JP2011184423A - Dipeptide derived from cladosiphon okamuranus, l-isoleucyl-l-triptophan and antiangiogenic agent - Google Patents
Dipeptide derived from cladosiphon okamuranus, l-isoleucyl-l-triptophan and antiangiogenic agent Download PDFInfo
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Abstract
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本発明は、オキナワモズク由来のジペプチド及び血管新生阻害剤に関する。The present invention relates to a dipeptide derived from Okinawa mozuku and an angiogenesis inhibitor.
オキナワモズク由来のジペプチド、Ｌ−イソロイシル−Ｌ−トリプトファンは、血管新生阻害剤及び抗癌剤としての利点を持つ。
血管新生（ａｎｇｉｏｇｅｎｅｓｉｓ）は、既存の血管から新しい血管が形成される現象である。通常、血管新生の促進と抑制の両バランスは保たれているが、バランスが促進側に傾くと、血管新生が惹起される。血管新生と腫瘍の関わりとして、腫瘍がある一定以上の大きさ（１〜２ｎｍ３）になるには、腫瘍に栄養成分や酸素を供給するため血管新生が必要であることが唱えられ、血管新生の抑制による制ガンの可能性が始めて示唆された［非特許文献１］。その後、血管内皮細胞の培養系確立に伴い、血管新生研究が本格化し、種々の血管新生促進因子として、血管内皮増殖因子（ＶＥＧＦ：ｖａｓｃｕｌａｒ ｅｎｄｏｔｈｅｌｉａｌ ｇｒｏｗｔｈ ｆａｃｔｏｒ）、血小板由来内皮細胞増殖因子（ＰＤＥＣＧＦ）、繊維芽細胞増殖因子（ＦＧＦ：ｐｌａｔｅｌｅｔ ｄｅｒｉｖｅｄ ｅｎｄｏｔｈｅｌｉａｌ ｃｅｌｌ ｇｒｏｗｔｈ ｆａｃｔｏｒ）、腫瘍懐死因子（ＴＮＦ−α：ｔｕｍｏｒ ｎｅｃｒｏｔｉｃ ｆａｃｔｏｒ−α）、インターロイキン（ｉｎｔｅｒｌｅｕｋｉｎ８，ＩＬ８）等が次々に同定されてきた。腫瘍血管は、血管周皮細胞（ペリサイト）の欠如あるいは減少により、血管新生因子、特にＶＥＧＦの影響を受け易い状態にある。このため、未成熟な新生血管の形成が繰り返し行われている。腫瘍血管新生のメカニズムは、腫瘍などから分泌されたＶＥＧＦが内皮細胞、特にペリサイトが失われている内皮細胞膜上のＶＥＧＦ受容体（ＶＥＧＦＲ）に結合し、ＶＥＧＦＲのチロシンキナーゼドメインの活性化、自己リン酸化、細胞内シグナル伝達を経て、内皮細胞の増殖・遊走・管腔形成につながると考えられている［非特許文献２］。従って、血管新生抑制の観点からＶＥＧＦは最も有望な治療ターゲットといえる［非特許文献３］。このような背景のもとで開発されている血管新生抑制剤として特に注目を浴びている、例えば、組み換えヒト抗ＶＥＧＦであるアバスチン（ベバシズマブ；米国ジェネンテック社製）［非特許文献４］やＥＧＦＲ（上皮成長因子受容体）チロシンキナーゼ阻害剤であるイレッサ（ゲフィチニブ；アストラゼネカ社製）［非特許文献５］等が使用されてきてはいるが、細胞毒性が高く、副作用の発生が懸念されており、このような疾病の予防又は治療にあたっては、長期間に継続して行うことが必要なため、より効果的な血管新生の抑制と副作用の回避との両方を達成し得る血管新生抑制剤の開発が望まれている。本発明者等はこれまでオキナワモズク由来のフコイダンの免疫賦活作用［特許文献１］を報告してきてはいるが、オキナワモズク由来のジペプチドに血管新生阻害活性を有することは未だ知られていない。Angiogenesis is a phenomenon in which new blood vessels are formed from existing blood vessels. Normally, the balance between promotion and suppression of angiogenesis is maintained, but when the balance is tilted toward the promotion side, angiogenesis is induced. In relation to angiogenesis and tumors, it is advocated that angiogenesis is necessary to supply nutrients and oxygen to the tumor in order for the tumor to become a certain size (1-2 nm 3 ), and suppression of angiogenesis For the first time, the possibility of anti-cancer was suggested by [Non-Patent Document 1]. Then, with the establishment of a culture system for vascular endothelial cells, angiogenesis research has been in full swing, and various angiogenesis promoting factors such as vascular endothelial growth factor (VEGF), platelet-derived endothelial cell growth factor (PDECGF), Fibroblast growth factor (FGF), tumor necrosis factor (TNF-α), interleukin (IL8), etc. have been identified one after another. Tumor blood vessels are susceptible to angiogenic factors, particularly VEGF, due to the absence or reduction of vascular pericytes (pericytes). For this reason, immature new blood vessels are repeatedly formed. The mechanism of tumor angiogenesis is that VEGF secreted from tumors or the like binds to VEGF receptor (VEGFR) on endothelial cells, particularly endothelial cells where perisite is lost, activation of the tyrosine kinase domain of VEGFR, self It is thought to lead to proliferation / migration / luminal formation of endothelial cells through phosphorylation and intracellular signal transduction [Non-patent Document 2]. Therefore, VEGF can be said to be the most promising therapeutic target from the viewpoint of angiogenesis suppression [Non-Patent Document 3]. For example, Avastin (bevacizumab; manufactured by Genentech, USA) [Non-patent Document 4] or EGFR (recombinant human anti-VEGF), which has received particular attention as an angiogenesis inhibitor developed under such circumstances. Although epidermal growth factor receptor) tyrosine kinase inhibitor Iressa (gefitinib; manufactured by AstraZeneca) [Non-Patent Document 5] has been used, there is concern about the occurrence of side effects due to high cytotoxicity. In order to prevent or treat such diseases, it is necessary to continue for a long period of time. Therefore, the development of an angiogenesis inhibitor that can achieve both more effective suppression of angiogenesis and avoidance of side effects. Is desired. The present inventors have so far reported the immunostimulatory action of Fucoidan derived from Okinawa Mozuku [Patent Document 1], but it is not yet known that dipeptides derived from Okinawa Mozuku have angiogenesis inhibitory activity.
本発明者は、前記の課題を解決するために鋭意研究した結果、オキナワモズク由来のジペプチドが血管新生阻害効果を有することを見出し、本発明を完成するに至った。即ち、オキナワモズクから薬理作用を有する物質を検索したところ、オキナワモズク由来のジペプチドが強い血管新生阻害作用を有することを見出した。そして、オキナワモズク由来のジペプチドを医薬として実用化するための研究を鋭意行い、その結果、オキナワモズク由来のジペプチドが抗癌効果を有し、天然物由来の血管新生阻害剤としての有用性を見出した。本発明は係る知見に基づくものである。本発明に係るオキナワモズク由来のペプチドは、Ｌ−イソロイシル−Ｌ−トリプトファンで示されるジペプチド構造を有し、常温における性状は白色の粉末である。As a result of diligent research to solve the above-mentioned problems, the present inventor found that a dipeptide derived from Okinawa mozuku has an angiogenesis inhibitory effect, and has completed the present invention. That is, when a substance having a pharmacological action was searched from Okinawa mozuku, it was found that a dipeptide derived from Okinawa mozuku has a strong angiogenesis inhibitory action. As a result, we diligently researched the practical application of Okinawa Mozuku-derived dipeptides as pharmaceuticals. As a result, Okinawa Mozuku-derived dipeptides have anticancer effects and found usefulness as an angiogenesis inhibitor derived from natural products. It was. The present invention is based on such knowledge. The peptide derived from Okinawa Mozuku according to the present invention has a dipeptide structure represented by L-isoleucyl-L-tryptophan, and is white powder at room temperature.
本発明に係るオキナワモズク由来のジペプチドは、化学的に合成する方法、又はオキナワモズクから分離精製する方法を挙げることができる。本発明に係るオキナワモズク由来のジペプチドを化学的に合成する場合には、液相法または固相法等の通常の合成方法によって行うことができるが、好ましくは、固相法によってポリマー性の固相支持体へ当該ペプチドのカルボキシル末端側からそのアミノ酸残基に対応したＬ体のアミノ酸を順次ペプチド結合によって結合して行くのが良い。そして、そのようにして得られた合成ジペプチドは、トリフルオロメタンスルホン酸、フッ化水素などを用いてポロマー性の固相支持体から切断した後、アミノ酸側鎖の保護基を除去し、逆相系のカラムを用いた高速液体クロマトグラフィー（以下、ＨＰＬＣと略記する）などを用いた通常の方法で精製することができる。Examples of the dipeptide derived from Okinawa mozuku according to the present invention include a method of chemically synthesizing or a method of separating and purifying from Okinawa mozuku. In the case of chemically synthesizing the Okinawa-derived dipeptide according to the present invention, it can be carried out by an ordinary synthesis method such as a liquid phase method or a solid phase method. The L-form amino acid corresponding to the amino acid residue may be sequentially bound to the phase support by a peptide bond from the carboxyl terminal side of the peptide. The synthetic dipeptide thus obtained is cleaved from the porous solid support using trifluoromethanesulfonic acid, hydrogen fluoride, etc., and then the amino acid side chain protecting group is removed, and the reverse phase system is removed. And purified by a conventional method using high performance liquid chromatography (hereinafter abbreviated as HPLC).
上記したように、本発明に係るオキナワモズク由来のジペプチドは、オキナワモズクから分離精製することができるが、その場合には、例えば以下のようにして行うことができる。採集してきたオキナワモズクを、水で良く洗浄後、天日乾燥後、高速粉砕器を用いて微粉砕化してオキナワモズクの乾燥粉体を得る。この乾燥粉体に水、塩水溶液又は弱アルカリ水溶液を加えて湿式磨砕しながら可溶性成分を抽出し、この抽出液からタンパク質分離法によりオキナワモズク由来のタンパク質を得る。本発明で用いるタンパク質分離法としては、例えばエタノール、ポリエチレングリコールその他の有機溶剤や硫酸アンモニウムやトリクロロ酢酸を用いるタンパク質沈殿法、イオン交換体吸着法、等電点沈殿法、膜分離法などがあり、これらを単独又は併用して行うことができる。更にオキナワモズク由来のタンパク質の抽出率を高めるために、予めアルギン酸リアーゼ等の酵素による分解や洗浄などにより粘質多糖類を除去しておくのもよい。このようにして分離精製されたオキナワモズク由来のタンパク質はブロモシアン分解、プロテアーゼ消化などの常法によりペプチド鎖に切断した後、逆相系のカラムを用いたＨＰＬＣでペプチドフラグメントに単離精製することにより、オキナワモズク由来のジペプチドを得ることができる。As described above, the dipeptide derived from Okinawa mozuku according to the present invention can be separated and purified from Okinawa mozuku, and in that case, for example, it can be carried out as follows. The collected Okinawa mozuku is thoroughly washed with water, dried in the sun, and then finely pulverized using a high-speed pulverizer to obtain a dry powder of Okinawa mozuku. To this dry powder, water, a salt aqueous solution or a weak alkaline aqueous solution is added and a soluble component is extracted while wet grinding, and a protein derived from Okinawa mozuku is obtained from this extract by a protein separation method. Examples of protein separation methods used in the present invention include protein precipitation methods using ethanol, polyethylene glycol and other organic solvents, ammonium sulfate and trichloroacetic acid, ion exchanger adsorption methods, isoelectric precipitation methods, membrane separation methods, etc. Can be used alone or in combination. Furthermore, in order to increase the extraction rate of Okinawa mozuku-derived protein, it is also possible to remove the viscous polysaccharide in advance by degradation or washing with an enzyme such as alginate lyase. The Okinawa mozuku-derived protein thus separated and purified is cleaved into peptide chains by conventional methods such as bromocyanolysis and protease digestion, and then isolated and purified into peptide fragments by HPLC using a reversed-phase column. A dipeptide derived from Okinawa mozuku can be obtained.
本発明に係るオキナワモズク由来のジペプチドの製法において用いる藻類としては、本発明の目的を達成できる限りいかなる海藻及び微細藻類を用いても良いが、好ましくは褐藻類としてオキナワモズク、ワカメ、アラメ、コンブ、ヒジキ、ホンダワラ、アカモク、紅藻類としてノリ、及び緑藻類としてクロレラ、藍藻類としてスピルリナを用いるのが良い。以上のようにして得られた本発明に係るオキナワモズク由来のジペプチドは、静脈内へ繰り返し投与を行った場合、抗体産生を惹起せず、アナフィラキシーショックを起こさせない。又、本発明に係るオキナワモズク由来のジペプチドはＬ−アミノ酸のみの配列構造からなり、投与後、生体内のプロテアーゼにより徐々に分解される為、毒性は極めて低く、安全性は極めて高い（ＬＤ５０＞５０００ｍｇ／ｋｇ；ラット経口投与）。本発明に係るオキナワモズク由来のジペプチドは、通常用いられる賦形剤等の添加物を用いて注射剤、錠剤、カプセル剤、顆粒剤、散剤等に調製することができる。投与方法としては、通常は哺乳類（例えば、ヒト、イヌ、ラット等）に注射すること、あるいは経口投与することがあげられる。投与量は、例えば、動物体重当たり本発明に係るオキナワモズク由来のジペプチド０．０１〜１００ｍｇの量である。投与回数は、通常１日１〜４回程度であるが、投与経路によって、適宜、調製することができる。As the algae used in the method for producing the dipeptide derived from Okinawa mozuku according to the present invention, any seaweed and microalgae may be used as long as the object of the present invention can be achieved. , Hijiki, hondawala, akamoku, laver as red algae, chlorella as green algae, and spirulina as cyanobacteria. The Okinawa mozuku-derived dipeptide according to the present invention obtained as described above does not cause antibody production and does not cause anaphylactic shock when repeatedly administered intravenously. In addition, the Okinawa Mozuku-derived dipeptide according to the present invention has a sequence structure consisting of only L-amino acids, and is gradually degraded by protease in vivo after administration. Therefore, toxicity is extremely low and safety is very high (LD 50 > 5000 mg / kg; rat oral administration). The dipeptide derived from Okinawa mozuku according to the present invention can be prepared into injections, tablets, capsules, granules, powders, and the like using additives such as commonly used excipients. Examples of the administration method include injection into a mammal (eg, human, dog, rat, etc.) or oral administration. The dose is, for example, an amount of 0.01 to 100 mg of the dipeptide derived from Okinawa mozuku according to the present invention per animal weight. The number of administration is usually about 1 to 4 times a day, but can be appropriately prepared depending on the administration route.
上記の各種製剤において用いられる賦形剤、結合剤、潤沢剤の種類は、とくに限定されず、通常の注射剤、散剤、顆粒剤、錠剤あるいはカプセル剤に用いられるものを使用することができる。錠剤、カプセル剤、顆粒剤、散剤に用いる添加物としては、下記のものをあげることができる。賦形剤としては、結晶セルロース等の糖類、マンニトール等の糖アルコール類、デンプン類、無水リン酸カルシウム等；結合剤としてはでんぷん類、ヒドロキシプロピルメチルセルローズ等；崩壊剤としてはカルボキシメチルセルロースおよびそのカリウム塩類；潤滑剤としてはステアリン酸およびその塩類、タルク、ワックス類を挙げることができる。又、製剤の調整にあたっては必要に応じメントール、クエン酸およびその塩類、香料等の矯臭剤を用いることができる。注射用の無菌組成物は、常法により、本発明に係るオキナワモズク由来のジペプチドを、注射用水、生理食塩水およびキシリトールやマンニトールなどの糖アルコール注射液、プロピレングリコールやポリエチレングリコール等のグリコールに溶解または懸濁させて注射剤とすることができる。この際、緩衝液、防腐剤、酸化防止剤等を必要に応じて添加することができる。本発明に係るオキナワモズク由来のジペプチドを含有する製剤は凍結乾燥品又は乾燥粉末の形とし、用時、通常の溶解剤、例えば水または生理食塩液に溶解して用いることもできる。The types of excipients, binders, and lubricants used in the various preparations are not particularly limited, and those used for ordinary injections, powders, granules, tablets, or capsules can be used. Examples of additives used in tablets, capsules, granules, and powders include the following. Examples of excipients include sugars such as crystalline cellulose, sugar alcohols such as mannitol, starches, anhydrous calcium phosphate and the like; binders such as starches and hydroxypropylmethylcellulose; and disintegrants such as carboxymethylcellulose and potassium salts thereof; Examples of the lubricant include stearic acid and its salts, talc, and waxes. In preparation of the preparation, flavoring agents such as menthol, citric acid and salts thereof, and fragrance can be used as necessary. Aseptic composition for injection, the Okinawa Mozuku-derived dipeptide according to the present invention is dissolved in water for injection, physiological saline, sugar alcohol injection solution such as xylitol and mannitol, glycol such as propylene glycol and polyethylene glycol, etc. by a conventional method. Or it can be suspended and used as an injection. At this time, a buffer solution, a preservative, an antioxidant and the like can be added as necessary. The preparation containing the dipeptide derived from Okinawa mozuku according to the present invention is in the form of a lyophilized product or a dry powder, and can be used by dissolving it in a normal solubilizing agent such as water or physiological saline at the time of use.
本発明に係るオキナワモズク由来のジペプチドは、強力な血管新生抑制作用を有することにより抗癌活性を十分に発現させると同時に副作用の少ない抗癌剤として有用である。The dipeptide derived from Okinawa mozuku according to the present invention is useful as an anticancer agent with sufficient side effects while having sufficient anticancer activity due to its potent anti-angiogenic activity.
本発明に係るオキナワモズク由来のペプチドは、Ｌ−イソロイシル−Ｌ−トリプトファンで示されるジペプチド構造を有し、常温における性状は白色の粉末である。以下に実施例として、製造例及び試験例を記載し、本発明を更に詳細に説明するが、本発明はこれら実施例に限定されるものではない。The peptide derived from Okinawa Mozuku according to the present invention has a dipeptide structure represented by L-isoleucyl-L-tryptophan, and is white powder at room temperature. EXAMPLES Examples and test examples will be described below as examples, and the present invention will be described in more detail. However, the present invention is not limited to these examples.
製造例１
オキナワモズク（褐藻綱、ナガマツモ目、ナガマツモ科、オキナワモズク属）を乾燥させて調製した乾燥オキナワモズクを、高速粉砕器で３０メッシュに微粉砕化した。この微粉砕粉末３５ｇに０．１Ｎ水酸化ナトリウム溶液１Ｌ加えホモジナイズした。２４時間、室温にて撹拌後、遠心分離（１５０００ｒｐｍ、２０分間）し、更に珪藻土とろ紙（東洋ろ紙Ｎｏ．２）を用いて吸引ろ過しオキナワモズクエキス６３０ｍＬを得た。この溶液にエタノール６Ｌ添加し、低温室（−５℃）で１８時間放置してたんぱく画分を沈殿させ、再度、遠心分離（１５０００ｒｐｍ、２０分間）してオキナワモズク由来のたんぱく質の沈殿物を得た。この沈殿物を常法に準じてブロモシアン分解法又はプロテアーゼ消化法にてオキナワモズク由来のたんぱく質をペプチド鎖に切断した後、逆相ＨＰＬＣを行った。カラムとしては野村化学社製Ｄｅｖｅｌｏｓｉｌ ＯＤＳ−５（４．５ｍｍＩＤ×２５ｃｍＬ）を使用し、移動相としては０．０５％ＴＦＡから２５％アセトニトリル／０．０５％ＴＦＡの濃度勾配法を行い、流速１．０ｍＬ／ｍｉｎ検出波長２２０ｎｍでＨＰＬＣを行い、プロリルエンドペプチダーゼ阻害活性を有するペプチドフラグメントを単離精製することにより、オキナワモズク由来のペプチドを得ることができた。このようにして得られた血管新生阻害活性を有するペプチドフラグメントのアミノ酸配列は、ＡＢＩ社製のプロテインシークエンサー４７７Ａ型を用いて決定された。その結果、本発明に係るオキナワモズク由来のペプチドは、Ｌ−イソロイシル−Ｌ−トリプトファンで示されるジペプチド構造を有し、常温における性状は白色の粉末である。Production Example 1
A dried Okinawa mozuku prepared by drying Okinawa mozuku (Brown algae, Nagamatsumo, Nagamatsumo, Okinawa mozuku) was finely pulverized to 30 mesh with a high-speed pulverizer. 1 L of 0.1N sodium hydroxide solution was added to 35 g of this finely pulverized powder and homogenized. After stirring at room temperature for 24 hours, the mixture was centrifuged (15000 rpm, 20 minutes), and suction filtered using diatomaceous earth and filter paper (Toyo Filter Paper No. 2) to obtain 630 mL of Okinawa mozuku extract. 6 L of ethanol was added to this solution, and the protein fraction was allowed to stand for 18 hours in a low-temperature chamber (−5 ° C.), and centrifuged again (15000 rpm, 20 minutes) to obtain a protein precipitate derived from Okinawa Mozuku. It was. A protein derived from Okinawa mozuku was cleaved into peptide chains by bromocyan decomposition or protease digestion according to a conventional method, followed by reverse phase HPLC. As the column, Develosil ODS-5 (4.5 mm ID × 25 cmL) manufactured by Nomura Chemical Co., Ltd. was used, and as the mobile phase, a concentration gradient method of 0.05% TFA to 25% acetonitrile / 0.05% TFA was performed, and a flow rate of 1 By conducting HPLC at a detection wavelength of 220 nm at 0.0 mL / min and isolating and purifying a peptide fragment having prolyl endopeptidase inhibitory activity, a peptide derived from Okinawa mozuku could be obtained. The amino acid sequence of the thus obtained peptide fragment having angiogenesis inhibitory activity was determined using protein sequencer 477A manufactured by ABI. As a result, the Okinawa mozuku-derived peptide according to the present invention has a dipeptide structure represented by L-isoleucyl-L-tryptophan, and is white powder at room temperature.
尚、本発明に係るオキナワモズク由来のジペプチドを血管新生阻害剤として、例えば錠剤に製剤する場合には、常法に従って、例えば次のように処理すればよい：▲１▼オキナワモズク由来のジペプチド８ｇ、▲２▼乳糖７０ｇ、▲３▼コーンスターチ３２ｇ、▲４▼ステアリン酸マグネシウム１．４ｇを原料とし、先ず▲１▼、▲２▼及び１３ｇのコーンスターチを混和し、７ｇのコーンスターチから作ったペーストとともに顆粒化し、この顆粒に１４ｇのコーンスターチと▲４▼とを加え、得られた混合物を圧縮錠剤機で打錠し、錠剤１０００個を製造する。In addition, when the dipeptide derived from Okinawa mozuku according to the present invention is formulated as an angiogenesis inhibitor, for example, into a tablet, it may be treated according to a conventional method, for example, as follows: (1) 8 g dipeptide derived from Okinawa mozuku (2) 70g of lactose, (3) 32g of corn starch, (4) 1.4g of magnesium stearate After granulating, 14 g of corn starch and (4) are added to this granule, and the resulting mixture is compressed with a compression tablet machine to produce 1000 tablets.
製造例２
本例は、合成法による製造例である。
Ｌ−イソロイシル−Ｌ−トリプトファンの合成法
アプライドバイオシステム社製のペプチド自動合成装置４３０Ａ型を用いた固相法によって当該ペプチドを合成した。固相担体としては、スチレンジビニルベンゼン共重合体（ポリスチレン樹脂）をクロロメチル化した樹脂を使用した。まず、当該ペプチドのアミノ酸配列に従って、常法どおり、そのＣ末端側のトリプトファンからクロロメチル樹脂に反応させペプチド結合樹脂を得た。この時のアミノ酸は、ｔ−ブトキシカルボニル（以下、ｔ−Ｂｏｃと略記する）基で保護されたｔ−Ｂｏｃアミノ酸を使用した。次に、このペプチド結合樹脂をエタンジチオールとチオアニソールからなる混合液に懸濁し、室温で１０分間撹拌後、氷冷下でトリフルオロ酢酸（以下、ＴＦＡと略記する）を加え、更に１０分間撹拌した。この混合液にトリフルオロメタンスルホン酸を滴下し、室温で３０分間撹拌した後、無水エーテルを加えてその生成物を沈澱させて分離し、その沈澱物を無水エーテルで数回洗浄した後、減圧下で乾燥した。このようにして得られた未精製の合成ペプチドは蒸留水に溶解した後、逆相系のカラムＣ１８（５μｍ）を用いたＨＰＬＣにより精製した。移動相として（Ａ）０．１％ＴＦＡ含有蒸留水、（Ｂ）０．１％ＴＦＡ含有アセトニトリル溶液を使用し、（Ａ）液が９３分間で８６％→３４％の濃度勾配法により流速１．２ｍＬ／ｍｉｎでクロマトグラフィーを行った。紫外部波長２１８ｎｍで検出し、最大の吸収を示した溶出画分を分取し、これを凍結乾燥することによって目的とする合成ペプチド（Ｌ−イソロイシル−Ｌ−トリプトファン）を得た。Production Example 2
This example is an example of production by a synthesis method.
Synthesis Method of L-Isoleucil-L-Tryptophan The peptide was synthesized by a solid phase method using an automatic peptide synthesizer type 430A manufactured by Applied Biosystems. As the solid support, a resin obtained by chloromethylating a styrene divinylbenzene copolymer (polystyrene resin) was used. First, according to the amino acid sequence of the peptide, as usual, the C-terminal tryptophan was reacted with chloromethyl resin to obtain a peptide-bonded resin. As the amino acid at this time, a t-Boc amino acid protected with a t-butoxycarbonyl (hereinafter abbreviated as t-Boc) group was used. Next, this peptide-bonded resin is suspended in a mixed solution composed of ethanedithiol and thioanisole, stirred at room temperature for 10 minutes, added with trifluoroacetic acid (hereinafter abbreviated as TFA) under ice cooling, and further stirred for 10 minutes. did. Trifluoromethanesulfonic acid was added dropwise to the mixture, and the mixture was stirred at room temperature for 30 minutes. Then, anhydrous ether was added to precipitate the product, and the precipitate was washed several times with anhydrous ether. And dried. The crude synthetic peptide thus obtained was dissolved in distilled water and then purified by HPLC using a reverse phase system column C 18 (5 μm). (A) 0.1% TFA-containing distilled water and (B) 0.1% TFA-containing acetonitrile solution were used as the mobile phase, and (A) liquid flow rate was 1 by a concentration gradient method of 86% → 34% in 93 minutes. Chromatography was performed at 2 mL / min. The eluate fraction that was detected at the ultraviolet wavelength of 218 nm and showed the maximum absorption was collected and lyophilized to obtain the desired synthetic peptide (L-isoleucil-L-tryptophan).
この合成ペプチドをアミノ酸分析により分析した結果、アミノ酸配列が前記で示したアミノ酸配列構造を有するジペプチドであることが確認された。このような合成によって得られた本発明に係るオキナワモズク由来のジペプチドは、以下に示す試験によって薬理効果が確認された。As a result of analyzing this synthetic peptide by amino acid analysis, it was confirmed that the amino acid sequence was a dipeptide having the amino acid sequence structure shown above. As for the dipeptide derived from Okinawa mozuku according to the present invention obtained by such synthesis, the pharmacological effect was confirmed by the following test.
試験例１
（血管新生抑制作用）Ｔ．Ｂｉｓｈｏｐ等の方法［非特許文献６］により開発された倉敷紡績（株）製血管新生キット（Ａｎｇｉｏｇｅｎｅｓｉｓ Ｋｉｔ）を用いた。培養操作：ウェル培地中の本発明に係るフコキサンチンの最終濃度が３５μｇ／ｍＬとなるよう、及びウェル培地中ＶＥＧＦ（血管内皮成長因子：Ｖａｓｃｕｌａｒ Ｅｎｄｏｔｈｅｌｉａｌ Ｇｒｏｗｔｈ Ｆａｃｔｏｒ）の最終濃度が１０ｎｇ／ｍＬとなるよう調製した培地を３７℃、５％ＣＯ２雰囲気下で培養し、４日目、７日目、及び９日目で新しい培地への交換を行った。細胞層の固定と染色方法：培養開始１１日目に、各ウェルに対し、管腔染色キット（ＣＤ３１染色用）を用いて染色を行った。即ち、１次抗体添加後インキュベート（３７℃、６０分間）、次に２次抗体添加後インキュベート（３７℃、６０分間）した後、ＢＣＩＰ／ＮＢＴ（ブロモクロロインドリン酸／ニトロブルーテトラゾリウム）基質溶液を用いて染色し、管腔が深紫色になるまでインキュベート（３７℃）した。顕微鏡観察と画像の解析：管腔の染色画像を倉敷紡績製血管新生ソフトウェアＶｅｒ２を用いて、管腔の面積、長さ、管腔ジョイント数（分岐点の数）、管腔パス数（分岐して得られた枝の数）を、陽性対照ＶＥＧＦ添加区（コントロール）を１００％として再算出した。本発明に係るオキナワモズク由来のジペプチドのコントロールに対する％（ｎ＝３）を表１に示した。Test example 1
(Angiogenesis inhibitory effect) An angiogenesis kit (Angiogenesis Kit) manufactured by Kurashiki Boseki Co., Ltd. developed by the method of Bishop et al. Culturing procedure: final concentration of fucoxanthin according to the present invention in the well medium is 35 μg / mL, and final concentration of VEGF (Vascular Endothelial Growth Factor) in the well medium is 10 ng / mL The prepared medium was cultured at 37 ° C. in a 5% CO 2 atmosphere, and the medium was replaced with a new medium on the 4th, 7th, and 9th days. Cell layer fixation and staining method: On the 11th day from the start of the culture, each well was stained with a luminal staining kit (for CD31 staining). That is, after incubation (37 ° C., 60 minutes) after the addition of the primary antibody, and then incubation (37 ° C., 60 minutes) after the addition of the secondary antibody, a BCIP / NBT (bromochloroindophosphate / nitroblue tetrazolium) substrate solution was added. And stained (37 ° C.) until the lumen became deep purple. Microscopic observation and image analysis: Staining images of lumens using an angiogenesis software Ver2 made by Kurashikibo, lumen area, length, number of lumen joints (number of branch points), number of lumen paths (branching) The number of branches obtained) was recalculated with the positive control VEGF added group (control) as 100%. Table 1 shows the percentage (n = 3) of the Okinawa Mozuku-derived dipeptide according to the present invention relative to the control.
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