施例1に記載の方法により製造した化1~4を濃度10mMになるようにジメチルスルホキシドに溶解し、これをDMSO溶液とした。50%(v/v)エタノールを用いてDMSO溶液を希釈することにより、化合物濃度が30、60および120μMとなるよう調製し、これを化合物溶液とした。トロロックス溶液を化合物溶液に代えて同様の操作を行い、吸光度を測定した。続いて、下記の式2により化合物吸光値を算出し、検量線を用いて化1~4のトロロックス等価活性(トロロックス相当量)を算出した。同様の試験を3回行い、トロロックス相当量の平均値および標準偏差を算出した。その結果を表3および図2に示す。
式2:化合物吸光値= Aa-Ac
Compounds 1 to 4 prepared by the method described in Example 1 were dissolved in dimethyl sulfoxide to a concentration of 10 mM, and this was used as a DMSO solution. The DMSO solution was diluted with 50% (v/v) ethanol to prepare compound concentrations of 30, 60, and 120 μM, and this was used as a compound solution. The same operation was performed using Trolox solution instead of the compound solution, and the absorbance was measured. Subsequently, the compound absorbance was calculated according to the following formula 2, and the Trolox equivalent activity (Trolox equivalent amount) of compounds 1 to 4 was calculated using the calibration curve. The same test was performed three times, and the average value and standard deviation of the Trolox equivalent amount were calculated. The results are shown in Table 3 and Figure 2.
Equation 2: Compound absorbance value = Aa - Ac

表3および図2に示すように、ケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシド(化3)およびケルセチン3-O-(6″-O-マロニル)-β-D-グルコシド(化4)は、強い抗酸化能を有することで知られるクロロゲン酸よりもトロロックス相当量が顕著に大きかった。特に、ケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシド(化3)のトロロックス相当量は、クロロゲン酸と比較して1.41倍、ケルセチン3-O-ネオヘスペリジン(化2)と比較して1.5倍、ケルセチン3-O-(6″-O-マロニル)-β-D-グルコシド(化4)と比較して1.26倍であり、顕著に高い鉄イオン還元能を有することが明らかになった。 As shown in Table 3 and Figure 2, quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside (chemical formula 3) and quercetin 3-O-(6"-O-malonyl)-β-D-glucoside (chemical formula 4) had significantly higher trolox equivalents than chlorogenic acid, which is known to have strong antioxidant properties. In particular, the trolox equivalent of quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside (chemical formula 3) was 1.41 times that of chlorogenic acid, 1.5 times that of quercetin 3-O-neohesperidin (chemical formula 2), and 1.26 times that of quercetin 3-O-(6"-O-malonyl)-β-D-glucoside (chemical formula 4), demonstrating that it has a significantly higher iron ion reducing ability.

(2)ABTS法:ABTSラジカルに対する還元能
本実施例3(1)において顕著に高い鉄イオン還元能が示されたケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシド(化3)について、ABTSラジカルの消去能を、広く知られた抗酸化物質であるクロロゲン酸(化1)と比較した。ABTS試薬は、7mMの2,2'-azino-bis(3-ethy1benzothiazo1ine-6-su1fonic acid)(ABTS)水溶液と7.35mMのペルオキソ二硫酸カリウム水溶液とを2:1となるように混和し、常温暗所に12~16時間攪拌放置した後、溶液の735nmにおける吸光度が0.7±0.02となるように99.5%(v/v)エタノールで希釈したものを用いた。
(2) ABTS method: Reducing ability for ABTS radicals The ABTS radical scavenging ability of quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside (Chemical formula 3), which showed a remarkably high iron ion reducing ability in Example 3(1), was compared with that of chlorogenic acid (Chemical formula 1), a widely known antioxidant. The ABTS reagent was prepared by mixing a 7 mM aqueous solution of 2,2'-azino-bis(3-ethy1benzothiazo1ine-6-sulfonic acid) (ABTS) with a 7.35 mM aqueous solution of potassium peroxodisulfate in a ratio of 2:1, leaving the mixture in a dark place at room temperature with stirring for 12 to 16 hours, and then diluting the mixture with 99.5% (v/v) ethanol so that the absorbance at 735 nm of the solution became 0.7±0.02.

まず、トロロックスを50%(v/v)エタノールに溶解することにより、濃度37.5、75、150および300μMのトロロックス溶液を調製した。96穴プレートにトロロックス溶液(サンプルa、b)または50%(v/v)エタノール(サンプルc、d)を各30μLずつ分注した。続いて、この96穴プレートに、ABTS試薬(サンプルa、c)または超純水(MilliQ水)(サンプルb、d)を各150μLずつ分注した。室温、暗所で5分間反応させた後、735nmでの吸光度を測定した。続いて、下記の式3によりトロロックス吸光値を算出し、トロロックス溶液の濃度に対してプロットすることにより、検量線を作成した。
式3:トロロックス吸光値= (Ac-Ad) -(Aa-Ab)
First, Trolox solutions with concentrations of 37.5, 75, 150 and 300 μM were prepared by dissolving Trolox in 50% (v/v) ethanol. 30 μL each of Trolox solution (samples a, b) or 50% (v/v) ethanol (samples c, d) was dispensed into a 96-well plate. Then, 150 μL each of ABTS reagent (samples a, c) or ultrapure water (MilliQ water) (samples b, d) was dispensed into the 96-well plate. After reacting at room temperature in the dark for 5 minutes, the absorbance at 735 nm was measured. Next, the Trolox absorbance value was calculated according to the following formula 3, and a calibration curve was created by plotting it against the concentration of the Trolox solution.
Equation 3: Trolox absorbance value = (Ac-Ad) - (Aa-Ab)

実施例1に記載の方法により製造した化1および化3を濃度10mMになるようにジメチルスルホキシドに溶解し、これをDMSO溶液とした。50%(v/v)エタノールを用いてDMSO溶液を希釈することにより、化合物濃度が37.5、75、150および300μMとなるよう調製し、これを化合物溶液とした。トロロックス溶液を化合物溶液に代えて同様の操作を行い、吸光度を測定した。続いて、下記の式4により化合物吸光値を算出し、検量線を用いて化1および化3のトロロックス相当量を算出した。同様の試験を3回行い、トロロックス相当量の平均値および標準偏差を算出した。その結果を表4および図3に示す。
式4:化合物吸光値= Ac-Aa
Chemical formula 1 and Chemical formula 3 prepared by the method described in Example 1 were dissolved in dimethyl sulfoxide to a concentration of 10 mM, and this was used as a DMSO solution. The DMSO solution was diluted with 50% (v/v) ethanol to prepare compound concentrations of 37.5, 75, 150 and 300 μM, and this was used as a compound solution. The same operation was performed using Trolox solution instead of the compound solution, and the absorbance was measured. Subsequently, the compound absorbance was calculated according to the following formula 4, and the Trolox equivalent amount of Chemical formula 1 and Chemical formula 3 was calculated using the calibration curve. The same test was performed three times, and the average value and standard deviation of the Trolox equivalent amount were calculated. The results are shown in Table 4 and Figure 3.
Equation 4: Compound absorbance value = Ac - Aa

表4および図3に示すように、ケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシド(化3)のトロロックス相当量は、クロロゲン酸(化1)と比較して顕著に大きかった。すなわち、ケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシド(化3)は、顕著に高いABTSラジカルの消去能を有することが明らかになった。 As shown in Table 4 and Figure 3, the trolox equivalent amount of quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside (chemical formula 3) was significantly greater than that of chlorogenic acid (chemical formula 1). In other words, it was revealed that quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside (chemical formula 3) has a significantly high ability to scavenge ABTS radicals.

(3)DPPH法:DPPHラジカルに対する還元能
本実施例3(1)において顕著に高い鉄イオン還元能が示されたケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシド(化3)について、DPPHラジカルの消去能を、広く知られた抗酸化物質であるクロロゲン酸(化1)と比較した。DPPH試薬は、2,2-diphenyl-1-picrylhydrazyl(DPPH)を99.5%(v/v)エタノールに0.20mMとなるよう溶解した後、使用時まで4℃の暗所に保管したものを用いた。
(3) DPPH method: Reducing ability of DPPH radical The DPPH radical scavenging ability of quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside (Chemical formula 3), which showed a significantly high iron ion reducing ability in this Example 3(1), was compared with that of chlorogenic acid (Chemical formula 1), a widely known antioxidant. The DPPH reagent used was prepared by dissolving 2,2-diphenyl-1-picrylhydrazyl (DPPH) in 99.5% (v/v) ethanol to a concentration of 0.20 mM and storing the solution in the dark at 4°C until use.

まず、トロロックスを50%(v/v)エタノールに溶解することにより、濃度12.5、25、50および100μMのトロロックス溶液を調製した。96穴プレートにトロロックス溶液(サンプルa、b)または50%(v/v)エタノール(サンプルc、d)を各100μLずつ分注した。続いて、この96穴プレートに、DPPH試薬(サンプルa、c)または99.5%(v/v)エタノール(サンプルb、d)を各100μLずつ分注した。室温、暗所で30分間反応させた後、517nmでの吸光度を測定した。続いて、上記の式3によりトロロックス吸光値を算出し、トロロックス溶液の濃度に対してプロットすることにより、検量線を作成した。 First, Trolox solutions with concentrations of 12.5, 25, 50, and 100 μM were prepared by dissolving Trolox in 50% (v/v) ethanol. 100 μL each of Trolox solution (samples a and b) or 50% (v/v) ethanol (samples c and d) was dispensed into a 96-well plate. Next, 100 μL each of DPPH reagent (samples a and c) or 99.5% (v/v) ethanol (samples b and d) was dispensed into the 96-well plate. After reacting for 30 minutes at room temperature in the dark, the absorbance at 517 nm was measured. Next, the Trolox absorbance value was calculated using the above formula 3, and a calibration curve was created by plotting it against the concentration of the Trolox solution.

実施例1に記載の方法により製造した化1および化3を濃度10mMになるようにジメチルスルホキシドに溶解し、これをDMSO溶液とした。50%(v/v)エタノールを用いてDMSO溶液を希釈することにより、化合物濃度が12.5、25、50および100μMとなるよう調製し、これを化合物溶液とした。トロロックス溶液を化合物溶液に代えて同様の操作を行い、吸光度を測定した。続いて、上記の式4により化合物吸光値を算出し、検量線を用いて化1および化3のトロロックス相当量を算出した。同様の試験を3回行い、トロロックス相当量の平均値および標準偏差を算出した。その結果を表5および図4に示す。
Chemical formula 1 and Chemical formula 3 prepared by the method described in Example 1 were dissolved in dimethyl sulfoxide to a concentration of 10 mM, and this was used as a DMSO solution. The DMSO solution was diluted with 50% (v/v) ethanol to prepare compound concentrations of 12.5, 25, 50 and 100 μM, and this was used as a compound solution. The same operation was performed using Trolox solution instead of the compound solution, and the absorbance was measured. Subsequently, the compound absorbance was calculated according to the above formula 4, and the equivalent amount of Trolox of Chemical formula 1 and Chemical formula 3 was calculated using the calibration curve. The same test was performed three times, and the average value and standard deviation of the equivalent amount of Trolox were calculated. The results are shown in Table 5 and Figure 4.

表5および図4に示すように、ケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシド(化3)のトロロックス相当量は、クロロゲン酸(化1)と比較して顕著に大きかった。すなわち、ケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシド(化3)は、顕著に高いDPPHラジカルの消去能を有することが明らかになった。 As shown in Table 5 and Figure 4, the trolox equivalent amount of quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside (chemical formula 3) was significantly greater than that of chlorogenic acid (chemical formula 1). In other words, it was revealed that quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside (chemical formula 3) has a significantly high ability to scavenge DPPH radicals.

Claims (5)

ケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシドまたはその塩からなる、抗酸化剤。 An antioxidant comprising quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside or a salt thereof. キンセンカ属からケルセチン3-O-(2″-O-α-ラムノシル-6″-O-マロニル)-β-D-グルコシドまたはその塩を抽出する工程を有する、請求項1に記載の抗酸化剤を製造する方法。 2. A method for producing the antioxidant according to claim 1 , comprising the step of extracting quercetin 3-O-(2"-O-α-rhamnosyl-6"-O-malonyl)-β-D-glucoside or a salt thereof from Calendula officinalis. 経口摂取で用いられる医薬品、医薬部外品、健康食品、食品、飲料または飼料に、請求項1に記載の抗酸化剤を配合する工程を有する、生体内で酸化反応の進行を抑制する方法(医療行為を除く)。13. A method for inhibiting the progression of an oxidation reaction in a living body (excluding medical procedures), comprising the step of incorporating the antioxidant according to claim 1 into a medicine, quasi-drug, health food, food, beverage, or feed for oral ingestion. 経皮的に用いられる医薬品、医薬部外品または化粧品に、請求項1に記載の抗酸化剤を配合する工程を有する、皮膚において酸化反応の進行を抑制する方法(医療行為を除く)。A method for inhibiting the progression of an oxidation reaction in the skin (excluding medical procedures), comprising the step of incorporating the antioxidant according to claim 1 into a drug, quasi-drug, or cosmetic for transdermal application. 前記キンセンカ属が、トウキンセンカ(Calendula officinalis)である、請求項に記載の方法。 The method according to claim 2 , wherein the Calendula is Calendula officinalis.
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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Phytochemistry,62,2003年,p.229-237,DOI:10.1016/S0031-9422(02)00486-7
RSC Advances,5,2015年,p.17751-17767,DOI:10.1039/c4ra13155j

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