JP2015027958A - Reaction product of resveratrol - Google Patents
Reaction product of resveratrol Download PDFInfo
- Publication number
- JP2015027958A JP2015027958A JP2013157658A JP2013157658A JP2015027958A JP 2015027958 A JP2015027958 A JP 2015027958A JP 2013157658 A JP2013157658 A JP 2013157658A JP 2013157658 A JP2013157658 A JP 2013157658A JP 2015027958 A JP2015027958 A JP 2015027958A
- Authority
- JP
- Japan
- Prior art keywords
- resveratrol
- novel compound
- pharmaceutically acceptable
- acceptable salt
- present
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
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Images
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- Coloring Foods And Improving Nutritive Qualities (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
Description
本発明は、非常に簡便な方法であり、食品でも応用可能な合成方法によって得られる、抗癌活性を有する新規化合物、該新規化合物を含む抗癌剤、遅筋形成促進剤、持久力向上剤、食品、医薬品及び医薬部外品に関する。また、本発明は、前記新規化合物の製造方法に関する。 The present invention is a very simple method, obtained by a synthesis method applicable to foods, a novel compound having anticancer activity, an anticancer agent containing the novel compound, a slow muscle formation promoter, an endurance improver, a food , Pharmaceuticals and quasi drugs. The present invention also relates to a method for producing the novel compound.
ブドウ果皮に含有されるスチルベン誘導体であるレスベラトロールについて、画期的な研究成果が明らかにされつつある。レスベラトロールは本来ブドウが病原菌から自己を守るファイトアレキシンとして存在する抗菌作用を有する化合物であり、赤系、白系を問わずブドウ果皮に含まれることが知られているが、最近の研究で、レスベラトロールは哺乳動物に対しても有用な効果を有していることが明らかになりつつある。いわゆる「フレンチパラドックス」と言われる赤ワインの有用な生理効果は、レスベラトロールの抗酸化能を始めとして各種の生理活性機能が一因であるとされている。さらに、レスベラトロールは多くの疾病に効果があることが明らかにされつつあり（非特許文献１）、その一つにレスベラトロールは強い抗癌作用を有することが報告されている（非特許文献２）。 Breakthrough research results are being revealed for resveratrol, a stilbene derivative contained in grape skin. Resveratrol is an antibacterial compound that originally exists as a phytoalexin that protects grapes from self-causing bacteria, and is known to be contained in grape skins regardless of whether they are red or white. It is becoming clear that resveratrol has a useful effect on mammals. The useful physiological effect of red wine called so-called “French paradox” is attributed to various physiologically active functions including the antioxidant ability of resveratrol. Furthermore, resveratrol is being clarified to be effective for many diseases (Non-Patent Document 1), and one of them is reported that resveratrol has a strong anticancer activity (Non-patent Document 1). Reference 2).
またレスベラトロールには運動機能や筋肉のエネルギー代謝に有用な効果を示すことが報告されている。筋肉組織は体内最大のエネルギー消費器官であるため、そのエネルギー代謝を亢進することは、肥満症、糖尿病、循環器系疾患の予防や治療に効果がある。そのため予防医学において筋肉のエネルギー代謝を向上させるために推奨されるのが有酸素運動であるが、その効果をより高めるためには遅筋とよばれるタイプの筋肉を発達させることが重要である。遅筋は、脂肪を主なエネルギー源とするタイプの筋肉であり、持久力に富む筋肉である。レスベラトロールにはレスベラトロールを有効成分とする持久力向上剤（特許文献１）、レスベラトロールを有効成分とするミトコンドリア生合成増強剤（特許文献２）が先行技術として報告されていることから、筋肉の持久力やエネルギー代謝に対して有用な効果を有するものとして期待されている。 Resveratrol has been reported to have useful effects on motor function and muscle energy metabolism. Since muscle tissue is the body's largest energy consuming organ, enhancing its energy metabolism is effective in preventing and treating obesity, diabetes and cardiovascular diseases. Therefore, aerobic exercise is recommended in order to improve muscle energy metabolism in preventive medicine, but it is important to develop a type of muscle called slow muscle in order to increase the effect. The slow muscle is a type of muscle that uses fat as a main energy source, and is a muscle that is rich in endurance. Resveratrol has been reported in the prior art as an endurance improver (Patent Document 1) containing resveratrol as an active ingredient and a mitochondrial biosynthesis enhancer (Patent Document 2) containing resveratrol as an active ingredient. Therefore, it is expected to have a useful effect on muscle endurance and energy metabolism.
しかしながら、レスベラトロールを含有する食可能な植物としてはブドウや落花生等が知られているがその数は限られている。また、ブドウ中でも特にブドウ果皮に多く含まれているが、それでもその含有量は極めて低く、５０〜１００μｇ／ｇ程度といわれている。 However, grapes and peanuts are known as edible plants containing resveratrol, but the number is limited. Moreover, although it is contained a lot in grape skin especially in grapes, the content is still very low, and is said to be about 50 to 100 μg / g.
そこで、食品中のレスベラトロール濃度を高める取り組みもされており、紫外線照射によりレスベラトロール濃度を高めた後にレスベラトロール含有抽出物を得て、その抽出物を食品に添加した食品が提案されている（特許文献３）。また、レスベラトロールの腸管吸収効率を高めるための技術として、腸管吸収促進剤の提案もなされている（特許文献４）。このようにレスベラトロールは、抗癌作用、抗菌作用、抗酸化作用、色調保持作用、皮膚老化防止作用、口腔疾患治癒作用等の様々な有用な性質を有する化合物であるが、希少成分であるが故に、高コストな商品となってしまい、サプリメント等で販売はされているものの今現在では十分に社会に浸透しているとは言いがたい。 Therefore, efforts are also being made to increase the resveratrol concentration in foods, and after increasing the resveratrol concentration by ultraviolet irradiation, a resveratrol-containing extract was obtained, and a food in which the extract was added to the food was proposed. (Patent Document 3). In addition, as a technique for increasing the intestinal absorption efficiency of resveratrol, an intestinal absorption promoter has been proposed (Patent Document 4). Thus, resveratrol is a compound having various useful properties such as anticancer action, antibacterial action, antioxidant action, color tone retention action, skin aging prevention action, oral disease healing action, etc., but it is a rare component. Therefore, although it becomes a high-cost product and it is sold as a supplement, it is hard to say that it is sufficiently infiltrated into society now.
また、前記レスベラトロールの誘導体として、天然にはレスベラトロールの重合体、例えばε−ビニフェリン（二量体）、α−ビニフェリン（三量体）、バチカノールＣ（四量体）等が報告されているが、いずれもレスベラトロールと同様に天然の希少成分であり、現在のところ十分量を供給することは困難である。 In addition, as a derivative of resveratrol, a resveratrol polymer such as ε-viniferin (dimer), α-viniferin (trimer), vaticanol C (tetramer) and the like have been reported in nature. However, both are natural rare components like resveratrol, and it is difficult to supply a sufficient amount at present.
そこで、医薬品分野では、化学合成によりレスベラトロールの作用機序を参考にした新規化合物を作製する試みもある（非特許文献３）が、これらは主に医薬品としての開発対象物であり、安全性の面から未だに多くの課題が残されている。
また、非天然型のレスベラトロール誘導体についての報告もある（特許文献５）。
Therefore, in the pharmaceutical field, there is an attempt to produce a new compound by referring to the action mechanism of resveratrol by chemical synthesis (Non-patent Document 3), but these are mainly development objects as pharmaceuticals and are safe. Many challenges still remain from the sexual aspect.
There is also a report on a non-natural type resveratrol derivative (Patent Document 5).
このように、レスベラトロールは食経験の豊かな天然物質であり、また、生体調節機能に優れた安全な化合物でもあることから、前記のように原料やリード化合物としてのこれらを効率的に製造する技術開示もなされている。 In this way, resveratrol is a natural substance with a rich dietary experience and is a safe compound with excellent bioregulatory functions. Technical disclosure has also been made.
本発明者らもこれまでに、レスベラトロール重合化合物を報告している（特許文献６）。
しかしながら、本発明のレスベラトロールの反応生成物は確認されておらず、他にも報告した例は見られない。
The present inventors have also reported the resveratrol polymerization compound so far (patent document 6).
However, the reaction product of resveratrol of the present invention has not been confirmed, and no other reported examples are found.
また、厚生労働省の調べによると、平成２０年の日本人の死亡原因の３０％が悪性新生物つまり癌である。現在の抗癌剤の研究では、日常的に摂取できる天然物由来の化合物としては、ケルセチンをはじめとするフラボノイド類等が知られている（非特許文献４）。 According to a survey by the Ministry of Health, Labor and Welfare, 30% of Japanese deaths in 2008 are malignant neoplasms or cancer. In current research on anticancer agents, flavonoids such as quercetin are known as compounds derived from natural products that can be ingested on a daily basis (Non-Patent Document 4).
従って、有効性の認められている素材をさらに高機能化し、現実的に使用可能な素材を開発することが強く求められている。 Therefore, there is a strong demand to develop materials that can be used practically by further enhancing the functionality of materials that have been recognized as being effective.
本発明者らは、レスベラトロールに関する前記の状況を鑑みて、新規な生理活性又は強力な生理活性を有するレスベラトロール誘導体の探索と、その製造方法を確立すべく鋭意検討した結果、意外にもレスベラトロールを塩基性条件下で加熱するという簡便且つ安全な方法により、レスベラトロールと比べて優れた抗癌活性や遅筋形成促進作用を有する新規化合物を製造することに成功し、本発明を完成するに至った。 In view of the above-mentioned situation regarding resveratrol, the present inventors have surprisingly studied to search for a novel or active resveratrol derivative having a strong physiological activity and to establish a production method thereof. Succeeded in producing a novel compound with superior anticancer activity and slow muscle formation promoting action compared to resveratrol by a simple and safe method of heating resveratrol under basic conditions. The invention has been completed.
したがって、本発明は、レスベラトロールよりも優れた抗癌活性や遅筋形成促進作用を有する新規化合物を提供し、さらに該新規化合物を効率よく、安全に製造する方法を提供することを目的とする。
また、本発明は、前記新規化合物を含有することを特徴とする抗癌剤、遅筋形成促進剤、持久力向上剤、さらには、食品、医薬品及び医薬部外品を提供することを目的とする。
Accordingly, an object of the present invention is to provide a novel compound having an anticancer activity superior to resveratrol and a slow muscle formation promoting action, and further to provide a method for producing the novel compound efficiently and safely. To do.
Another object of the present invention is to provide an anticancer agent, a slow muscle formation promoter, an endurance improver characterized by containing the above-mentioned novel compound, as well as foods, pharmaceuticals and quasi drugs.
本発明の要旨は、
〔１〕レスベラトロールの反応生成物であって、式（１）：
The gist of the present invention is as follows:
[1] A reaction product of resveratrol having the formula (1):
で表される新規化合物又はその薬学的に許容可能な塩、
〔２〕前記〔１〕記載の新規化合物又はその薬学的に許容可能な塩を含有する抗癌剤、
〔３〕前記〔１〕記載の新規化合物又はその薬学的に許容可能な塩を含有する遅筋形成促進剤、
〔４〕前記〔１〕記載の新規化合物又はその薬学的に許容可能な塩を含有する持久力向上剤、
〔５〕前記〔１〕記載の新規化合物又はその薬学的に許容可能な塩を含有する食品、医薬品又は医薬部外品、
〔６〕レスベラトロールを塩基性条件下で加熱することにより、目的の化合物を生成することを特徴とする前記〔１〕記載の新規化合物又はその薬学的に許容可能な塩の製造方法、
に関する。
Or a pharmaceutically acceptable salt thereof,
[2] An anticancer agent comprising the novel compound according to [1] or a pharmaceutically acceptable salt thereof,
[3] A slow muscle formation promoter containing the novel compound according to [1] or a pharmaceutically acceptable salt thereof,
[4] Endurance improver containing the novel compound or the pharmaceutically acceptable salt thereof according to [1],
[5] A food, pharmaceutical or quasi drug containing the novel compound or the pharmaceutically acceptable salt thereof according to [1],
[6] A method for producing a novel compound or a pharmaceutically acceptable salt thereof according to the above [1], wherein the target compound is produced by heating resveratrol under basic conditions,
About.
本発明である前記式（１）で表される新規化合物又はその薬学的に許容可能な塩（以下、「新規化合物」という。）は、レスベラトロールと比べて、抗癌活性に優れていることから、新規な抗癌剤として有用である。またレスベラトロールに比べて、遅筋形成促進作用に優れており、新規な持久力向上剤として有用である。
また、本発明の新規化合物は、前記のような生理活性に優れることから、食品、医薬品及び医薬部外品に配合することで、抗癌活性、遅筋形成促進作用に優れた食品、医薬品及び医薬部外品を提供することができる。
The novel compound represented by the formula (1) or a pharmaceutically acceptable salt thereof (hereinafter referred to as “novel compound”) according to the present invention is superior in anticancer activity compared to resveratrol. Therefore, it is useful as a novel anticancer agent. Moreover, compared with resveratrol, it is excellent in the action of promoting slow muscle formation and is useful as a novel endurance improver.
In addition, since the novel compound of the present invention is excellent in the physiological activity as described above, it is incorporated into foods, pharmaceuticals and quasi drugs, thereby providing foods, pharmaceuticals and anti-cancer activities and slow muscle formation promoting effects. Quasi-drugs can be provided.
以下、本発明について詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明の新規化合物は、レスベラトロールの反応生成物であって、式（１）： The novel compound of the present invention is a reaction product of resveratrol having the formula (1):
で示される新規化合物又はその薬学的に許容可能な塩である。 Or a pharmaceutically acceptable salt thereof.
前記新規化合物の薬学的に許容可能な塩としては、例えば、リチウム塩、ナトリウム塩、カリウム塩等のアルカリ金属塩；マグネシウム塩、カルシウム塩、バリウム塩等のアルカリ土類金属塩；アルミニウム塩；アルミニウムヒドロキシド塩等の金属ヒドロキシド塩；アルキルアミン塩、ジアルキルアミン塩、トリアルキルアミン塩、アルキレンジアミン塩、シクロアルキルアミン塩、アリールアミン塩、アラルキルアミン塩、複素環式アミン塩等のアミン塩；α−アミノ酸塩、ω−アミノ酸塩等のアミノ酸塩；ペプチド塩又はそれらから誘導される第１級、第２級、第３級若しくは第４級アミン塩等が挙げられる。これらの薬学的に許容可能な塩は、単独で又は２種以上を混合して用いることができる。 Examples of the pharmaceutically acceptable salt of the novel compound include alkali metal salts such as lithium salt, sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt, calcium salt and barium salt; aluminum salt; aluminum Metal hydroxide salts such as hydroxide salts; amine salts such as alkylamine salts, dialkylamine salts, trialkylamine salts, alkylenediamine salts, cycloalkylamine salts, arylamine salts, aralkylamine salts, heterocyclic amine salts; Amino acid salts such as α-amino acid salts and ω-amino acid salts; peptide salts or primary, secondary, tertiary, or quaternary amine salts derived from them. These pharmaceutically acceptable salts can be used alone or in admixture of two or more.
本発明の新規化合物は、当該分野で周知の方法に従って化学合成することも可能ではあるが、反応工程が複雑となり、人体に有害な試薬、触媒、溶媒等を必要とする。また、化学合成では不純物を除去する煩雑さもあり、さらに安全性の観点から、前記新規化合物の精製を徹底する必要もあり、製造コストの面で工業的には不向きな方法である。 Although the novel compound of the present invention can be chemically synthesized according to a method well known in the art, the reaction process is complicated, and reagents, catalysts, solvents, etc. harmful to the human body are required. In addition, chemical synthesis involves the complexity of removing impurities, and from the viewpoint of safety, it is necessary to thoroughly purify the novel compound, which is an industrially unsuitable method in terms of production cost.
そこで、本発明者らは、鋭意検討した結果、レスベラトロールを塩基性条件下で加熱することで、前記の化学合成法のように有害な試薬等や煩雑な工程を必要とせずに、本発明の新規化合物を効率的かつ安全に製造することができることを見出した。以下に、本発明の新規化合物の製造方法（以下、「本発明の製造方法」という）について具体的に説明する。 Therefore, as a result of intensive studies, the present inventors have heated resveratrol under basic conditions, so that the present invention does not require harmful reagents and complicated steps as in the chemical synthesis method described above. It has been found that the novel compounds of the invention can be produced efficiently and safely. Below, the manufacturing method (henceforth "the manufacturing method of this invention") of the novel compound of this invention is demonstrated concretely.
本発明の製造方法では、前駆体としてレスベラトロールを用いる。レスベラトロールにはトランス体とシス体の構造異性体が存在するが、加熱や紫外線によってトランス体とシス体の変換が一部生じる。したがって、レスベラトロールとしては、トランス体でもシス体でも、あるいはトランス体とシス体の混合物であってもよい。レスベラトロールは、ブドウ果皮・茎・葉、ピーナッツの渋皮、メリンジョ、イタドリ、リンゴンベリー等の原料から抽出・精製した天然由来のものであっても、化学合成された純度の高い化成品であってもよい。天然由来のレスベラトロールを用いる場合は、完全に精製されたものである必要はなく、レスベラトロール以外の成分を含む混合物も使用できる。また、レスベラトロールには、塩等も含まれる。
ただし、本発明の新規化合物の回収率の観点からは、レスベラトロール換算で５重量％以上含有された混合物が原料として望ましい。
前記レスベラトロールとしては、ブドウ果皮・茎・葉、ピーナッツの渋皮、メリンジョ、イタドリ、リンゴンベリー等の原料からの抽出物、凍結乾燥品等を使用してもよい
In the production method of the present invention, resveratrol is used as a precursor. Resveratrol has structural isomers of trans form and cis form, but some conversion of trans form and cis form occurs by heating or ultraviolet rays. Therefore, resveratrol may be a trans isomer, a cis isomer, or a mixture of a trans isomer and a cis isomer. Resveratrol is a chemically synthesized, high-purity chemical product, even if it is naturally derived and extracted from raw materials such as grape skins / stems / leaves, peanut astringent skin, meringo, itadori and lingonberry. May be. When natural resveratrol is used, it does not have to be completely purified, and a mixture containing components other than resveratrol can also be used. Resveratrol also includes salts and the like.
However, from the viewpoint of the recovery rate of the novel compound of the present invention, a mixture containing 5% by weight or more in terms of resveratrol is desirable as a raw material.
As the resveratrol, grape skins / stems / leaves, peanut astringent skin, extracts from raw materials such as meringo, itadori, lingonberry, freeze-dried products, etc. may be used.
本発明の製造方法では、レスベラトロールを適切な溶媒に溶解させる。この際、溶媒が水のみであれば、レスベラトロールの水への溶解度が著しく低いために、水と有機溶媒の混合液や、有機溶媒のみに溶解させればよい。水と有機溶媒の配合比や、有機溶媒の種類については特に制限はなく、レスベラトロールが十分に溶解すればよい。中でも、メタノールやエタノールのみの溶媒、水とメタノール、水とエタノール等の混合液を使用することが、安全性やコスト面から好ましい。特に、本発明の新規化合物を含む反応後の組成物を、十分な精製をせずに食品等の原料として使用する場合には、安全性や法規面から溶媒としてエタノールや含水エタノールを使用することが望ましい。 In the production method of the present invention, resveratrol is dissolved in a suitable solvent. At this time, if the solvent is only water, the solubility of resveratrol in water is remarkably low, and therefore, it may be dissolved in a mixed solution of water and an organic solvent or only in an organic solvent. There is no restriction | limiting in particular about the compounding ratio of water and an organic solvent, and the kind of organic solvent, Resveratrol should just fully melt | dissolve. Among them, it is preferable from the viewpoint of safety and cost to use a solvent only of methanol or ethanol, or a mixed solution of water and methanol, water and ethanol, or the like. In particular, when using the post-reaction composition containing the novel compound of the present invention as a raw material for foods without sufficient purification, ethanol or hydrous ethanol should be used as a solvent for safety and legal purposes. Is desirable.
得られるレスベラトロールを含有する溶液中のレスベラトロールの濃度については特に制限はないが、レスベラトロールの濃度が高いほど溶媒使用量が少ない等のメリットがあるため、レスベラトロールの濃度は溶媒に対しレスベラトロールが飽和する濃度近くが好ましい。 The concentration of resveratrol in the resulting solution containing resveratrol is not particularly limited, but the higher the resveratrol concentration, the lower the amount of solvent used. Near the concentration at which resveratrol is saturated with respect to the solvent is preferred.
次に、前記レスベラトロールを含有する溶液（以下、レスベラトロール含有溶液）を塩基性に調整する。具体的には、レスべラトロール含有溶液のｐＨを８以上に調整することが好ましい。調整方法として、例えば、レスベラトロール含有溶液を調製した後にｐＨ調整剤を添加してｐＨを調整してもよいし、前記レスべラトロール含有溶液の調製時に前もって溶媒のｐＨを調整しておいてもよい。レスベラトロール含有溶液の反応開始時のｐＨは１３．０以上であれば、他の反応や目的化合物の分解も一方で生じるために最終的な本発明の新規化合物の回収量が低下する傾向がある。したがって、本発明の新規化合物の回収量の観点から、反応開始時のレスべラトロール含有溶液のｐＨは８以上１３未満が望ましい。 Next, the solution containing resveratrol (hereinafter referred to as resveratrol-containing solution) is adjusted to be basic. Specifically, it is preferable to adjust the pH of the resveratrol-containing solution to 8 or more. As an adjustment method, for example, after preparing a resveratrol-containing solution, a pH adjuster may be added to adjust the pH, or the pH of the solvent is adjusted in advance when preparing the resveratrol-containing solution. Also good. If the pH at the start of the reaction of the resveratrol-containing solution is 13.0 or more, other reactions and decomposition of the target compound also occur on the other hand, so that the final recovered amount of the novel compound of the present invention tends to decrease. is there. Therefore, from the viewpoint of the recovered amount of the novel compound of the present invention, the pH of the resveratrol-containing solution at the start of the reaction is preferably 8 or more and less than 13.
前記ｐＨ調整剤としては、特に制限はないが、安全性、効率及びコスト面からは、水酸化ナトリウム、水酸化カリウム、炭酸水素ナトリウムが望ましい。また、必ずしも必要な手法ではないが、反応時のｐＨ変化を極力抑える場合が生じた際には、緩衝溶液を用いてもよい。 The pH adjuster is not particularly limited, but sodium hydroxide, potassium hydroxide, and sodium hydrogen carbonate are desirable from the viewpoint of safety, efficiency, and cost. In addition, although not necessarily a necessary technique, a buffer solution may be used when a case where the pH change during the reaction is suppressed as much as possible occurs.
次に、前記のように塩基性条件にしたレスベラトロール含有溶液を加熱処理する。この加熱処理により、本発明の新規化合物の生成反応を行う。生成反応を効率的に進ませるために、レスベラトロール含有溶液の加熱温度は１１０℃以上に調整することが好ましい。例えば、開放容器にレスベラトロール含有溶液を入れ、溶媒の沸点を超える高温で前記容器を加熱する、密閉容器にレスベラトロール含有溶液を入れて前記容器を加熱する、レトルト装置、オートクレーブ、超臨界装置、プレッシャークッカー等を用いて加圧加熱する等、少なくとも部分的に溶液温度が１１０℃以上に達するように加熱することが好ましい。また、使用する溶媒の沸点から考え、加圧加熱が望ましい。回収効率面から、溶液温度が均一に１１０℃〜１５０℃になることが、さらに好ましい。加熱時間も加熱温度と同様に限られたものではなく、効率的に目的の反応が進行する時間条件とすればよい。特に、加熱時間は加熱温度との兼ね合いによるものであり、加熱温度に応じた加熱時間にすることが望ましい。例えば、１３０℃付近に加熱する場合は、５分〜３６０分の加熱時間が望ましい。特に、生成の効率からすると、なるべく長めの加熱が好ましく、例えば、１１０℃〜１５０℃の加熱を行う場合６０分を超える加熱を行うことがより好ましい。また、加熱は、一度でもよいし、複数回に分けて繰り返し加熱してもよい。複数回に分けて加熱する場合、蒸発した溶媒を補うために溶媒を新たに追加して行うことでより効率よく本発明の新規化合物が生成できるので好ましい。 Next, the resveratrol containing solution made into basic conditions as mentioned above is heat-processed. By this heat treatment, the formation reaction of the novel compound of the present invention is carried out. In order to advance the production reaction efficiently, the heating temperature of the resveratrol-containing solution is preferably adjusted to 110 ° C. or higher. For example, a resveratrol-containing solution is put in an open container and the container is heated at a high temperature exceeding the boiling point of the solvent. A resveratrol-containing solution is put in a sealed container and the container is heated, a retort apparatus, an autoclave, supercritical It is preferable to heat the solution so that the solution temperature reaches 110 ° C. or higher, for example, by heating under pressure using an apparatus, a pressure cooker, or the like. Also, considering the boiling point of the solvent used, pressure heating is desirable. From the viewpoint of recovery efficiency, it is more preferable that the solution temperature be 110 ° C. to 150 ° C. uniformly. The heating time is not limited as in the case of the heating temperature, and may be a time condition in which the target reaction efficiently proceeds. In particular, the heating time depends on the heating temperature, and it is desirable to set the heating time according to the heating temperature. For example, when heating near 130 ° C., a heating time of 5 minutes to 360 minutes is desirable. In particular, from the viewpoint of production efficiency, heating as long as possible is preferable. For example, when heating at 110 ° C. to 150 ° C. is performed, it is more preferable to perform heating exceeding 60 minutes. Further, the heating may be performed once or may be repeated repeatedly in a plurality of times. In the case of heating in a plurality of times, the addition of a new solvent to supplement the evaporated solvent is preferable because the novel compound of the present invention can be produced more efficiently.
前記加熱処理による本発明の新規化合物の生成反応の終了は、例えば、ＨＰＬＣによる成分分析により本発明の新規化合物の生成量を確認して判断すればよい。 The completion of the production reaction of the novel compound of the present invention by the heat treatment may be judged by, for example, confirming the production amount of the novel compound of the present invention by component analysis by HPLC.
得られる反応溶液中には、生成した本発明の新規化合物が含有されている。
また、安全な原料のみを用いた工程で本発明の新規化合物を製造した場合には、本発明の新規化合物を含む混合物の状態で食品、医薬品又は医薬部外品に使用することが可能である。例えば、天然由来のレスベラトロールを含水エタノール溶媒に溶解し、炭酸水素ナトリウム等を添加してｐＨを調整し、加熱処理した場合には、得られる反応溶液を食品、医薬品又は医薬部外品の原料の一つとして使用することが可能である。
The resulting reaction solution contains the produced novel compound of the present invention.
In addition, when the novel compound of the present invention is produced by a process using only safe raw materials, it can be used in foods, pharmaceuticals or quasi drugs in the state of a mixture containing the novel compound of the present invention. . For example, when resveratrol derived from nature is dissolved in a water-containing ethanol solvent, the pH is adjusted by adding sodium hydrogencarbonate or the like, and the resulting reaction solution is treated with a food, drug or quasi drug. It can be used as one of the raw materials.
また、風味面での改良やさらなる高機能化を望む場合は、前記反応溶液を濃縮して本発明の新規化合物の濃度を高める、あるいは前記反応溶液を精製し本発明の新規化合物の純品を得ることができる。濃縮、精製は、公知の方法で実施可能である。例えば、クロロホルム、酢酸エチル、エタノール、メタノール等を用いた溶媒抽出法や炭酸ガスによる超臨界抽出法等で抽出して本発明の新規化合物を濃縮できる。また、カラムクロマトグラフィーを利用して濃縮や精製を施すことや、再結晶法や限外ろ過膜等の膜処理法も適用可能である。 In addition, if it is desired to improve the flavor and further enhance the functionality, the reaction solution is concentrated to increase the concentration of the novel compound of the present invention, or the reaction solution is purified to obtain a pure product of the novel compound of the present invention. Can be obtained. Concentration and purification can be performed by a known method. For example, the novel compound of the present invention can be concentrated by extraction with a solvent extraction method using chloroform, ethyl acetate, ethanol, methanol or the like, a supercritical extraction method with carbon dioxide gas, or the like. Further, concentration and purification using column chromatography, membrane treatment methods such as a recrystallization method and an ultrafiltration membrane can be applied.
また、前記反応溶液から本発明の新規化合物を分離して回収する場合には、カラムクロマトグラフィー、ＨＰＬＣ等を用いてもよい。 In addition, when the novel compound of the present invention is separated and recovered from the reaction solution, column chromatography, HPLC or the like may be used.
前記濃縮物や精製物を、必要に応じて、減圧乾燥や凍結乾燥して溶媒除去することで、粉末状の本発明の新規化合物を得ることができる。 If necessary, the concentrated or purified product can be dried under reduced pressure or lyophilized to remove the solvent, whereby the powdered novel compound of the present invention can be obtained.
以上のようにして得られる本発明の新規化合物は、レスベラトロールに比べて、優れた抗癌活性を有する。したがって、本発明の新規化合物を有効成分として含有する抗癌剤を提供することができる。またレスベラトロールに比べて、優れた遅筋形成促進作用を有する。したがって、本発明の新規化合物を有効成分として含有する遅筋形成促進剤及び持久力向上剤を提供することができる。 The novel compound of the present invention obtained as described above has an excellent anticancer activity compared to resveratrol. Therefore, an anticancer agent containing the novel compound of the present invention as an active ingredient can be provided. Moreover, it has a superior slow muscle formation promoting action compared to resveratrol. Therefore, the slow muscle formation promoter and endurance improver which contain the novel compound of this invention as an active ingredient can be provided.
特に、本発明の新規化合物の生理活性分野を考慮すると、癌予防・治療等の健康増進を目的とした抗癌剤や、遅筋形成促進剤、持久力の向上などを目的とした持久力向上剤として用いることが好ましい。 In particular, considering the field of physiological activity of the novel compound of the present invention, as an anticancer agent for the purpose of promoting health such as cancer prevention and treatment, a slow muscle formation promoter, an endurance improver for the purpose of improving endurance, etc. It is preferable to use it.
なお、本発明の新規化合物が持つさらなる効果効能は、得られた生理活性データより類推できる範囲で使用できる。 In addition, the further effect efficacy which the novel compound of this invention has can be used in the range which can be estimated from the obtained bioactivity data.
本発明の新規化合物の原料であるレスベラトロールは、その重合体も含めて食経験豊かな化合物であり、安全性にも優れるといえるため、本発明の新規化合物の安全性も同様に優れたものであると考えられる。
したがって、本発明の新規化合物は、食品、医薬品、医薬部外品等に配合して使用することができる。このような食品、医薬品、医薬部外品は、抗癌作用を有する食品、医薬品、医薬部外品となる。特に、抗癌作用が知られていない材料のみからなる抗癌作用のない食品、医薬品、医薬部外品でも、本発明の新規化合物を配合することで、簡単に抗癌作用を付与することができる。
Resveratrol, which is a raw material for the novel compound of the present invention, is a compound with abundant experience including its polymer, and it can be said that it is also excellent in safety. Therefore, the safety of the novel compound of the present invention is also excellent. It is thought to be a thing.
Therefore, the novel compound of the present invention can be used by blending with foods, pharmaceuticals, quasi drugs and the like. Such foods, pharmaceuticals, and quasi drugs are foods, pharmaceuticals, and quasi drugs that have an anticancer effect. In particular, foods, pharmaceuticals, and quasi-drugs consisting only of materials whose anti-cancer activity is not known can be easily imparted with anti-cancer activity by blending the novel compound of the present invention. it can.
前記食品としては、例えば、飲料、アルコール飲料、ゼリー、菓子等、どのような形態でもよく、菓子類の中でも、その容量等から保存や携帯性に優れた、ハードキャンディ、ソフトキャンディ、グミキャンディ、タブレット等が挙げられるが、特に限定はない。例えば、本発明の新規化合物をワインに添加することで、ワインの健康機能効果をさらに増強した新規なワインとすることもできる。この新規なワインのように、嗜好性と健康機能効果の双方を兼ね備えた飲食品は、社会ニーズの非常に高い分野の飲食品であり、この社会ニーズに十分応えることが可能である。また、本発明の新規化合物は、優れた抗癌活性を有することから、癌に対する予防を目的に、容易に摂取できるキャンディー、グミキャンディ、タブレット等にすることができる。なお、食品には、機能性食品、健康食品、健康志向食品等も含まれる。 As the food, for example, any form such as beverage, alcoholic beverage, jelly, confectionery, etc., among confectionery, hard candy, soft candy, gummy candy, which is excellent in storage and portability due to its capacity, etc. A tablet or the like is included, but there is no particular limitation. For example, by adding the novel compound of the present invention to wine, it is possible to obtain a new wine that further enhances the health function effect of the wine. Like this new wine, foods and drinks that have both taste and health function effects are foods and drinks in fields with very high social needs, and can fully meet these social needs. Moreover, since the novel compound of the present invention has an excellent anticancer activity, it can be made into a candy, gummy candy, tablet or the like that can be easily taken for the purpose of preventing cancer. The food includes functional food, health food, health-oriented food, and the like.
また、前記食品には、ヒトが食べる食品だけでなく、例えば、非ヒト動物、例えば、ラット、マウス、モルモット、ウサギ、ヒツジ、ブタ、ウシ、ウマ、ネコ、イヌ、サル、チンパンジー等の哺乳類、鳥類、両生類、爬虫類等の治療剤又は飼料に配合してもよい。飼料としては、例えばヒツジ、ブタ、ウシ、ウマ、ニワトリ等に用いる家畜用飼料、ウサギ、ラット、マウス等に用いる小動物用飼料、ウナギ、タイ、ハマチ、エビ等に用いる魚介類用飼料、イヌ、ネコ、小鳥、リス等に用いるペットフードが挙げられる。 In addition, the food includes not only foods eaten by humans, but also non-human animals such as rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, and other mammals, You may mix | blend with therapeutic agents or feed, such as birds, amphibians, and reptiles. As feed, for example, livestock feed used for sheep, pigs, cattle, horses, chickens, etc., feed for small animals used for rabbits, rats, mice, etc. The pet food used for a cat, a small bird, a squirrel, etc. is mentioned.
前記医薬品としては、散剤、錠剤、丸剤、カプセル剤、顆粒剤等の固形製剤、懸濁剤、乳剤等の液剤、ゲル剤等が挙げられる。錠剤、丸剤、顆粒剤、顆粒を含有するカプセル剤等の顆粒は、必要により、ショ糖等の糖類、マルチトール等の糖アルコールで糖衣を施したり、ゼラチン、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース等でコーティングを施してもよいし、胃溶性若しくは腸溶性物質のフィルムで被覆してもよい。また、製剤の溶解性を向上させるために、前記の製剤に公知の可溶化処理を施すこともできる。常法に基づいて、前記液剤を注射剤又は点滴剤に配合して使用してもよい。 Examples of the pharmaceuticals include solid preparations such as powders, tablets, pills, capsules and granules, liquids such as suspensions and emulsions, and gels. If necessary, granules such as tablets, pills, granules, capsules containing granules may be sugar-coated with sugars such as sucrose, sugar alcohols such as maltitol, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, etc. It may be coated with a film of gastric or enteric material. Moreover, in order to improve the solubility of a formulation, the said formulation can also be given a known solubilization process. Based on a conventional method, the liquid preparation may be used in an injection or infusion.
医薬部外品としては、口腔に用いられる医薬部外品、例えば、歯磨き、マウスウォッシュ、マウスリンス、ドリンク剤が挙げられる。 Examples of quasi-drugs include quasi-drugs used in the oral cavity, such as toothpaste, mouthwash, mouth rinse, and drink.
本発明の新規化合物を用いて食品、医薬品又は医薬部外品を調製する場合、本発明の効果が損なわれない範囲内で食品、医薬品又は医薬部外品に通常用いられる成分を適宜任意に配合することができる。
例えば、食品の場合には、水、アルコール、澱粉質、蛋白質、繊維質、糖質、脂質、ビタミン、ミネラル、着香料、着色料、甘味料、調味料、安定剤、防腐剤のような食品に通常配合される原料又は素材と組み合わせることができる。
医薬品や医薬部外品の場合には、主剤、基材、界面活性剤、起泡剤、湿潤剤、増粘剤、透明剤、着香料、着色料、安定剤、防腐剤、殺菌剤等に組み合わせ、常法に基づいて、液状、軟膏状又はスプレー噴射可能な最終形態等にすることができる。
When preparing foods, pharmaceuticals or quasi-drugs using the novel compounds of the present invention, the ingredients normally used in foods, pharmaceuticals or quasi-drugs are arbitrarily combined arbitrarily within a range that does not impair the effects of the present invention. can do.
For example, in the case of food, food such as water, alcohol, starch, protein, fiber, carbohydrate, lipid, vitamin, mineral, flavoring, coloring, sweetener, seasoning, stabilizer, preservative Can be combined with raw materials or materials usually blended in
In the case of pharmaceuticals and quasi-drugs, the main ingredients, base materials, surfactants, foaming agents, wetting agents, thickeners, clearing agents, flavoring agents, coloring agents, stabilizers, preservatives, bactericides, etc. Based on a combination and a conventional method, it can be made into a liquid, ointment-like or sprayable final form.
また、本発明の新規化合物を食品に添加する場合には、該食品中に、通常は０．００１〜２０重量％添加することが好ましい。 Moreover, when adding the novel compound of this invention to a foodstuff, it is usually preferable to add 0.001-20 weight% in this foodstuff.
本発明の新規化合物を医薬用途で使用する場合、例えば、その摂取量は、所望の改善、治療又は予防効果が得られるような量であれば特に制限されず、通常、薬剤の態様、患者の年齢、性別、体質その他の条件、疾患の種類並びにその程度等に応じて適宜選択される。１日当たり約０．１ｍｇ〜１，０００ｍｇ程度とするのがよく、これを１日に１〜４回に分けて摂取することができる。 When the novel compound of the present invention is used for pharmaceutical purposes, for example, the amount of intake thereof is not particularly limited as long as the desired improvement, treatment or prevention effect can be obtained. It is appropriately selected according to age, sex, constitution and other conditions, the type and degree of disease. About 0.1 mg to about 1,000 mg per day is preferable, and this can be taken in 1 to 4 times a day.
本発明の新規化合物を医薬部外品に添加する場合には、該医薬部外品中に、通常０．００１〜３０重量％添加するのが好ましい。 When the novel compound of the present invention is added to a quasi drug, it is usually preferable to add 0.001 to 30% by weight in the quasi drug.
次に、本発明を実施例に基づいて詳細に説明するが、本発明はかかる実施例にのみ限定されるものではない。 EXAMPLES Next, although this invention is demonstrated in detail based on an Example, this invention is not limited only to this Example.
（実施例１：レスベラトロールの反応生成方法の検討）
トランス−レスベラトロール（東京化成工業（株）製）１ｇをエタノール２０ｍＬに溶解し、５％炭酸水素ナトリウム水溶液を２０ｍＬ加えて、レスベラトロール含有溶液（ｐＨ９．９）を調製した。このレスベラトロール含有溶液をオートクレーブ（三洋電機（株）製、「ＳＡＮＹＯ ＬＡＢＯ ＡＵＴＯＣＬＡＶＥ」）にて１３０℃、９０分間加熱した。得られた反応溶液のうち１ｍＬを取り出して、メタノールにて５０ｍＬにメスアップし、このうちの１０μＬをＨＰＬＣにより分析した。
(Example 1: Examination of reaction production method of resveratrol)
1 g of trans-resveratrol (manufactured by Tokyo Chemical Industry Co., Ltd.) was dissolved in 20 mL of ethanol, and 20 mL of 5% aqueous sodium hydrogen carbonate solution was added to prepare a resveratrol-containing solution (pH 9.9). This resveratrol-containing solution was heated in an autoclave (manufactured by Sanyo Electric Co., Ltd., “SANYO LABO AUTOCLAVE”) at 130 ° C. for 90 minutes. 1 mL of the obtained reaction solution was taken out, made up to 50 mL with methanol, and 10 μL of this was analyzed by HPLC.
ＨＰＬＣ分析は以下条件にて行った。
カラム：逆相用カラム「Ｄｅｖｅｌｏｓｉｌ（登録商標）Ｃ−３０−ＵＧ−５」（４．６ｍｍｉ．ｄ．×２５０ｍｍ）
移動相：Ａ・・・Ｈ2Ｏ（０．１％トリフルオロ酢酸（ＴＦＡ））， Ｂ・・・アセトニトリル（０．１％ＴＦＡ）
流速：１ｍＬ／ｍｉｎ
注入：１０μＬ
検出：２５４ｎｍ
勾配（容量％）：８０％Ａ／２０％Ｂから２０％Ａ／８０％Ｂまで３０分間、２０％Ａ／８０％Ｂから１００％Ｂまで５分間、１００％Ｂで１０分間（全て直線）
HPLC analysis was performed under the following conditions.
Column: Column for reverse phase “Develosil (registered trademark) C-30-UG-5” (4.6 mm.d. × 250 mm)
Mobile phase: A: H 2 O (0.1% trifluoroacetic acid (TFA)), B: Acetonitrile (0.1% TFA)
Flow rate: 1 mL / min
Injection: 10 μL
Detection: 254 nm
Gradient (% by volume): 30 minutes from 80% A / 20% B to 20% A / 80% B, 5 minutes from 20% A / 80% B to 100% B, 10 minutes at 100% B (all linear)
得られたクロマトグラムを図１に示す。 The obtained chromatogram is shown in FIG.
（実施例２：新規化合物の単離・構造決定）
得られた反応物のうち、図１のＡで示したピーク（溶出時間：約１８分付近）に含まれる化合物を分取ＨＰＬＣにより単離し、常法により乾燥したところ、褐色粉末状の物質を７２．６ｍｇ得た。この物質をＵＨＡ４０２２と命名した。
(Example 2: Isolation and structure determination of a novel compound)
Among the obtained reactants, a compound contained in the peak shown by A in FIG. 1 (elution time: about 18 minutes) was isolated by preparative HPLC and dried by a conventional method. As a result, a brown powdery substance was obtained. 72.6 mg was obtained. This material was named UHA4022.
次いで、前記ＵＨＡ４０２２の分子量を高分解能Ｎｅｇａｔｉｖｅ−ＦＡＢ−ＭＳ（Ｆａｓｔ Ａｔｏｍ Ｂｏｍｂａｒｄｍｅｎｔ−Ｍａｓｓ Ｓｐｅｃｔｒｏｍｅｔｒｙ）にて測定したところ、測定値は６４７．２１４４であり、理論値との比較から、以下の分子式を得た。
理論値Ｃ42Ｈ31Ｏ7（Ｍ−Ｈ-）：６４７．２１４８
分子式Ｃ42Ｈ32Ｏ7
Subsequently, when the molecular weight of the UHA4022 was measured with a high resolution negative-FAB-MS (Fast Atom Bombardment-Mass Spectrometry), the measured value was 647.2144, and the following molecular formula was obtained from comparison with the theoretical value. .
Theoretical value C 42 H 31 O 7 (M−H − ): 647.2148
Molecular formula C 42 H 32 O 7
次に、前記ＵＨＡ４０２２を核磁気共鳴（ＮＭＲ）測定に供し、1Ｈ−ＮＭＲ、13Ｃ−ＮＭＲ及び各種２次元ＮＭＲデータの解析から、前記ＵＨＡ４０２２が前記式（１）で表される構造を有する新規化合物であることを確認した。したがって、前記式（１）で表される新規化合物は本発明の方法で効率的に生成できることが示された。 Next, the UHA4022 is subjected to nuclear magnetic resonance (NMR) measurement, and from analysis of 1 H-NMR, 13 C-NMR and various two-dimensional NMR data, the UHA4022 has a structure represented by the formula (1). It was confirmed to be a new compound. Therefore, it was shown that the novel compound represented by the formula (1) can be efficiently produced by the method of the present invention.
なお、前記ＮＭＲ測定値については、ＵＨＡ４０２２を For the NMR measurement value, UHA4022 is used.
として、その1Ｈ核磁気共鳴スペクトル、13Ｃ核磁気共鳴スペクトルを表１に示す。
値はδ、ｐｐｍで、溶媒はＤＭＳＯ−ｄ6で測定した。
Table 1 shows the 1 H nuclear magnetic resonance spectrum and 13 C nuclear magnetic resonance spectrum.
The values were δ and ppm, and the solvent was measured with DMSO-d 6 .
また、ＵＨＡ４０２２の物理化学的性状は、以下のようになった。
（性状）
褐色粉末
（溶解性）
水：難溶
メタノール：溶解
エタノール：溶解
ＤＭＳＯ：溶解
クロロホルム：難溶
酢酸エチル：難溶
The physicochemical properties of UHA4022 were as follows.
(Properties)
Brown powder (soluble)
Water: Slightly soluble methanol: Soluble ethanol: Dissolved DMSO: Dissolved chloroform: Slightly soluble ethyl acetate: Slightly soluble
（実施例３：ＵＨＡ４０２２のヒト骨髄球性白血病細胞に対する抗癌作用）
次に癌細胞に対する実施例２で得られたＵＨＡ４０２２の効果を見るため、ＨＬ−６０細胞（ヒト骨髄球性白血病細胞）を用いた癌細胞増殖抑制作用について試験した。
(Example 3: Anticancer effect of UHA4022 on human myeloid leukemia cells)
Next, in order to see the effect of UHA4022 obtained in Example 2 on cancer cells, the cancer cell proliferation inhibitory action using HL-60 cells (human myeloid leukemia cells) was tested.
ＨＬ−６０細胞の培養には、４ｍＭグルタミン（Ｌ−Ｇｌｕｔａｍｉｎｅ、シグマアルドリッチジャパン社製）、１％アンチバイオティック−アンチマイコティック（Ａｎｔｉｂｉｏｔｉｃ−Ａｎｔｉｍｙｃｏｔｉｃ、ギブコ（ＧＩＢＣＯ）社製）、１０％ウシ胎児血清（ＦＢＳ、Ｂｉｏｌｏｇｉｃａｌ ｉｎｄｕｓｔｒｉｅｓ社製）を含む高栄養培地「ＲＰＭＩ−１６４０」（シグマアルドリッチジャパン社製）を使用した。試験には細胞培養用９６ウェルプレート（コーニングジャパン（株）製）を用い、５×１０5ｃｅｌｌｓ／ｍＬとなるように細胞数を調整したＨＬ−６０細胞を１ウェルあたり１００μＬずつ播種して試験に使用した。 For culture of HL-60 cells, 4 mM glutamine (L-Glutamine, manufactured by Sigma-Aldrich Japan), 1% antibiotic-antimycotic (manufactured by Antibiotic-Antimycotic, Gibco (GIBCO)), 10% fetal bovine serum The high nutrient medium “RPMI-1640” (manufactured by Sigma Aldrich Japan) containing FBS (manufactured by Biological industries) was used. For the test, a 96-well plate for cell culture (manufactured by Corning Japan Co., Ltd.) was used and seeded with 100 μL per well of HL-60 cells adjusted to a cell number of 5 × 10 5 cells / mL. Used for.
試料は、実施例２で得られたＵＨＡ４０２２、原料のレスベラトロールの２種類を用いた。試料調製は、各々の化合物をジメチルスルホキシド（Ｄｉｍｅｔｈｙｌ ｓｕｌｆｏｘｉｄｅ：ＤＭＳＯ、和光純薬工業（株）製）にて溶解し、ＨＬ−６０細胞培養液中の最終濃度がそれぞれ６．３μＭ、１２．５μＭ、２５μＭ、５０μＭ及び1００μＭとなるように添加して、３７℃、５％ＣＯ2の培養条件下で試験を開始した。なお、溶媒であるＤＭＳＯのみを同量添加したものをネガティブコントロールとした。 Two types of samples were used: UHA4022 obtained in Example 2 and raw material resveratrol. In the sample preparation, each compound was dissolved in dimethyl sulfoxide (DMSO, manufactured by Wako Pure Chemical Industries, Ltd.), and the final concentrations in the HL-60 cell culture solution were 6.3 μM, 12.5 μM, The test was started under the culture conditions of 37 ° C. and 5% CO 2 by adding 25 μM, 50 μM and 100 μM. A negative control was prepared by adding the same amount of DMSO as a solvent.
生存細胞数の定量は「Ｃｅｌｌ ｃｏｕｎｔｉｎｇ ｋｉｔ−８」（（株）同人化学研究所製）を用いたＭＴＴ法にて行った。つまり、試験開始より２４時間後、各ウェルにＣｅｌｌ ｃｏｕｎｔｉｎｇ ｋｉｔ−８溶液を１０μＬ添加し、よく攪拌した。３７℃、５％ＣＯ2条件下で１時間の遮光反応を行った。その後にプレートリーダー（「ＭＵＬＴＩＳＫＡＮ ＦＣ」、サーモフィッシャーサイエンティフィック株式会社製）を用いて測定波長４５０ｎｍの吸光度測定を行い、得られたデータをもとに細胞生存率を算出した。細胞生存率とは、溶媒であるＤＭＳＯのみを添加した培養液の生存細胞数を１００％とし、各化合物の濃度下における細胞の生存細胞数を相対値として算出した値である。各化合物濃度と細胞生存率の関係から、細胞増殖を５０％抑制する濃度ＩＣ50（５０％阻害濃度）を算出した。その結果を表２に示す。
表２に示す結果から、ＵＨＡ４０２２に優れた癌細胞増殖抑制能が認められた。この抗癌作用は、レスベラトロールよりも高い抗癌活性を示した。したがってレスベラトロールを前記式（１）で表される新規化合物に変換する高い有意性が示された。
The number of viable cells was quantified by the MTT method using “Cell counting kit-8” (manufactured by Dojin Chemical Laboratory). That is, 24 hours after the start of the test, 10 μL of the Cell counting kit-8 solution was added to each well and stirred well. The light-shielding reaction was performed for 1 hour at 37 ° C. and 5% CO 2 . Thereafter, absorbance was measured at a measurement wavelength of 450 nm using a plate reader (“MULTISKAN FC”, manufactured by Thermo Fisher Scientific Co., Ltd.), and the cell viability was calculated based on the obtained data. The cell viability is a value calculated by setting the number of viable cells in a culture solution to which only DMSO as a solvent is added as 100% and the number of viable cells in each compound concentration as a relative value. From the relationship between the concentration of each compound and the cell viability, the concentration IC 50 (50% inhibitory concentration) that suppresses cell proliferation by 50% was calculated. The results are shown in Table 2.
From the results shown in Table 2, the cancer cell growth suppressing ability superior to UHA4022 was recognized. This anticancer activity showed higher anticancer activity than resveratrol. Therefore, the high significance which converts resveratrol into the novel compound represented by said Formula (1) was shown.
（実施例４：ＵＨＡ４０２２の遅筋型ミオシンの定量）
次に、ＵＨＡ４０２２の遅筋形成促進作用を評価するために、培養筋管細胞を用いた遅筋型ミオシンの定量を行った。
(Example 4: Quantification of slow-muscle myosin of UHA4022)
Next, in order to evaluate the slow muscle formation promoting action of UHA4022, slow muscle myosin was quantified using cultured myotube cells.
レスベラトロール及び本発明品であるＵＨＡ４０２２はＤＭＳＯに溶解させたものを使用した。培養筋管細胞は培養筋芽細胞（Ｃ２Ｃ１２細胞，ＤＳファーマ株式会社製）を分化させることによって得た。つまり、Ｃ２Ｃ１２細胞を１０％ウシ胎児血清（ＦＢＳ、Ｂｉｏｌｏｇｉｃａｌ ｉｎｄｕｓｔｒｉｅｓ社製）、１％アンチバイオティック−アンチマイコティック（Ａｎｔｉｂｉｏｔｉｃ−Ａｎｔｉｍｙｃｏｔｉｃ、Ａｎｔｉ−Ａｎｔｉ、ギブコ（ＧＩＢＣＯ）社製）を含むＤＭＥＭ（Ｄｕｌｂｅｃｃｏ’ｓ ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ ｍｅｄｉｕｍ、シグマ社製）で５ｘ１０4ｃｅｌｌ／Ｄｉｓｈに調整し、１２ｗｅｌｌプレート（コーニング社製）に播種した。３７℃、５％ＣＯ2条件下で４８時間培養した。これにレスベラトロール及び本発明品であるＵＨＡ４０２２を１、０．３μＭ（ＤＭＳＯ ０．５％）添加した２％馬血清（ＨＳ、ライフテクノロジージャパン社製）、１％Ａｎｔｉ−Ａｎｔｉ含有ＤＭＥＭに交換し、１週間培養した。その際、陰性対象としてＤＭＳＯを用いた。培養終了後、ＲＩＰＡバッファーで細胞を溶解させてタンパク質を抽出した。 Resveratrol and UHA4022 of the present invention were dissolved in DMSO. Cultured myotube cells were obtained by differentiating cultured myoblasts (C2C12 cells, manufactured by DS Pharma Co., Ltd.). That is, DMEM (Dulbecco ') containing C2C12 cells containing 10% fetal bovine serum (FBS, manufactured by Biological Industries), 1% antibiotic-antimycotic (manufactured by Antibiotic-Antilytic, Anti-Anti, Gibco). s modified Eagle medium (manufactured by Sigma) to adjust to 5 × 10 4 cells / Dish, and seeded on a 12-well plate (manufactured by Corning). The cells were cultured for 48 hours at 37 ° C. and 5% CO 2 . 2% horse serum (HS, manufactured by Life Technology Japan Co., Ltd.) supplemented with 1,0.3 μM (DMSO 0.5%) of resveratrol and UHA4022 of the present invention was replaced with DMEM containing 1% Anti-Anti. And cultured for 1 week. At that time, DMSO was used as a negative target. After completion of the culture, the cells were lysed with RIPA buffer and the protein was extracted.
抽出したタンパク質はＰｉｅｒｃｅ ＢＣＡ Ｐｒｏｔｅｉｎ Ａｓｓａｙ Ｋｉｔ（サーモサイエンティフィック社製）にて定量を行い、定法に従いＳＤＳ−ポリアクリルアミド電気泳動に供した。つまり、タンパク質５μｇに１／５量のＬａｅｍｍｌｉバッファー（１０％ドデシル硫酸ナトリウム、１００ｍＭジチオトレイトール、３０％グリセロール、５０ｍＭＴｒｉｓ−ＨＣｌ、ｐＨ６．８）を添加し、９６℃で５分加熱変性させた。ゲルにはＣｒｉｔｅｒｉｏｎ ＴＧＸ Ｇｅｌ（Ａｎｙ ｋＤ、バイオ・ラッド・ラボラトリーズ社製）を使用した。
変性させたタンパク質サンプルを全量ゲルに供し、２００Ｖ、４０分間電気泳動した。電気泳動後、セミドライ式ブロッティング装置（ＴＲＡＮＳ−ＢＬＯＴ Ｓ−Ｄ ＳＥＭＩ−ＤＲＹ ＴＲＡＮＳＦＥＲ ＣＥＬＬ，バイオ・ラッド・ラボラトリーズ社製）でＰＶＤＦ膜（ミリポア社製）に転写し、イムノブロック（ＤＳファーマ社製）にてブロッキングした。ここから、抗遅筋型ミオシン抗体（アバカム社製）と抗マウスＩｇＧＨＲＰ結合抗体（ＣＳＴジャパン社製）を用いて遅筋型ミオシンを検出した。同時に抗速筋型ミオシン抗体（アバカム社製）と抗マウスＩｇＧＨＲＰ結合抗体を用いて速筋型ミオシンの検出も行った。検出されたバンドを画像解析ソフトＩｍａｇｅＪにてバンド強度を算出し、遅筋型ミオシンと速筋型ミオシンの比を算出した。結果は図２に示した。
The extracted protein was quantified by Pierce BCA Protein Assay Kit (manufactured by Thermo Scientific) and subjected to SDS-polyacrylamide electrophoresis according to a conventional method. That is, 1/5 amount of Laemmli buffer (10% sodium dodecyl sulfate, 100 mM dithiothreitol, 30% glycerol, 50 mM Tris-HCl, pH 6.8) was added to 5 μg of protein, and heat-denatured at 96 ° C. for 5 minutes. Criterion TGX Gel (Any kD, manufactured by Bio-Rad Laboratories) was used as the gel.
The entire amount of the denatured protein sample was applied to a gel and electrophoresed at 200 V for 40 minutes. After electrophoresis, it was transferred to a PVDF membrane (Millipore) using a semi-dry blotting device (TRANS-BLOT S-D SEMI-DRY TRANSFER CELL, manufactured by Bio-Rad Laboratories) and transferred to an immunoblock (DS Pharma). And blocked. From here, slow muscle myosin was detected using an anti-slow muscle myosin antibody (manufactured by Abercam) and an anti-mouse IgG HRP-conjugated antibody (manufactured by CST Japan). At the same time, fast muscle myosin was also detected using an anti-fast muscle myosin antibody (Avacam) and an anti-mouse IgG HRP-conjugated antibody. The band intensity of the detected band was calculated by image analysis software ImageJ, and the ratio of slow muscle type myosin to fast muscle type myosin was calculated. The results are shown in FIG.
図２に示す結果より、ＵＨＡ４０２２においてレスベラトロールよりも遅筋型ミオシン量の比が増加していることが示された。この作用はレスベラトロールよりも高かった。従って、本発明品であるＵＨＡ４０２２はレスベラトロールよりも優れた遅筋形成促進作用を有することが示された。 From the results shown in FIG. 2, it was shown that the ratio of the amount of slow muscle myosin was increased in UHA4022 compared to resveratrol. This effect was higher than resveratrol. Therefore, it was shown that UHA4022 which is the product of the present invention has a slow muscle formation promoting action superior to resveratrol.
（実施例５：遅筋関連の遺伝子発現の定量）
次にミオシンタイプにおいて遅筋形成が認められたＵＨＡ４０２２で遅筋に関連する遺伝子の発現量を評価するために、培養筋管細胞を用いてペルオキシゾーム増殖剤応答性受容体δ（ＰＰＡＲδ）、遅筋型トロポニン１（Ｔｎｎｉ１）遺伝子発現の定量を行った。
(Example 5: Quantification of slow muscle-related gene expression)
Next, in order to evaluate the expression level of the gene related to slow muscle in UHA4022 in which slow muscle formation was observed in the myosin type, peroxisome proliferator-responsive receptor δ (PPARδ), delayed using cultured myotube cells Muscle type troponin 1 (Tnni1) gene expression was quantified.
試料及び細胞培養は実施例４に準じて行った。培養終了後、Ｔｒｉｚｏｌ試薬（ライフテクノロジージャパン社製）を用いてＴｏｔａｌ ＲＮＡを抽出した。得られたＲＮＡを２ステップリアルタイムＲＴ−ＰＣＲ用逆転写試薬（商品名：ＰｒｉｍｅＳｃｒｉｐｔ（登録商標）ＲＴ Ｍａｓｔｅｒ Ｍｉｘ、タカラバイオ（株）製）の取扱説明書に準じて逆転写反応を行った。 Samples and cell culture were performed according to Example 4. After completion of the culture, total RNA was extracted using Trizol reagent (manufactured by Life Technology Japan). The obtained RNA was subjected to a reverse transcription reaction in accordance with the instruction manual of a reverse transcription reagent for 2-step real-time RT-PCR (trade name: PrimeScript (registered trademark) RT Master Mix, manufactured by Takara Bio Inc.).
つまり５×（Ｐｒｉｍｅｓｃｒｉｐｔ（登録商標） ＲＴ Ｍａｓｔｅｒ Ｍｉｘ）４μＬ及び全量ＲＮＡ １μｇを混合し、ＲＮａｓｅ Ｆｒｅｅ ｄＨ2Ｏで全量を１０μＬにした。ＰＣＲ用サーマルサイクラー（商品名：ＧｅｎｅＡｍｐ（登録商標）ＰＣＲ Ｓｙｓｔｅｍ ９７００、Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍ社製）を使用して１サイクルが「３７℃×１５分→８５℃×５秒」であるプログラムにて逆転写反応を行った。逆転写反応液を滅菌超純水にて１０倍希釈した希釈液をリアルタイムＲＴ−ＰＣＲ解析に使用した。 Specifically, 4 μL of 5 × (Primescript (registered trademark) RT Master Mix) and 1 μg of total RNA were mixed, and the total volume was adjusted to 10 μL with RNase Free dH 2 O. Using a thermal cycler for PCR (trade name: GeneAmp (registered trademark) PCR System 9700, manufactured by Applied Biosystem), reverse transcription reaction with a program in which one cycle is “37 ° C. × 15 minutes → 85 ° C. × 5 seconds” Went. A diluted solution obtained by diluting the reverse transcription reaction solution 10-fold with sterilized ultrapure water was used for real-time RT-PCR analysis.
リアルタイムＲＴ−ＰＣＲ解析は定法に従って行った。解析には、ＥＣＯ Ｒｅａｌｔｉｍｅ ＲＴ―ＰＣＲ ｓｙｓｔｅｍ」（商品名、イルミナ（株）製）を使用した。ＰＧＣ−１βプライマーには、ＰＰＡＲδフォワードプライマー（プライマーＩＤ：ＭＡ１２７４９９−Ｆ）及びＰＰＡＲδリバースプライマー（プライマーＩＤ：ＭＡ１２７４９９−Ｒ）を使用した。ＴｎｎｉプライマーにはＴｎｎｉ１フォワードプライマー（プライマーＩＤ：ＭＡ１１０７７９−Ｆ）及びＴｎｎｉ１リバースプライマー（プライマーＩＤ: ＭＡ１１０７７９−Ｒ）を使用した。細胞内遺伝子の内部標準はβ−アクチンとし、そのプライマーとして、ＡＣＴＢフォワードプライマー（プライマーＩＤ：ＨＡ０６７８０３−Ｆ）及びＡＣＴＢリバースプライマー（プライマーＩＤ：ＨＡ０６７８０３−Ｒ）（前記６種のプライマーはいずれもタカラバイオ（株）製）を使用した。 Real-time RT-PCR analysis was performed according to a conventional method. For the analysis, ECO Realtime RT-PCR system (trade name, manufactured by Illumina) was used. As the PGC-1β primer, a PPARδ forward primer (primer ID: MA127499-F) and a PPARδ reverse primer (primer ID: MA127499-R) were used. Tnni1 forward primer (primer ID: MA1107979-F) and Tnni1 reverse primer (primer ID: MA1107979-R) were used as Tnni primers. The internal standard of the intracellular gene is β-actin, and as its primer, an ACTB forward primer (primer ID: HA067803-F) and an ACTB reverse primer (primer ID: HA067803-R) (all of the above six primers are Takara Bio). Used).
反応にはリアルタイムＲＴ−ＰＣＲ試薬（商品名：ＳＹＢＲ Ｓｅｌｅｃｔ Ｍａｓｔｅｒ Ｍｉｘ、ライフテクノロジージャパン社製）を使用した。反応液は４８ウェルＰＣＲプレート（イルミナ（株）製）中に、２×ＳＹＢＲ Ｓｅｌｅｃｔ ２．５μＬ、フォワードプライマー（５０μＭ）０．０４μＬ、リバースプライマー（５０μＭ）０．０４μＬ、逆転写反応液１μＬ及び（ｄＨ2Ｏ）１．４２μＬ（総量５μＬ）を混合した。各遺伝子の解析には『５０℃×２分→９５℃×３０秒→「９５℃×５秒→６５℃×３０秒」×４０サイクル→９５℃×１５秒→５５℃×１５秒→９５℃×１５秒』のプログラムにてＰＣＲ反応を行った。
A real-time RT-PCR reagent (trade name: SYBR Select Master Mix, manufactured by Life Technology Japan) was used for the reaction. In a 48-well PCR plate (manufactured by Illumina), the reaction solution was 2 × SYBR Select 2.5 μL, forward primer (50 μM) 0.04 μL, reverse primer (50 μM) 0.04 μL, reverse
得られた各細胞中のβ−アクチンと各遺伝子のＣｔ値（Ｔｈｒｅｓｈｏｌｄ Ｃｙｃｌｅ：一定の増幅量（閾値）に達するサイクル数）から各遺伝子発現量の相対値を算出した。結果を図３に示した。 The relative value of each gene expression level was calculated from the obtained β-actin in each cell and the Ct value of each gene (Threshold Cycle: the number of cycles reaching a certain amplification level (threshold)). The results are shown in FIG.
図３に示す結果より、本発明の新規化合物により遅筋関連遺伝子がレスベラトロールによるよりも有意に増加していた。従って、本発明の新規化合物がレスベラトロールよりも優れた遅筋形成促進作用を有することが遺伝子発現レベルからも示された。 From the results shown in FIG. 3, the slow muscle-related gene was significantly increased by the novel compound of the present invention than by resveratrol. Therefore, it was also shown from the gene expression level that the novel compound of the present invention has a slow muscle formation promoting action superior to resveratrol.
（実施例６：加熱温度によるＵＨＡ４０２２の生成量の違い）
レスベラトロール１ｇ、エタノール２０ｍＬ、５％炭酸水素ナトリウム水溶液２０ｍＬの混合溶液（ｐＨ＝９．９）を、オートクレーブにて７０℃、９０℃、１１０℃、１３０℃の各温度条件で９０分間加熱した。それぞれの温度条件で得られた反応後組成物１ｍＬをメタノールにて５０ｍＬにメスアップし、実施例１と同様にＨＰＬＣにより分析した。
(Example 6: Difference in production amount of UHA4022 depending on heating temperature)
A mixed solution (pH = 9.9) of resveratrol 1 g,
その結果、１１０℃以上でＵＨＡ４０２２の生成は確認できた。レスベラトロール量からの生成比率（重量％）は、７０℃、９０℃が非生成、１１０℃が極微量、１３０℃が７．１％となり、１３０℃での加熱によりもっとも多くのＵＨＡ４０２２が生成していた。 As a result, generation of UHA4022 was confirmed at 110 ° C. or higher. The production ratio (% by weight) from the amount of resveratrol is 70 ° C, 90 ° C is not produced, 110 ° C is extremely small, 130 ° C is 7.1%, and heating at 130 ° C produces the most UHA4022. Was.
（実施例７：ＵＨＡ４０２２含有エキスの調製）
レスベラトロール〔（株）Ｔｅｃｈｎｏ ｓｃｉｅｎｃｅ製〕１ｇ、５％炭酸水素ナトリウム（和光純薬工業（株）製、食品添加物）水溶液２０ｍＬ、エタノール２０ｍＬを調製した混合溶液（ｐＨ＝９．９）を、オートクレーブにて１３０℃、９０分間加熱した。得られた反応溶液を減圧加熱させて乾固し、ＵＨＡ４０２２含有エキスを１．７ｇ得た。得られたＵＨＡ４０２２含有エキス１．７ｇ中には、実施例２と同様の手法で確認したところＵＨＡ４０２２が０．０７０ｇ含有されていた。必要に応じてこの作業を繰り返した。
(Example 7: Preparation of UHA4022 containing extract)
Resveratrol (manufactured by Techno science Co., Ltd.) 1 g, 5% sodium hydrogen carbonate (Wako Pure Chemical Industries, Ltd., food additive)
（実施例８：ＵＨＡ４０２２を含有する食品）
実施例７で得たＵＨＡ４０２２含有エキス１ｇをあらかじめ１００ｍＬのエタノールに溶解させ、これに砂糖５００ｇ、水飴４００ｇを混合溶解し、生クリーム１００ｇ、バター２０ｇ、練乳７０ｇ、乳化剤１．０ｇを混合した後、真空釜にて−５５０ｍｍＨｇ減圧させ、１１５℃の条件下で濃縮し、水分値３．０重量％のミルクハードキャンディを得た。このミルクハードキャンディは、菓子として食べ易いものであることはもちろん、癌患者における癌の拡散のリスクを低減したり、癌の発症のリスクを低減したり、癌の予防を期待した機能性食品や持久力向上を期待した機能性食品としても利用できる。
(Example 8: Food containing UHA4022)
1 g of UHA4022-containing extract obtained in Example 7 was dissolved in 100 mL of ethanol in advance, 500 g of sugar and 400 g of starch syrup were mixed and dissolved in this, and after mixing 100 g of fresh cream, 20 g of butter, 70 g of condensed milk, and 1.0 g of emulsifier, The pressure was reduced by −550 mmHg in a vacuum kettle and concentrated under a condition of 115 ° C. to obtain a milk hard candy having a moisture value of 3.0% by weight. This milk hard candy is easy to eat as a confectionery, as well as functional foods that reduce the risk of cancer spread in cancer patients, reduce the risk of developing cancer, and prevent cancer. It can also be used as a functional food that is expected to improve endurance.
（実施例９：ＵＨＡ４０２２を含有する医薬品）
実施例１，２の方法で得たＵＨＡ４０２２をエタノールに溶解し、これを微結晶セルロースに添加して吸着させた後に、減圧乾燥させた。この吸着物を用いて常法に従い、打錠品を得た。処方はＵＨＡ４０２２を１０重量部、コーンスターチ２３重量部、乳糖１２重量部、カルボキシメチルセルロース８重量部、微結晶セルロース３２重量部、ポリビニルピロリドン４重量部、ステアリン酸マグネシウム３重量部、タルク８重量部である。本打錠品は、癌の治癒を目的とする医薬品や脂質代謝能の高い遅筋形成を目的とした医薬品として有効に利用できる。
(Example 9: Drug containing UHA4022)
UHA4022 obtained by the methods of Examples 1 and 2 was dissolved in ethanol, added to microcrystalline cellulose and adsorbed, and then dried under reduced pressure. Using this adsorbent, a tableted product was obtained according to a conventional method. The formulation is 10 parts by weight of UHA4022, 23 parts by weight of corn starch, 12 parts by weight of lactose, 8 parts by weight of carboxymethylcellulose, 32 parts by weight of microcrystalline cellulose, 4 parts by weight of polyvinylpyrrolidone, 3 parts by weight of magnesium stearate, and 8 parts by weight of talc. . This tableted product can be effectively used as a pharmaceutical for the purpose of healing cancer or a pharmaceutical for the purpose of forming slow muscles with high lipid metabolism ability.
（実施例１０：ＵＨＡ４０２２を含有する医薬部外品）
実施例１，２の方法で得たＵＨＡ４０２２ １．２ｇを１０ｍＬのエタノールに溶解し、これにタウリン２０ｇ、ビタミンＢ１硝酸塩０．１２ｇ、安息香酸ナトリウム０．６ｇ、クエン酸４ｇ、砂糖６０ｇ、ポリビニルピロリドン１０ｇを溶解させた精製水を混合し、さらに精製水で１０００ｍＬにメスアップした。なお、ｐＨは、希塩酸を用いて３．２に調整した。得られた溶液１０００ｍＬのうち５０ｍＬをガラス瓶に充填し、８０℃で３０分間滅菌して、医薬部外品であるドリンク剤を完成させた。本ドリンク剤は、栄養補給の目的に加えて、癌患者における癌の拡散のリスクを低減したり、癌の発症のリスクを低減したり、癌の予防を目的とする医薬部外品や持久力向上を期待した機能性食品として有効に利用できる。
(Example 10: Quasi-drug containing UHA4022)
1.2 g of UHA4022 obtained by the method of Examples 1 and 2 was dissolved in 10 mL of ethanol, to which 20 g of taurine, 0.12 g of vitamin B1 nitrate, 0.6 g of sodium benzoate, 4 g of citric acid, 60 g of sugar, polyvinylpyrrolidone Purified water in which 10 g was dissolved was mixed, and further made up to 1000 mL with purified water. The pH was adjusted to 3.2 using dilute hydrochloric acid. 50 ml of 1000 ml of the obtained solution was filled in a glass bottle and sterilized at 80 ° C. for 30 minutes to complete a quasi-drug drink. In addition to the purpose of nutritional supplements, this drink reduces the risk of cancer spread in cancer patients, reduces the risk of developing cancer, and quasi drugs and endurance for the purpose of preventing cancer. It can be effectively used as a functional food that is expected to improve.
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| JP2011251914A (en) * | 2010-05-31 | 2011-12-15 | Uha Mikakuto Co Ltd | New polymerized compound of resveratrol, or pharmacologically acceptable salt, ester or ether thereof |
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| JP2016069326A (en) * | 2014-09-30 | 2016-05-09 | ユーハ味覚糖株式会社 | Spontaneity enhancer composition |
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