JP2006022094A - NEW 3,4-seco-LUPANE TYPE TRITERPENOID SAPONIN COMPOUND - Google Patents
NEW 3,4-seco-LUPANE TYPE TRITERPENOID SAPONIN COMPOUND Download PDFInfo
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- JP2006022094A JP2006022094A JP2005171553A JP2005171553A JP2006022094A JP 2006022094 A JP2006022094 A JP 2006022094A JP 2005171553 A JP2005171553 A JP 2005171553A JP 2005171553 A JP2005171553 A JP 2005171553A JP 2006022094 A JP2006022094 A JP 2006022094A
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- compound
- lipase
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Images
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Abstract
Description
本発明は、医薬、特に、リパーゼ阻害剤として有用な新規3,4-seco-lupane型トリテルペノイドサポニン化合物の用途に関する。さらに、本発明は、3,4-seco-lupane型トリテルペノイドサポニン化合物を含む、リパーゼ阻害剤に関し、更に詳しくは、生体内での脂質の消化吸収をにない肥満症、高脂血症の鍵を握る膵リパーゼを有効に阻害して肥満や高脂血症の抑制や予防に寄与し得ると共に、細菌性リパーゼに起因するニキビ、皮膚炎、ふけなどの皮膚疾患の予防、治療に有効な安全性の高いリパーゼ阻害剤に関する。
The present invention relates to the use of
3,4-seco-lupane型トリテルペノイドサポニンは、落葉性小低木であるウコギ科の、智異山ウコギ(Acanthopanax chiisanensis)、ケヤマウコギ(Acanthopanax divaricatus)及びAcanthopanax senticosus forma inermisの葉から抽出単離される化合物として、非特許文献1〜4に報告されている。また、リパーゼ阻害作用のような生理活性については、肝毒性予防作用、糖尿病予防作用などが、非特許文献5、6に報告されているに過ぎない。本発明の化合物は、これら文献に記載されておらず、新規の化合物である。
3,4-seco-lupane-type triterpenoid saponins are extracted and isolated from the leaves of the deciduous small shrub, Acanthopanax chiisanensis, Acanthopanax divaricatus, and Acanthopanax senticosus forma inermis.
また、従来、からだの脂肪組織及び種々の臓器に異常な脂肪沈着を来し、その結果起こる肥満、あるいは血清脂質が異常に高い症状を示す高脂血症は、高血圧、動脈硬化、糖尿病などの各種生活習慣病の発症に密接に関与していると考えられている。肥満は、体質的因子、食餌性因子、精神的因子、中枢性因子、代謝性因子、運動不足などが要因となり、結果的にカロリー摂取が消費カロリーを上回り、脂肪が蓄積して起こると言われている。食餌中の脂質は、膵臓のリパーゼで分解されて小腸から吸収される。そこで脂質の吸収を抑制するべく、リパーゼ作用の活性阻害機能を有するリパーゼ阻害剤を用い、肥満を防止したり、あるいは高脂血症状を抑制することが可能であると考えられている。 Conventionally, abnormal fat deposition in the body adipose tissue and various organs, resulting in obesity, or hyperlipidemia with abnormally high serum lipids, such as hypertension, arteriosclerosis, diabetes, etc. It is thought to be closely involved in the development of various lifestyle-related diseases. Obesity is said to be caused by constitutional factors, dietary factors, mental factors, central factors, metabolic factors, lack of exercise, etc., resulting in caloric intake exceeding calorie consumption and fat accumulation. ing. Dietary lipids are broken down by pancreatic lipase and absorbed from the small intestine. Therefore, it is thought that obesity can be prevented or hyperlipidemia can be suppressed by using a lipase inhibitor having a function of inhibiting the activity of lipase in order to suppress lipid absorption.
この様な観点から、特に日常的に摂取しうる種々の天然物由来の成分で、強いリパーゼ阻害活性を有する成分、すなわち脂質吸収抑制活性を有する天然成分の探索が精力的に行われている。これまでに、ホスファチジルコリン(非特許文献7)、大豆蛋白(非特許文献8、9)、タンニン(非特許文献10)、シャクヤク、オウレン、オウバク、ボタンピ、ゲンノショウコ、チャ、クジンなどの生薬の溶媒抽出エキス(特許文献1)、ピーマン、かぼちゃ、しめじ、まいたけ、ふじき、緑茶、紅茶及びウーロン茶の水抽出物(特許文献2)、ドッカツ、リョウキョウ、ビンロウシ、ヨウバイヒ、ケツメイシの抽出物(特許文献3)などがリパーゼ阻害活性を有するものとして報告されているが、物性や効果の面で需要性に乏しい。さらに、このような探索においては、原料中の含有量が多く、工業的生産に適した物質が期待されているが、これまでに条件を十分に満たす物質は未だ見出されていない。 From such a point of view, various natural products that can be ingested on a daily basis are vigorously searched for components having strong lipase inhibitory activity, that is, natural components having lipid absorption inhibitory activity. So far, solvent extraction of herbal medicines such as phosphatidylcholine (Non-patent document 7), soy protein (Non-patent documents 8 and 9), tannin (Non-patent document 10), peonies, auren, apricot, button pi, gennoshouko, tea, and kudin. Extract (Patent Document 1), Pepper, Pumpkin, Shimeji, Maitake, Fujiki, Green tea, Black tea and Oolong tea water extract (Patent Document 2), Dokatsu, Ryokyo, Betel wax, Yobaihi, Ketsumeishi extract (Patent Document 3) ) And the like have been reported as having lipase inhibitory activity, but they are poor in demand in terms of physical properties and effects. Furthermore, in such a search, a substance that has a high content in the raw material and is suitable for industrial production is expected, but no substance that satisfies the conditions has been found yet.
また、人体における細菌性リパーゼには、皮膚表層に常在する微生物(プロピオニバクテリウム アクネス:Propionibacterium acnes、ピティロスポラム オバール:Pityrosporum ovale、マイクロコッカス属:Micrococcus sp.など)が産生するリパーゼがあり、これらのリパーゼが皮脂中に含まれるトリグリセライドを分解し遊離脂肪酸を産生する。遊離脂肪酸は、皮膚に対して刺激性の炎症反応を起こし、ニキビ、皮膚炎、ふけなどの皮膚疾患の要因の一つとして考えられている。特に、ニキビの原因とされるプロピオニバクテリウム アクネスの菌数と産生する遊離脂肪酸量には相関関係があり、毛包壁に対して、刺激性の炎症反応とそれに伴う過角化、コメドの形成を引き起こすと考えられている(非特許文献11)。 In addition, bacterial lipases in the human body include lipases produced by microorganisms resident on the skin surface (Propionibacterium acnes, Pityrosporum ovale, Micrococcus sp., Etc.). Lipase decomposes triglyceride contained in sebum to produce free fatty acids. Free fatty acids cause an irritating inflammatory reaction to the skin and are considered as one of the causes of skin diseases such as acne, dermatitis and dandruff. In particular, there is a correlation between the number of Propionibacterium acnes, the cause of acne, and the amount of free fatty acids produced. It is thought to cause formation (Non-Patent Document 11).
しかし、細菌性リパーゼを阻害して疾患を抑制または予防する薬剤の開発は未だあまり進められておらず、2-Pyridylmethyl-2-(p-(2-methylpropyl)-phenyl)propinate(慣用名:イブプロフェンピコノール)(非特許文献12)、テトラサイクリンおよび金属塩(特許文献4)や植物抽出物としてビワ葉抽出物(特許文献5)やコラ・デ・カバロ抽出物(特許文献6)などが報告されているが、他の配合成分との関係からリパーゼ阻害作用を発揮できなかったり、局所適用における安全性、有効性の点で必ずしも満足し得るものではない。 However, the development of a drug that inhibits bacterial lipase to suppress or prevent diseases has not been advanced yet, and 2-Pyridylmethyl-2- (p- (2-methylpropyl) -phenyl) propinate (common name: ibuprofen) Piconol) (Non-patent document 12), tetracycline and metal salt (patent document 4), loquat leaf extract (patent document 5), cora de caballo extract (patent document 6) and the like as plant extracts have been reported. However, the lipase inhibitory action cannot be exhibited due to the relationship with other compounding components, and it is not always satisfactory in terms of safety and effectiveness in topical application.
本研究者はリパーゼ阻害剤について、鋭意研究を続けており、ウコギ科のヒメウコギから抽出したオレアナン型トリテルペノイドサポニンを有効成分とするリパーゼ阻害剤を特許出願した(特許文献7)。本発明は、さらに研究開発を続けた結果、新たにリパーゼ阻害作用を奏する化合物を開発したものである。 The present investigator has continued intensive research on lipase inhibitors, and has filed a patent application for a lipase inhibitor containing oleanane-type triterpenoid saponins extracted from urchinaceae (Patent Document 7). As a result of further research and development, the present invention has newly developed a compound that exhibits a lipase inhibitory action.
本発明の課題は、連用しても副作用の恐れがない、効果的で安全性の高い、リパーゼ作用の活性阻害機能を有する化合物を開発し、肥満防止、あるいは高脂血症抑制、又はプロピオニバクテリウム アクネスなどの皮膚常在菌が産生するリパーゼを阻害することによるニキビなどの皮膚疾患の予防あるいは治療に有効な当該化合物を含有するリパーゼ阻害剤、抗肥満剤、高脂血症改善剤、ニキビ用皮膚改善剤を提供することにある。 An object of the present invention is to develop an effective and safe compound having a function of inhibiting the activity of lipase activity without causing a side effect even if used continuously, to prevent obesity, suppress hyperlipidemia, or propioni. Lipase inhibitors, anti-obesity agents, hyperlipidemia-improving agents containing the compounds effective in the prevention or treatment of skin diseases such as acne by inhibiting lipases produced by skin resident bacteria such as bacteria acnes The object is to provide a skin improvement agent for acne.
本発明者らは、鋭意研究を進めた結果、マンシュウウコギから抽出単離される化合物で式(1)で示される3,4−seco−lupane型トリテルペノイドサポニンの一種で、新規化合物である3, 4−seco−4 (23), 20 (29)−lupadiene−4, 28−dioic acid 28−O −α−L−rhamnopyranosyl(1→4)−β−D−glucopyranosyl(1→6)−β−D−glucopyranosideを見出し、さらに、本化合物がリパーゼ阻害活性を有する素材として医薬、食品、化粧品に使用できる性質を有することを見出し、本発明を完成させた。
As a result of diligent research, the inventors of the present invention are compounds that are extracted and isolated from Manchuriagi and are a kind of 3,4-seco-lupane-type triterpenoid saponins represented by the formula (1), which are
本発明の主な構成は、次のとおりである。1.下記式(1) The main configuration of the present invention is as follows. 1. Following formula (1)
本発明の化合物は、リパーゼ阻害活性を有し、医薬、食品、化粧料として使用できる。さらに、リパーゼ活性を強力に阻害することから、肥満や高脂血症の抑制や予防に寄与し得ると共に、細菌性リパーゼに起因するニキビや皮膚炎、ふけなどの皮膚疾患の予防、治療に有効である。このリパーゼ阻害剤は、長期に連用しても安全で効果的であるから、健康食品のような形態の食品としても提供できる。 The compound of the present invention has lipase inhibitory activity and can be used as a medicine, food or cosmetic. In addition, since it strongly inhibits lipase activity, it can contribute to the suppression and prevention of obesity and hyperlipidemia, and also effective in the prevention and treatment of skin diseases such as acne, dermatitis and dandruff caused by bacterial lipase. It is. Since this lipase inhibitor is safe and effective even when used for a long period of time, it can also be provided as a food in the form of a health food.
マンシュウウコギ(Acanthopanax sessiliflorus)は、ウコギ科の落葉低木で、中国では五加とよばれ、葉および根皮を乾燥させたものは、滋養強壮、鎮痛剤として腹痛、疲労回復、冷え症などに用いられている。 Acanthopanax sessiliflorus is a deciduous shrub of the family Argiaceae, which is called Goka in China, and its dried leaves and root bark are used for nourishing tonic and pain relief as abdominal pain, fatigue recovery, coldness, etc. ing.
本発明の化合物は、マンシュウウコギ(Acanthopanax sessiliflorus)の葉を適当な溶媒で抽出し、その抽出溶媒より精製して得ることができる。抽出溶媒としては、水又は水と混和する有機溶媒の混合液、メタノール、エーテル、アセトン、アセトニトリル、n−ブタノール、酢酸エチル、クロロホルムなどを用いることができる。好適な例としては0〜30 重量%の含水エタノールが挙げられる。原料と抽出
溶媒の割合は、1:0.5〜1:10(重量比)が望ましく、また抽出は常温、加圧、攪拌下で1〜10時間程度行うことが望ましい。ろ過により抽出液を得ることができるが、その際必要があればアンバーライトなどのろ過助剤を用いることもできる。
The compound of the present invention can be obtained by extracting leaves of Acanthopanax sessiliflorus with a suitable solvent and purifying from the extraction solvent. As the extraction solvent, water or a mixed solution of an organic solvent miscible with water, methanol, ether, acetone, acetonitrile, n-butanol, ethyl acetate, chloroform and the like can be used. Preferable examples include 0 to 30% by weight of water-containing ethanol. The ratio of the raw material to the extraction solvent is desirably 1: 0.5 to 1:10 (weight ratio), and the extraction is desirably performed for about 1 to 10 hours at room temperature, pressure, and stirring. An extract can be obtained by filtration, and if necessary, a filter aid such as amberlite can be used.
こうして得られた抽出液は、減圧濃縮、凍結乾燥など通常の方法により濃縮する。本発明の化合物は水溶性であるが酢酸エチルあるいはn−ブタノールで抽出される性質を有することから、この性質を利用して、次のような精製を行うことができる。抽出溶媒が水などの場合、濃縮する前にエーテル、石油エーテル、ヘキサン、クロロホルムなどの酢酸エチルあるいはn−ブタノールより脂溶性の高い溶媒で洗浄し、余剰物を除くこともできる。抽出溶媒に有機溶媒が含まれる場合は、濃縮後、残渣を水に転溶し、エーテル、石油エーテル、ヘキサン、クロロホルムなどの酢酸エチルあるいはn−ブタノールより脂溶性の高い溶媒で洗浄し、余剰物を除くことが望ましい。 The extract thus obtained is concentrated by an ordinary method such as concentration under reduced pressure or lyophilization. Since the compound of the present invention is water-soluble but has the property of being extracted with ethyl acetate or n-butanol, the following purification can be performed using this property. When the extraction solvent is water or the like, the excess can be removed by washing with ethyl acetate such as ether, petroleum ether, hexane and chloroform or a solvent having higher lipophilicity than n-butanol before concentration. If the extraction solvent contains an organic solvent, after concentration, the residue is dissolved in water, washed with a solvent that is more lipophilic than ethyl acetate such as ether, petroleum ether, hexane, or chloroform, or n-butanol. It is desirable to exclude.
洗浄後の水溶液は減圧濃縮、凍結乾燥など通常の方法により濃縮し、そのまま分画精製に供するか、水溶液を酢酸エチルあるいはn−ブタノールで抽出し、酢酸エチル画分あるいはn−ブタノール画分を濃縮後、分画精製することにより、本発明の化合物を得ることもできる。分画精製の方法としては、順相クロマトグラフィー、逆相クロマトグラフィーなど通常の方法を用いることができるが、高速液体クロマトグラフィー(HPLC)を用いた逆相クロマトグラフィーで分画することが望ましい。その際には、リパーゼ阻害活性を指標にして精製を進めることが望ましい。精製した画分は減圧濃縮、凍結乾燥など通常の方法により濃縮し、本発明の化合物を得ることができる。 The washed aqueous solution is concentrated by a conventional method such as concentration under reduced pressure or freeze-drying, and subjected to fraction purification as it is, or the aqueous solution is extracted with ethyl acetate or n-butanol, and the ethyl acetate fraction or n-butanol fraction is concentrated. Thereafter, the compound of the present invention can be obtained by fractional purification. As a method for fractional purification, usual methods such as normal phase chromatography and reverse phase chromatography can be used, but it is desirable to perform fractionation by reverse phase chromatography using high performance liquid chromatography (HPLC). In that case, it is desirable to proceed with purification using lipase inhibitory activity as an index. The purified fraction can be concentrated by a conventional method such as concentration under reduced pressure or lyophilization to obtain the compound of the present invention.
すなわち、乾燥させたマンシュウウコギ(Acanthopanax sessiliflorus)の葉を熱メタノールを用いて抽出する。エバポレーターを用いてメタノールを留去する。残った抽出物を蒸留水を用いて懸濁した後に、ヘキサンを用いて脱脂する。その後水層を高極性多孔質樹脂DIAION HP-20(三菱化学社製)に供し、蒸留水、メタノール、3-ペンタノンを用いて順次溶出させ、これにより得られたメタノール画分を減圧濃縮、凍結乾燥など通常の方法により濃縮することにより、本発明の化合物を得ることができる。 That is, the dried leaves of Acanthopanax sessiliflorus are extracted using hot methanol. Methanol is distilled off using an evaporator. The remaining extract is suspended using distilled water and then degreased using hexane. The aqueous layer was then applied to a highly polar porous resin DIAION HP-20 (manufactured by Mitsubishi Chemical Corporation), and eluted sequentially using distilled water, methanol, and 3-pentanone. The resulting methanol fraction was concentrated under reduced pressure and frozen. The compound of the present invention can be obtained by concentrating by a usual method such as drying.
本発明の化合物を含有する抽出物をそのままリパーゼ阻害剤、脂質吸収阻害剤、抗肥満剤、高脂血症改善剤、ニキビ用皮膚改善剤として用いても良く、さらに高速液体クロマトグラフィー(HPLC)、シリカゲルカラムクロマトグラフィーなど慣用の精製手段を用いて精製したものを用いても良い。 The extract containing the compound of the present invention may be used as it is as a lipase inhibitor, lipid absorption inhibitor, anti-obesity agent, hyperlipidemia improving agent, acne skin improving agent, and high performance liquid chromatography (HPLC). Alternatively, a product purified by a conventional purification means such as silica gel column chromatography may be used.
本発明の化合物は、新規の化合物であり、さらにリパーゼ阻害活性のような生理活性を有することは、本発明により得られた新知見である。 It is a new finding obtained by the present invention that the compound of the present invention is a novel compound and further has physiological activity such as lipase inhibitory activity.
本発明における化合物は、卓越したリパーゼ阻害活性を有しており、これを含有する食品、医薬及び化粧料として、さらに、抗肥満剤、高脂血症改善剤、ニキビ用皮膚改善剤として使用可能である。 The compounds in the present invention have excellent lipase inhibitory activity, and can be used as foods, pharmaceuticals and cosmetics containing them, and further as anti-obesity agents, hyperlipidemia improving agents, and acne skin improving agents. It is.
本発明の化合物を、リパーゼ阻害剤、抗肥満剤、高脂血症改善剤、ニキビ用皮膚改善剤及びこれらを含有する食品、医薬及び化粧料として製造することができる。 The compounds of the present invention can be produced as lipase inhibitors, anti-obesity agents, hyperlipidemia-improving agents, acne skin-improving agents, and foods, medicines and cosmetics containing these.
医薬としての適用方法は、経口投与又は非経口投与のいずれも採用することができる。投与に際しては、有効成分を経口投与、直腸内投与、注射などの投与方法に適した固体又は液体の医薬用無毒性担体と混合して、慣用の医薬製剤の形態で投与することができる。このような製剤としては、例えば、錠剤、顆粒剤、散剤、カプセル剤などの固形剤、溶液剤、懸濁剤、乳剤などの液剤、凍結乾燥製剤などが挙げられ、これらの製剤は製剤上の常法手段により調製することができる。上記の医薬用無毒性担体としては、例えば、グルコース、乳糖、ショ糖、澱粉、マンニトール、デキストリン、脂肪酸グリセリド、ポリエチレングルコール、ヒドロキシエチルデンプン、エチレングリコール、ポリオキシエチレンソルビタン脂肪酸エステル、アミノ酸、ゼラチン、アルブミン、水、生理食塩水などが挙げられる。また、必要に応じて、安定化剤、湿潤剤、乳化剤、結合剤、等張化剤などの慣用の添加剤を適宜添加することもできる。 As an application method as a medicine, either oral administration or parenteral administration can be adopted. In administration, the active ingredient can be mixed with a solid or liquid nontoxic pharmaceutical carrier suitable for administration methods such as oral administration, rectal administration, and injection, and administered in the form of a conventional pharmaceutical preparation. Examples of such preparations include solid preparations such as tablets, granules, powders and capsules, solutions such as solutions, suspensions and emulsions, freeze-dried preparations, and the like. It can be prepared by conventional means. Examples of the non-toxic pharmaceutical carrier include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, Examples include albumin, water, and physiological saline. Further, if necessary, conventional additives such as a stabilizer, a wetting agent, an emulsifier, a binder, and an isotonic agent can be appropriately added.
食品としては、そのまま、又は種々の栄養成分を加えて、若しくは飲食品中に含有せしめて、高脂血症あるいは肥満の治療及び予防に有用な保健用食品又は食品素材として食される。例えば、上述した適当な助剤を添加した後、慣用の手段を用いて、食用に適した形態、例えば、顆粒状、粒状、錠剤、カプセル、ペーストなどに成形して食用に供してもよく、また種々の食品、例えば、ハム、ソーセージなどの食肉加工食品、かまぼこ、ちくわなどの水産加工食品、パン、菓子、バター、粉乳、発酵乳製品に添加して使用したり、水、果汁、牛乳、清涼飲料などの飲料に添加して使用してもよい。 As a food, it is eaten as a health food or food material useful for the treatment and prevention of hyperlipidemia or obesity as it is, or with various nutritional components added or contained in food or drink. For example, after adding the above-mentioned appropriate auxiliaries, it may be used for edible by using conventional means, forming into an edible form, for example, granular, granular, tablet, capsule, paste, etc. It can also be used in various foods such as processed foods such as ham and sausage, processed fish foods such as kamaboko and chikuwa, bread, confectionery, butter, milk powder, fermented milk products, water, fruit juice, milk, You may add and use for drinks, such as a soft drink.
本発明の化合物の有効投与量は、患者の年齢、体重、症状、患者の程度、投与経路、投与スケジュール、製剤形態、素材の阻害活性の強さなどにより、適宜選択・決定されるが、例えば、経口投与の場合、一般に1日当たり0.001〜1000 mg/kg体重程度であり、1日に数回に分けて投与してもよい。 The effective dose of the compound of the present invention is appropriately selected and determined according to the patient's age, weight, symptoms, patient grade, administration route, administration schedule, formulation form, strength of the inhibitory activity of the material, etc. In the case of oral administration, it is generally about 0.001 to 1000 mg / kg body weight per day, and may be divided into several times a day.
また、本発明の化合物を含有せしめて、化粧料または化粧料素材として使用する場合、例えば、本発明の化合物を小麦胚芽油あるいはオリーブ油に添加してリパーゼ阻害剤含有組成物とし、これを化粧料素材として使用することができる。本発明の化合物の添加量は、特に限定されるものではないが、一例としてあげると、小麦胚芽油あるいはオリーブ油の重量に対して0.1 重量%以上60 重量%以下、好ましくは、0.5 重量%以上50 重量%以下が適当である。 When the compound of the present invention is contained and used as a cosmetic or a cosmetic material, for example, the compound of the present invention is added to wheat germ oil or olive oil to obtain a lipase inhibitor-containing composition, which is used as a cosmetic. Can be used as a material. The amount of the compound of the present invention is not particularly limited, but as an example, it is 0.1 wt% or more and 60 wt% or less, preferably 0.5 wt% or more and 50 wt% or less based on the weight of wheat germ oil or olive oil. Less than wt% is suitable.
さらに、直接、化粧料成分として使用し、リパーゼ阻害作用を有する化粧料を製造することができる。化粧料としては特に限定されるものではないが、機能面からは、例えば乳液、化粧液、フェイスクリーム、ハンドクリーム、ローション、エッセンス、シャンプー、リンスなどが好ましい。 Furthermore, it can be directly used as a cosmetic ingredient to produce a cosmetic having a lipase inhibitory action. Although it does not specifically limit as cosmetics, From a functional surface, for example, a milky lotion, a cosmetic liquid, a face cream, a hand cream, a lotion, an essence, a shampoo, a rinse etc. are preferable.
このような化粧料は、常法に従って製造することができる。化粧料における本発明の化合物の添加量は、特に限定されるものではないが、一例としてあげると、化粧料全重量の0.01 重量%以上20 重量%以下程度が適当である。 Such a cosmetic can be produced according to a conventional method. The amount of the compound of the present invention to be added to the cosmetic is not particularly limited, but for example, it is suitably about 0.01% by weight to 20% by weight of the total weight of the cosmetic.
本発明の化合物は、その毒性は低く、毎日1000 mg/kg、100 日間という長期間に亘ってラットに経口投与しても、死亡例は認められず、体重変化も観察されなかった。 The compound of the present invention has low toxicity, and even when it was orally administered to rats over a long period of 1000 mg / kg every day for 100 days, no death was observed and no change in body weight was observed.
以下に実施例を挙げて、具体的に説明するが、これに限定されるものではない。 Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.
製造例[各種サポニンの抽出] サポニンの抽出は、以下に示す方法に従って行った。 すなわち、乾燥したマンシュウウコギ(Acanthopanax sessiliflorus)の葉10 kgを水80 Lを用いて90 ℃で1 時間抽出した。得られた抽出液をエバポレーターを用いて60 ℃でBrix10になるまで濃縮した後、入口温度140 ℃、出口温度80 ℃にて噴霧乾燥することによりマンシュウウコギ葉熱水抽出物を2.2kg得た。本抽出法による収率は、22 %(重量%)であった。
Production Example [Extraction of various saponins] Extraction of saponins was performed according to the following method. That is, 10 kg of dried leaves of Acanthopanax sessiliflorus were extracted with 80 L of water at 90 ° C. for 1 hour. The obtained extract was concentrated to
次に、上述の操作により得たマンシュウウコギ葉熱水抽出物(1.64 kg)を、酢酸エチル、n-ブタノール及び水を用いて分配することにより、酢酸エチル可溶画分(21.27 g)、n-ブタノール可溶画分(263.93 g)、水画分(1373.94 g)を得た。酢酸エチル可溶画分(21.27 g)をShepadex LH-20 カラムクロマトグラフィー(カラムサイズ:φ 4 cm× 20 cm)に供し、メタノールを用いて溶出し、18 個の画分を得、TLCにて溶出パターンを確認しながら4つの画分(S-1:9.69 g、S-2:5.69 g、S-3:4.63 g、S-4:0.25 g)に分けた。次にS-3 画分(4.63 g)をメタノールにて結晶化することにより、フラボノイドの一種であるヒペリン(hyperin)1.21 gを得た。次に、S-1 画分(9.69 g)をシリカゲル カラムクロマトグラフィー(カラムサイズ:φ 9.5 cm× 30 cm)に供し、クロロホルム:メタノール:水=75:25:5(v/v/v)混液及びクロロホルム:メタノール:酢酸エチル:水=20:20:40:10(v/v/v/v)混液で順次溶出し、さらに、ODS カラムクロマトグラフィー(カラムサイズ:φ 5.5 cm× 30 cm)にて70%(v/v)メタノールにて溶出することにより110 個の画分を得、このうちTLCにて溶出パターンを確認しながら38 個目から63 個目の画分を濃縮することにより本発明の化合物0.25gを得た。 Next, the hot water extract (1.64 kg) of Shirasugi leaves obtained by the above-mentioned operation was distributed using ethyl acetate, n-butanol and water, so that an ethyl acetate soluble fraction (21.27 g), n -A butanol-soluble fraction (263.93 g) and a water fraction (1373.94 g) were obtained. The ethyl acetate soluble fraction (21.27 g) was subjected to Shepadex LH-20 column chromatography (column size: φ 4 cm × 20 cm) and eluted with methanol to obtain 18 fractions. While confirming the elution pattern, it was divided into four fractions (S-1: 9.69 g, S-2: 5.69 g, S-3: 4.63 g, S-4: 0.25 g). Next, the S-3 fraction (4.63 g) was crystallized from methanol to obtain 1.21 g of hyperin, a kind of flavonoid. Next, the S-1 fraction (9.69 g) was subjected to silica gel column chromatography (column size: φ9.5 cm × 30 cm) and mixed with chloroform: methanol: water = 75: 25: 5 (v / v / v). And chloroform: methanol: ethyl acetate: water = 20: 20: 40: 10 (v / v / v / v) elute sequentially, and further to ODS column chromatography (column size: φ5.5 cm × 30 cm) Elute with 70% (v / v) methanol to obtain 110 fractions, of which the 38th to 63rd fractions were concentrated by confirming the elution pattern with TLC. 0.25 g of the inventive compound was obtained.
また、初期に分離したn-ブタノール可溶画分からも本発明の化合物を抽出することができる。n-ブタノール可溶画分(263.93 gのうち28.34 gを以下処理した)をSephadex LH-20 カラムクロマトグラフィー(カラムサイズ:φ 4 cm× 20 cm)に供し、メタノールにて溶出することにより33 個の画分を得、TLCにて溶出パターンを確認しながら、4つの画分(A-1:14.11 g、A-2:2.08 g、A-3:0.73 g、A-4:0.20 g)に分けた。A-1 画分をさらにシリカゲル カラムクロマトグラフィー(カラムサイズ:φ 9.5 cm× 30 cm)に供し、クロロホルム:メタノール:水=75:25:3(v/v/v)混液にて溶出することにより117 個の画分を得、TLCにて溶出パターンを確認しながら3つの画分(B-1:0.23 g、B-2:2.93 g、B-3:6.64 g)に分けた。続いて、B-3 画分をODS カラムクロマトグラフィー(カラムサイズ:φ 5.5 cm× 30 cm)に供し、メタノール:水=65:35(v/v)混液にて溶出することにより81 個の画分を得、TLCにて溶出パターンを確認しながら5つの画分(C-1:0.07 g、C-2:0.59 g、C-3:0.18 g、C-4:0.58 g、C-5:0.56 g)を得た。C-5 画分をさらにODS カラムクロマトグラフィー(カラムサイズ:φ 5.5 cm× 30 cm)に供し、アセトニトリル:水=45:55(v/v)混液にて溶出することにより96 個の画分を得、TLCにて溶出パターンを確認しながら25 個目から41 個目の画分を濃縮することにより本発明の化合物0.18 gが得られた。 In addition, the compound of the present invention can be extracted from an n-butanol soluble fraction separated in the initial stage. The n-butanol soluble fraction (28.34 g of 263.93 g was treated below) was subjected to Sephadex LH-20 column chromatography (column size: φ 4 cm × 20 cm) and eluted with methanol to obtain 33 fractions. 4 fractions (A-1: 14.11 g, A-2: 2.08 g, A-3: 0.73 g, A-4: 0.20 g) while confirming the elution pattern by TLC. divided. The A-1 fraction was further subjected to silica gel column chromatography (column size: φ9.5 cm × 30 cm), and eluted with chloroform: methanol: water = 75: 25: 3 (v / v / v) mixture. 117 fractions were obtained and divided into three fractions (B-1: 0.23 g, B-2: 2.93 g, B-3: 6.64 g) while confirming the elution pattern by TLC. Subsequently, the B-3 fraction was subjected to ODS column chromatography (column size: φ5.5 cm × 30 cm) and eluted with methanol: water = 65: 35 (v / v) mixture to obtain 81 fractions. 5 fractions (C-1: 0.07 g, C-2: 0.59 g, C-3: 0.18 g, C-4: 0.58 g, C-5: 0.56 g) was obtained. The C-5 fraction was further subjected to ODS column chromatography (column size: φ5.5 cm × 30 cm) and eluted with acetonitrile: water = 45: 55 (v / v) mixture to obtain 96 fractions. Then, by concentrating the 25th to 41st fractions while confirming the elution pattern by TLC, 0.18 g of the compound of the present invention was obtained.
得られた本発明の化合物をHMBC(Heteronuclear Multiple−Bond Correlation)法により解析した。HMBC法は2あるいは3共有結合離れた水素-炭素間の相関シグナルを検出することにより、部分構造を結合し分子構造を決定するのに極めて有効な手法である。本発明者はこの本発明の化合物を「セシロサイド」と命名した。相関図(図2)に基づいた化学的特性を、以下に示す。[3,4-seco-4(23),20(29)-lupadiene-4,28-dioic acid 28-O-α-L-rhamnopyranosyl(1→4)-β-D-glucopyranosyl(1→6)-β-D-glucopyranoside]収率:0.42 %、白色粉末、13C NMR:表1参照、1H NMR:表2参照、HR-FAB-MS(positive)m/z:941.5165、TOF-MS m/z :963.7087 [M+Na]+、979.6440 [M+K]+ The obtained compound of the present invention was analyzed by the HMBC (Heteruclear Multiple-Bond Correlation) method. The HMBC method is a very effective method for determining a molecular structure by binding partial structures by detecting a correlation signal between hydrogen and carbon separated by 2 or 3 covalent bonds. The inventor has named this compound of the invention “Cesiloside”. The chemical properties based on the correlation diagram (FIG. 2) are shown below. [3,4-seco-4 (23), 20 (29) -lupadiene-4,28-dioic acid 28-O-α-L-rhamnopyranosyl (1 → 4) -β-D-glucopyranosyl (1 → 6) -β-D-glucopyranoside] Yield: 0.42%, white powder, 13 C NMR: see Table 1, 1 H NMR: see Table 2, HR-FAB-MS (positive) m / z: 941.5165, TOF-MS m / z: 963.7708 [M + Na] + , 979.6440 [M + K] +
薬理試験[リパーゼ阻害活性試験]{基質(10%大豆油/アラビアゴムエマルジョン)の調製} アラビアゴム1 gを約5 mlの蒸留水に溶かし、5 Lの蒸留水で一晩透析する。その後、透析チューブからアラビアゴムを20 mlメートルグラスに出し、共洗いをしながら、20 mlにする。また、大豆油1 gを50 mlビーカーに秤り取る。これに、前述の5 %アラビアゴム10 mlを加え、スターラーでよく攪拌した。 Pharmacological test [Lipase inhibitory activity test] {Preparation of substrate (10% soybean oil / gum arabic emulsion)} 1 g of gum arabic is dissolved in about 5 ml of distilled water and dialyzed overnight against 5 L of distilled water. Then, the gum arabic is taken out from the dialysis tube into a 20 ml metric glass. Also, weigh 1 g of soybean oil into a 50 ml beaker. To this, 10 ml of 5% gum arabic was added and stirred well with a stirrer.
{銅混合液の調製} 6.45 % 硝酸銅(II)三水和物100 ml、1M トリエタノールアミン90 ml、1N 酢酸 10 mlを加えて、銅混合液200 mlを調製した。 {Preparation of Copper Mixture} 100 ml of 6.45% copper (II) nitrate trihydrate, 90 ml of 1M triethanolamine, and 10 ml of 1N acetic acid were added to prepare 200 ml of a copper mixture.
{発色試薬(0.1 %DEDC)の調製} N,N-ジエチルジチオカルバミン酸ナトリウム三水和物10 mgを、10 mlのブタノールに溶解させた。 {Preparation of coloring reagent (0.1% DEDC)} 10 mg of sodium N, N-diethyldithiocarbamate trihydrate was dissolved in 10 ml of butanol.
{阻害活性試験} リパーゼ阻害活性の測定は、K.Satouchiらの方法(Agric. Biol.Chem. 40巻、889〜897頁(1976年))に従い、Duncombe法により測定した。 {Inhibitory Activity Test} The lipase inhibitory activity was measured by the Duncombe method according to the method of K. Satouchi et al. (Agric. Biol. Chem. 40, 889-897 (1976)).
すなわち、0.5M Tris-HCl (pH 7.4)緩衝液50 mlに、基質として10%大豆油エマルジョン50 ml、5mM 酢酸カルシウム100 ml、実施例1で得られたそれぞれのサポニンの各種濃度(1、0.75、0.5、0.25 mg/ml)の水溶液100 mlを加えた後、蒸留水を加えて450 mlとする。これを37 ℃、65 r.p.m.で5分間、プレインキュベーションした後、リパーゼ(シグマ社製、ブタ膵臓由来)(1 mg/ml)を50 ml加えて、37 ℃、125 r.p.m.で20 分間、インキュベートした。その後、クロロホルム3.5 mlを加え上層を除去し、残りの下層に銅混合液1.5 mlを加え、コンセントレーターで15 分間攪拌した後、上層を除去する。残りの下層に発色試薬(0.1 %DEDC)を0.5 ml加えた後、遊離した脂肪酸の量を440 nmでの吸光度を測定することにより、リパーゼ阻害活性を測定した。その結果を図1に示した。(なお、□はヒペリン、●は本発明の化合物である3,4-seco-4(23),20(29)-lupadiene-4,28-dioic acid 28-O-α-L-rhamnopyranosyl(1→4)-β-D-glucopyranosyl(1→6)-β-D- glucopyranosideを表わす)。 That is, 50 ml of 0.5 M Tris-HCl (pH 7.4) buffer, 50 ml of 10% soybean oil emulsion as a substrate, 100 ml of 5 mM calcium acetate, and various concentrations (1, 0.75) of each saponin obtained in Example 1. , 0.5, 0.25 mg / ml), and then add distilled water to make 450 ml. This was preincubated at 37 ° C. and 65 r.p.m. for 5 minutes, 50 ml of lipase (Sigma, porcine pancreas) (1 mg / ml) was added, and the mixture was incubated at 37 ° C. and 125 r.p.m. for 20 minutes. Then, add 3.5 ml of chloroform to remove the upper layer, add 1.5 ml of the copper mixture to the remaining lower layer, and stir for 15 minutes with a concentrator, and then remove the upper layer. After adding 0.5 ml of a coloring reagent (0.1% DEDC) to the remaining lower layer, the amount of liberated fatty acid was measured for absorbance at 440 nm to measure lipase inhibitory activity. The results are shown in FIG. (Note that □ is hyperin, and ● is 3,4-seco-4 (23), 20 (29) -lupadiene-4,28-dioic acid 28-O-α-L-rhamnopyranosyl (1 → 4) -β-D-glucopyranosyl (1 → 6) -β-D-glucopyranoside).
図1からもわかるように、マンシュウウコギ葉熱水抽出物由来のフラボノイドであるヒペリン(hyperin)にリパーゼ阻害活性は認められなかったものの、本発明のサポニン、すなわち、3,4-seco-4(23),20 (29)-lupadiene-4,28-dioic acid 28-O-α-L-rhamnopyranosyl(1→4) - β- D-glucopyranosyl(1→6)-β-D-glucopyranoside に高いリパーゼ阻害活性を有することがわかる。 As can be seen from FIG. 1, the saponin of the present invention, that is, 3,4-seco-4 (), although lipase inhibitory activity was not observed in hyperin, a flavonoid derived from the extract of hot water extract of manshukogi. 23), 20 (29) -lupadiene-4,28-dioic acid 28-O-α-L-rhamnopyranosyl (1 → 4)-β-D-glucopyranosyl (1 → 6) -β-D-glucopyranoside high lipase It can be seen that it has an inhibitory activity.
[溶血活性試験]
実験方法
赤血球懸濁液の調製
ラットより採取した血液10mlにACD溶液0.5mlを加えて遠心分離(3000rpm、5min)を行い、赤血球を取り出した。遠心分離後、沈査に等張食塩水(8.9g/L)20mlを加えて遠心分離(3000rpm、5min)を行った。等張食塩水で2回洗浄した後、沈査に等張食塩水を加えて20mlにメスアップした。この赤血球懸濁液0.5mlに1%界面活性剤、TRITON:10g/L、4.5mlを加え撹拌し、遠心分離(3000rpm、5min)を行った。遠心分離後、上清を540nmでの吸光度を測定し、吸光度が0.8〜0.9になるように赤血球懸濁液を調製した。
溶血活性の測定
等張食塩水4.5mlに上記で調製した赤血球懸濁液0.5mlを加えゆっくり撹拌し、その後本発明のサポニンを加えてゆっくり撹拌し、インキュベーション(37℃、20min)した。次いで遠心分離(3000rpm、5min)を行い、得られた上清を540nmでの吸光度を測定した。なお、本試験では陽性対照群として大豆由来のサポニンを用いた。
結果
図3に示したように、陽性対照群としての大豆サポニンは濃度依存的に溶血活性を示し、100μg/mlで約90%の赤血球が破壊された。
一方、本発明のサポニンであるセシロサイドは100μg/mlの濃度において、約30%の溶血活性を示したが、大豆サポニンに比較して非常に弱い値であった。
以上のことから、本発明のサポニンであるセシロサイドは溶血活性が弱いことがわかる。
[Hemolytic activity test]
Experimental Method Preparation of Erythrocyte Suspension 0.5 ml of ACD solution was added to 10 ml of blood collected from rats and centrifuged (3000 rpm, 5 min) to remove erythrocytes. After centrifugation, 20 ml of isotonic saline (8.9 g / L) was added to the sediment and centrifuged (3000 rpm, 5 min). After washing twice with isotonic saline, isotonic saline was added to the sediment to make up to 20 ml. 1% surfactant, TRITON: 10 g / L, 4.5 ml was added to 0.5 ml of this erythrocyte suspension, and the mixture was stirred and centrifuged (3000 rpm, 5 min). After centrifugation, the absorbance of the supernatant was measured at 540 nm, and an erythrocyte suspension was prepared so that the absorbance was 0.8 to 0.9.
Measurement of hemolytic activity 0.5 ml of the erythrocyte suspension prepared above was added to 4.5 ml of isotonic saline, followed by slow stirring, and then the saponin of the present invention was added and stirred slowly, followed by incubation (37 ° C., 20 min). Then, centrifugation (3000 rpm, 5 min) was performed, and the absorbance of the obtained supernatant was measured at 540 nm. In this test, soybean-derived saponin was used as a positive control group.
Results As shown in FIG. 3, soybean saponin as a positive control group showed hemolytic activity in a concentration-dependent manner, and about 90% of erythrocytes were destroyed at 100 μg / ml.
On the other hand, the saponin, saponin of the present invention, exhibited a hemolytic activity of about 30% at a concentration of 100 μg / ml, but was very weak compared to soybean saponin.
From the above, it can be seen that ceciloside which is a saponin of the present invention has weak hemolytic activity.
[アミラーゼ活性阻害作用試験]
アミラーゼ活性の阻害作用は、Bernfeld. Pの方法(MethodsEnzymol., 1, 147-158 (1955))に従って、3,5-ジニトロサリチル酸を用いて反応液中の還元糖の量を定量することにより行った。
すなわち、膵液10μgを酵素として用い、基質として澱粉2mgを用い、反応により生成した還元糖の量を535nmでの吸光度を測定することにより求めた。
なお、比較対照群として、一般的にタンパク質である酵素と水素結合することにより不溶化を起こすことで酵素活性を阻害することが知られているタンニン酸(Loomis, W. D., MethodsEnzymol., 16, 528-544 (1974))を用いた。
(結果)
図4(A)に示したように、タンニン酸はアミラーゼ及びリパーゼの何れにおいても活性阻害作用を有することがわかるが、図4(B)に示したように、本発明のサポニンの一つであるセシロサイドはリパーゼ活性は阻害するもののアミラーゼ活性は阻害しないことから、本発明のサポニンであるセシロサイドはリパーゼ活性を特異的に阻害していることがわかる。
[Amylase activity inhibition test]
Inhibition of amylase activity is performed by quantifying the amount of reducing sugar in the reaction solution using 3,5-dinitrosalicylic acid according to the method of Bernfeld. P (Methods Enzymol., 1, 147-158 (1955)). It was.
That is, 10 μg of pancreatic juice was used as an enzyme, 2 mg of starch was used as a substrate, and the amount of reducing sugar produced by the reaction was determined by measuring the absorbance at 535 nm.
As a comparative control group, tannic acid (Loomis, WD, MethodsEnzymol., 16, 528-, which is known to inhibit enzyme activity by insolubilization by hydrogen bonding with enzyme, which is a protein, in general) 544 (1974)).
(result)
As shown in FIG. 4 (A), tannic acid is found to have an activity-inhibiting action in both amylase and lipase. However, as shown in FIG. 4 (B), tannic acid is one of the saponins of the present invention. A certain ceciloside inhibits lipase activity but not amylase activity, which indicates that saponin, saponin of the present invention, specifically inhibits lipase activity.
以下に、本発明の有効成分を使用した処方例を記載する。
処方例1
[錠剤の製造]
実施例1で得られた本発明の化合物を用いて、常法に従って、下記の組成の錠剤を製造した。
(組 成) (配合:重量%)
本発明の化合物 24
乳糖 63
コーンスターチ 12
グァーガム 1
Below, the formulation example using the active ingredient of this invention is described.
Formulation Example 1
[Manufacture of tablets]
Using the compound of the present invention obtained in Example 1, tablets having the following composition were produced according to a conventional method.
(Composition) (Composition: Weight%)
Compounds of the
Lactose 63
Cornstarch 12
処方例2
[ジュースの製造]
実施例1で得られた本発明の化合物を用いて、常法に従って、下記の組成のジュースを製造した。
(組 成) (配合:重量%)
冷凍濃縮温州みかん果汁 5.0
果糖ブドウ糖液糖 11.0
クエン酸 0.2
L-アスコルビン酸 0.02
香料 0.2
色素 0.1
本発明の化合物 0.2
水 83.28
Formulation Example 2
[Manufacture of juice]
Using the compound of the present invention obtained in Example 1, a juice having the following composition was produced according to a conventional method.
(Composition) (Composition: Weight%)
Frozen concentrated Wenzhou orange juice 5.0
Fructose glucose liquid sugar 11.0
Citric acid 0.2
L-ascorbic acid 0.02
Fragrance 0.2
Dye 0.1
Compound of the present invention 0.2
Wed 83.28
処方例3
[フェイスクリームの製造]
実施例1で得られた本発明の化合物を用いて、常法に従って、下記の組成のフェイスクリームを製造した。
(組 成) (配合:重量%)
イソステアリン酸イソプロピル 8.0
ホホバ油 6.0
セタノール 8.0
ステアリルアルコール 2.0
ポリオキシエチレンラウリルエーテル 1.5
プロピレングリコール 6.0
ソルビトール 1.0
パラベン 0.4
本発明の化合物 0.5
ビタミンE 0.5
香料 0.1
精製水 66.0
Formulation Example 3
[Manufacture of face cream]
Using the compound of the present invention obtained in Example 1, a face cream having the following composition was produced according to a conventional method.
(Composition) (Composition: Weight%)
Isopropyl isostearate 8.0
Jojoba oil 6.0
Cetanol 8.0
Stearyl alcohol 2.0
Polyoxyethylene lauryl ether 1.5
Propylene glycol 6.0
Sorbitol 1.0
Paraben 0.4
Compounds of the invention 0.5
Vitamin E 0.5
Fragrance 0.1
Purified water 66.0
Claims (9)
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KR100813297B1 (en) * | 2006-07-07 | 2008-03-13 | 박원봉 | A cosmetic composition for preventing human skin wrinkle by ultraviolet radiation |
KR101494436B1 (en) | 2013-04-23 | 2015-02-24 | 대한민국 | Novel compound from the fruits of Acanthopanax sessiliflorus |
CN114195843A (en) * | 2021-11-19 | 2022-03-18 | 吉林农业大学 | Split-ring lupane derivative and application thereof in preparation of multi-target inhibitor |
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CN114195843A (en) * | 2021-11-19 | 2022-03-18 | 吉林农业大学 | Split-ring lupane derivative and application thereof in preparation of multi-target inhibitor |
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