JPH07224093A - Peptide - Google Patents
PeptideInfo
- Publication number
- JPH07224093A JPH07224093A JP6037707A JP3770794A JPH07224093A JP H07224093 A JPH07224093 A JP H07224093A JP 6037707 A JP6037707 A JP 6037707A JP 3770794 A JP3770794 A JP 3770794A JP H07224093 A JPH07224093 A JP H07224093A
- Authority
- JP
- Japan
- Prior art keywords
- ile
- seq
- pro
- peptide
- leu
- 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.)
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- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、生理活性を有する新規
ペプチド及び該ペプチドを含有する医薬組成物に関する
ものである。TECHNICAL FIELD The present invention relates to a novel peptide having physiological activity and a pharmaceutical composition containing the peptide.
【０００２】[0002]
【従来の技術】食品蛋白質からは多様な生理活性を有す
るペプチドが派生することが知られている。食品起源で
あるこれらペプチドには生体に対する安全性が期待でき
る。一般に食品蛋白質由来の生理活性ペプチドは、内因
性生理活性ペプチドとは異なり予想もつかないようなア
ミノ酸配列を持つものが多く、またその機能についても
複数あることから、今なお確認されていないものも数多
くあるものと思われる。ペプチドの持つ生理活性作用の
１つに貪食（ファゴサイトーシス）促進作用がある。こ
の作用は生体内に細菌等の外的異物が進入してきた場
合、好中球やマクロファージによる生体防御の初発反応
として重要である。本発明者は先にファゴサイトーシス
を活性化するペプチドとして、大豆グリシニンＡ1aサブ
ユニットに含まれるGln-Arg-Pro-Arg やHis-Cys-Gln-Ar
g-Pro-Arg がマクロファージの貪食能を高めることにつ
いて証明し、新規なペプチドとして報告してきた。2. Description of the Related Art It is known that peptides having various physiological activities are derived from food proteins. These peptides originating from foods can be expected to be safe for the living body. In general, many bioactive peptides derived from food proteins have an unexpected amino acid sequence unlike the endogenous bioactive peptides, and there are also multiple functions, so many have not been confirmed yet. It seems that there is. One of the physiologically active actions of peptides is a phagocytosis promoting action. This action is important as an initial reaction of the biological defense by neutrophils and macrophages when external foreign substances such as bacteria enter the living body. The present inventor previously identified Gln-Arg-Pro-Arg and His-Cys-Gln-Ar contained in soybean glycinin A 1a subunits as peptides that activate phagocytosis.
We have demonstrated that g-Pro-Arg enhances phagocytosis of macrophages and reported it as a novel peptide.
【０００３】[0003]
【発明が解決しようとする課題】その後、大豆蛋白質か
ら由来するペプチドについて更なる研究を行ったとこ
ろ、新たに大豆蛋白酵素分解物中にファゴサイトーシス
促進活性を初めとして、他にも優れた生理活性効果を持
つペプチドを見いだした。またその類似化合物について
も検討し、上記ペプチドと同様な効果を有することを見
いだして本発明を完成した。従って、本発明は上記生理
活性を有する新規ペプチド及び該ペプチドの新規な食品
及び医薬用途を提供せんとするものである。After that, further research was conducted on peptides derived from soybean protein, and it was found that phagocytosis-promoting activity was newly added to soybean protein enzymatic degradation products, and other excellent physiological properties were obtained. We have found peptides with active effects. Further, the similar compound was also examined, and the present invention was completed by finding out that it has the same effect as the above peptide. Therefore, the present invention provides a novel peptide having the above-mentioned physiological activity and a novel food and pharmaceutical use of the peptide.
【０００４】[0004]
【課題を解決するための手段】本発明は、次式で示され
る配列番号１〜10のペプチドに関するものである。 配列番号１：Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro-Gly-Arg 配列番号２：Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro-Gly 配列番号３：Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro 配列番号４：Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys 配列番号５：Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn 配列番号６：Met-Ile-Thr-Leu-Ala-Ile-Pro-Val 配列番号７：Met-Ile-Thr-Leu-Ala-Ile-Pro 配列番号８：Met-Ile-Thr-Leu-Ala-Ile 配列番号９：Met-Ile-Thr-Leu-Ala 配列番号10：Met-Ile-Thr-Leu さらに本発明は、上記配列番号１〜10で示されるペプチ
ド及びその医薬上許容される塩を有効成分とする免疫系
賦活作用を有する医薬組成物に関するものである。上記
配列番号１〜10で示されるペプチドは、配列の長さ13〜
４で、いづれも 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：ペプチド の性状を有する。The present invention relates to peptides of SEQ ID NOs: 1 to 10 represented by the following formula. SEQ ID NO: 1: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro-Gly-Arg SEQ ID NO: 2: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn -Lys-Pro-Gly SEQ ID NO: 3: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro SEQ ID NO: 4: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val -Asn-Lys SEQ ID NO: 5: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn SEQ ID NO: 6: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val SEQ ID NO: 7: Met- Ile-Thr-Leu-Ala-Ile-Pro SEQ ID NO: 8: Met-Ile-Thr-Leu-Ala-Ile SEQ ID NO: 9: Met-Ile-Thr-Leu-Ala SEQ ID NO: 10: Met-Ile-Thr-Leu Furthermore, the present invention relates to a pharmaceutical composition having an immune system activating action, which comprises the peptides represented by SEQ ID NOS: 1 to 10 and pharmaceutically acceptable salts thereof as active ingredients. The peptides represented by SEQ ID NOs: 1 to 10 have a sequence length of 13 to
4, each has a sequence type: amino acid topology: linear sequence type: peptide.
【０００５】本発明の有効成分である配列番号１のペプ
チドは、大豆蛋白質を酵素加水分解し、得られた消化物
を、ＤＥＡＥ−セルロースカラムによるクロマトグラフ
ィー、さらにオクタデシリル（ＯＤＳ）カラム及びフェ
ネチルカラムによる高速液体クロマトグラフィー（ＨＰ
ＬＣ）によって分画することによって得ることができ
る。The peptide of SEQ ID NO: 1, which is an active ingredient of the present invention, is obtained by enzymatically hydrolyzing soybean protein, and the obtained digestion product is chromatographed by DEAE-cellulose column and further by octadesilyl (ODS) column and phenethyl column. High performance liquid chromatography (HP
It can be obtained by fractionating by LC).
【０００６】本発明者らは、大豆蛋白質の酵素分解によ
って得られる配列番号１のペプチドがファゴサイトーシ
ス促進作用を有することを見いだし、該ペプチドに基づ
いて更に研究を進めた結果、配列番号１のペプチド及び
その類似体である配列番号２〜10のペプチドもファゴサ
イトーシス促進作用を示し、また配列番号１〜８のペプ
チドが活性酸素産出促進作用を持つことを見いだした。
従って配列番号１〜10のペプチドは免疫系賦活作用を有
する医薬組成物として使用することができる。The present inventors have found that the peptide of SEQ ID NO: 1 obtained by enzymatic decomposition of soybean protein has a phagocytosis-promoting action, and as a result of further research based on the peptide, the results of SEQ ID NO: 1 It was found that the peptides and the peptides of SEQ ID NOS: 2 to 10, which are analogs thereof, also have an action of promoting phagocytosis, and the peptides of SEQ ID NOS: 1 to 8 have an action of promoting active oxygen production.
Therefore, the peptides of SEQ ID NOS: 1 to 10 can be used as a pharmaceutical composition having an immune system activating action.
【０００７】本発明のペプチドは上記の方法によって配
列番号１のペプチドを得、これを更に加水分解し、分画
して配列番号２〜10のペプチドを得ることができるほ
か、ペプチド合成機を用いて公知の方法によって配列番
号１〜10のペプチドを得ることもできる。The peptide of the present invention can be obtained by the above-mentioned method to obtain the peptide of SEQ ID NO: 1, which can be further hydrolyzed and fractionated to obtain the peptides of SEQ ID NO: 2-10. The peptides of SEQ ID NOS: 1 to 10 can also be obtained by known methods.
【０００８】[0008]
【製造例】以下に本発明の配列番号１〜10のペプチド製
造の一例を示すが、これらの例に限定されるものではな
い。PRODUCTION EXAMPLES Examples of the production of the peptides of SEQ ID NOS: 1 to 10 of the present invention are shown below, but the invention is not limited to these examples.
【０００９】製造例１ 消化酵素による配列番号１のペ
プチドの調製 Ａ．消化酵素による調製法の概略を下記表−１に示す。 表−１ 大豆蛋白質からの調製法の概略 大豆蛋白質 ↓ トリプシン消化 ↓ カラムクロマト（ＤＥＡＥセルロースカラム） ↓ ＨＰＬＣ（ＯＤＳ−カラム） ↓ ＨＰＬＣ（フェネチルカラム） ↓ ＨＰＬＣ（ＯＤＳ−カラム） ↓ Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro-Gly-Arg Production Example 1 Preparation of peptide of SEQ ID NO: 1 by digestive enzyme A. The outline of the preparation method using digestive enzymes is shown in Table 1 below. Table-1 Outline of preparation method from soybean protein Soybean protein ↓ Trypsin digestion ↓ Column chromatography (DEAE cellulose column) ↓ HPLC (ODS-column) ↓ HPLC (phenethyl column) ↓ HPLC (ODS-column) ↓ Met-Ile-Thr -Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro-Gly-Arg
【００１０】Ｂ．大豆蛋白質消化物からの配列番号１の
ペプチドの調製 分離大豆蛋白質50ｇを 800mlの水に溶解し、3000rpm ×
20分の遠心により可溶性画分を分離した。これを30分煮
沸し、 500mgのトリプシンを加え１Ｎ−ＮａＯＨでpH＝
7.6 に調整して、37℃で消化を行った。５時間後煮沸に
より消化を停止させ、 10000rpm ×15分の遠心分離によ
って得た上澄み液の凍結乾燥によってトリプシン消化物
（固形物量23.8ｇ）を得た。このうちの 100mgをＤＥＡ
Ｅ−セルロースカラム（ＤＥ−５２、ワットマン製、担
体２ml）にロードし20mMのトリス−塩酸緩衝液（pH＝7.
8 ）によって展開し、非吸着画分を１mlずつ分取したと
ころ、活性ペプチドは２ml〜３mlの位置に溶出する画分
に含まれていた。次にこの画分を、ＯＤＳ−カラム（Co
smosil 5C18-AR, 20 × 250mm、ナカライテスク製）、
続いてフェネチルカラム（4.7 × 250mm、Develosil Ph
A-T-5,野村化学製）にロードし、 0.1％トリフルオロ酢
酸を含むアセトニトリルの直線的濃度勾配（１％／min
）により展開した。これらカラムによる活性ペプチド
の溶出は、各々アセトニトリル33〜34％（図１参照）、
36〜37％の位置であった（図２参照）。さらにこの活性
画分をＯＤＳ−カラム（Cosmosil 5C18-AR, 4.6× 150
mm、ナカライテスク製）にロードし、10mMのリン酸緩衝
液（pH＝7.4 ）を含むアセトニトリルの直線的濃度勾配
（１％／min ）により展開したところ、活性ペプチドは
アセトニトリル34〜35％の位置に単一のピークとして溶
出した（図３参照）。プロテインシーケンサーにて構造
決定したところ、活性ペプチドはMet-Ile-Thr-Leu-Ala-
Ile-Pro-Val-Asn-Lys-Pro-Gly-Arg であることが判明し
た。B. Preparation of peptide of SEQ ID NO: 1 from digest of soybean protein Dissolve 50 g of isolated soybean protein in 800 ml of water, 3000 rpm ×
The soluble fraction was separated by centrifugation for 20 minutes. Boil this for 30 minutes, add 500 mg of trypsin, and add 1N-NaOH to obtain pH =
It was adjusted to 7.6 and digested at 37 ° C. After 5 hours, the digestion was stopped by boiling, and the tryptic digest (solid content: 23.8 g) was obtained by freeze-drying the supernatant obtained by centrifugation at 10,000 rpm for 15 minutes. 100 mg of this is DEA
E-cellulose column (DE-52, Whatman, carrier 2 ml) was loaded and 20 mM Tris-HCl buffer (pH = 7.
When the non-adsorbed fraction was developed by 8) and 1 ml of the non-adsorbed fraction was collected, the active peptide was contained in the fraction eluted at a position of 2 to 3 ml. Next, this fraction was subjected to ODS-column (Co
smosil 5C 18 -AR, 20 × 250mm, made by Nakarai Tesque),
Followed by a phenethyl column (4.7 x 250 mm, Develosil Ph
AT-5, manufactured by Nomura Chemical Co., Ltd., and linear concentration gradient of acetonitrile containing 0.1% trifluoroacetic acid (1% / min)
). Elution of active peptides by these columns was carried out by using 33-34% acetonitrile (see FIG. 1),
The position was 36 to 37% (see FIG. 2). Further, this active fraction was subjected to ODS-column (Cosmosil 5C 18 -AR, 4.6 × 150).
mm, manufactured by Nacalai Tesque, Inc., and developed with a linear concentration gradient (1% / min) of acetonitrile containing 10 mM phosphate buffer (pH = 7.4). Eluted as a single peak (see FIG. 3). When the structure was determined with a protein sequencer, the active peptide was Met-Ile-Thr-Leu-Ala-
It was found to be Ile-Pro-Val-Asn-Lys-Pro-Gly-Arg.
【００１１】製造例２ 化学合成法（Fmoc法）による配
列番号１のペプチドの調製 置換率 0.70meq／ｇの Fmoc-Arg(Pmc)- 樹脂 0.3ｇをＳ
ＡＭ２ペプチド合成装置（バイオサーチ社）の反応容器
にセットし、デブロック液（ピペリジン：トルエン：ジ
メチルホルムアミド（ＤＭＦ）＝30：35：35）を加え攪
拌して９−フルオレニルメトキシカルボニル（Fmoc）基
を除去した。この樹脂をジクロロメタン（ＤＣＭ）：Ｄ
ＭＦ＝１：１で洗浄後、 Fmoc-Arg(Pmc)- 樹脂の４倍当
量のヒドロキシベンゾトリアゾール（ＨＯＢＴ）及びFm
oc-Glyを加えてカップリングを行った。反応終了後、Ｄ
ＣＭ：ＤＭＦ＝１：１で洗浄し、Fmoc-Gly-Arg(Pmc)-樹
脂を得た。以下同様にしてＮ末端の Metまで合成し、Ｄ
ＣＭ、メタノールで洗浄し、Met-Ile-Thr(Bzl)-Leu-Ala
-Ile-Pro-Val-Asn(Trt)-Lys(Boc)-Pro-Gly-Arg(Pmc)-樹
脂のペプチドを得た。上記樹脂に脱保護剤（トリフロロ
酢酸：水：チオアニソール：エタンジオール：エチルメ
チルサルファイド：フェノール＝82：５：５：３：２：
３）を加えて、室温で４時間放置した。ついで分離した
樹脂を濾過後、エーテルで洗浄、凍結乾燥し、粗Met-Il
e-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro-Gly-Arg を得
た。これをＯＤＳ−カラム（Cosmosil 5C18-AR, 20 ×
250mm、ナカライテスク製）にロードし、 0.1％トリフ
ロロ酢酸を含むアセトニトリルの直線的濃度勾配による
ＨＰＬＣにて精製したところ約 200mgの純品が得られ
た。なお上記において、Pmc は２，２，５，７，８−ペ
ンタメチルクローマン−６−スルホニル基、Bzl はベン
ジル基、Trt はトリチル基、Boc はｔ−ブトキシカルボ
ニル基を示す。Production Example 2 Preparation of peptide of SEQ ID NO: 1 by chemical synthesis method (Fmoc method) 0.3 g of Fmoc-Arg (Pmc) -resin having a substitution rate of 0.70 meq / g was added to S.
Set it in the reaction vessel of AM2 peptide synthesizer (Biosearch), add deblocking solution (piperidine: toluene: dimethylformamide (DMF) = 30: 35: 35) and stir to mix 9-fluorenylmethoxycarbonyl (Fmoc). ) Group was removed. This resin was added to dichloromethane (DCM): D
After washing with MF = 1: 1, 4-fold equivalents of Fmoc-Arg (Pmc) -resin of hydroxybenzotriazole (HOBT) and Fm
Coupling was performed by adding oc-Gly. After the reaction is completed, D
After washing with CM: DMF = 1: 1, Fmoc-Gly-Arg (Pmc) -resin was obtained. Similarly, synthesize up to the N-terminal Met,
Washed with CM and methanol, Met-Ile-Thr (Bzl) -Leu-Ala
A peptide of -Ile-Pro-Val-Asn (Trt) -Lys (Boc) -Pro-Gly-Arg (Pmc) -resin was obtained. Deprotection agent (trifluoroacetic acid: water: thioanisole: ethanediol: ethylmethylsulfide: phenol = 82: 5: 5: 3: 2:
3) was added and the mixture was allowed to stand at room temperature for 4 hours. The separated resin was then filtered, washed with ether and lyophilized to give crude Met-Il.
e-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro-Gly-Arg was obtained. This is an ODS-column (Cosmosil 5C 18 -AR, 20 ×
250 mm, manufactured by Nacalai Tesque, Inc., and purified by HPLC with a linear concentration gradient of acetonitrile containing 0.1% trifluoroacetic acid to obtain about 200 mg of a pure product. In the above, Pmc represents a 2,2,5,7,8-pentamethylchroman-6-sulfonyl group, Bzl represents a benzyl group, Trt represents a trityl group, and Boc represents a t-butoxycarbonyl group.
【００１２】製造例３ 化学合成法（Fmoc法）による配
列番号２のペプチドの調製 置換率 0.55meq／ｇの Fmoc-Gly-樹脂 0.4ｇをＳＡＭ２
ペプチド合成装置（バイオサーチ社）の反応容器にセッ
トし、２と同様な方法で合成し相当する12残基ペプチド
−樹脂を得た。上記樹脂に脱保護剤（トリフロロ酢酸：
エタンジオール：アニソール＝94：１：５）を加えて、
室温で１時間放置した。以下製造例２と同様にして、約
150mgの純品のMet-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-
Lys-Pro-Gly を得た。Production Example 3 Preparation of peptide of SEQ ID NO: 2 by chemical synthesis method (Fmoc method) 0.4 g of Fmoc-Gly-resin having a substitution rate of 0.55 meq / g was added to SAM2.
It was set in a reaction vessel of a peptide synthesizer (Biosearch) and synthesized in the same manner as in 2 to obtain a corresponding 12-residue peptide-resin. Deprotection agent (trifluoroacetic acid:
Ethanediol: anisole = 94: 1: 5),
It was left at room temperature for 1 hour. Hereinafter, in the same manner as in Production Example 2, about
150 mg of pure Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-
Lys-Pro-Gly was obtained.
【００１３】製造例４ 化学合成法（Fmoc法）による配
列番号３のペプチドの調製 置換率 0.71meq／ｇの Fmoc-Pro-樹脂 0.3ｇをＳＡＭ２
ペプチド合成装置（バイオサーチ社）の反応容器にセッ
トし、２と同様な方法で合成し相当する11残基ペプチド
−樹脂を得た。以下製造例３と同様にして、約 150mgの
純品のMet-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro
を得た。Production Example 4 Preparation of peptide of SEQ ID NO: 3 by chemical synthesis method (Fmoc method) 0.3 g of Fmoc-Pro-resin having a substitution rate of 0.71 meq / g was added to SAM2.
It was set in a reaction vessel of a peptide synthesizer (Biosearch) and synthesized in the same manner as in 2 to obtain a corresponding 11-residue peptide-resin. Then, in the same manner as in Production Example 3, about 150 mg of pure Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro was prepared.
Got
【００１４】製造例５ 化学合成法（Fmoc法）による配
列番号４のペプチドの調製 置換率 0.46meq／ｇの Fmoc-Lys-樹脂 0.4ｇをＳＡＭ２
ペプチド合成装置（バイオサーチ社）の反応容器にセッ
トし、２と同様な方法で合成し相当する10残基ペプチド
−樹脂を得た。以下製造例３と同様にして、約 150mgの
純品のMet-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys を得
た。Production Example 5 Preparation of peptide of SEQ ID NO: 4 by chemical synthesis method (Fmoc method) 0.4 g of Fmoc-Lys-resin having a substitution rate of 0.46 meq / g was added to SAM2.
It was set in a reaction vessel of a peptide synthesizer (Biosearch) and synthesized in the same manner as in 2 to obtain a corresponding 10-residue peptide-resin. Then, in the same manner as in Production Example 3, about 150 mg of pure Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys was obtained.
【００１５】製造例６ 化学合成法（Fmoc法）による配
列番号５のペプチドの調製 置換率 0.70meq／ｇの Fmoc-Asn-樹脂 0.3ｇをＳＡＭ２
ペプチド合成装置（バイオサーチ社）の反応容器にセッ
トし、２と同様な方法で合成し相当する９残基ペプチド
−樹脂を得た。以下製造例３と同様にして、約 150mgの
純品のMet-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn を得た。Production Example 6 Preparation of peptide of SEQ ID NO: 5 by chemical synthesis method (Fmoc method) 0.3 g of Fmoc-Asn-resin having a substitution rate of 0.70 meq / g was added to SAM2.
It was set in a reaction vessel of a peptide synthesizer (Biosearch) and synthesized in the same manner as in 2 to obtain a corresponding 9-residue peptide-resin. Then, in the same manner as in Production Example 3, about 150 mg of pure Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn was obtained.
【００１６】製造例７ 酵素による配列番号５のペプチ
ドの調製 配列番号５のペプチドは、配列番号１のペプチドの酵素
による加水分解によっても得ることができる。用いる酵
素は配列番号１のペプチドに含まれるAsn-Lysのペプチ
ド結合を特異的に切断するメタロエンドペプチダーゼ、
アスパラギニルエンドペプチダーゼ等を使用する。以下
にその調製法の一例を示すが、使用する酵素はこの例に
限定されるものではない。配列番号１のペプチド20mg
に、500Uのメタロエンドペプチダーゼ（生化学工業製）
を含む 100mMのグリシン−ＮａＯＨ緩衝液（pH＝10.0）
２mlを加えて、70℃で５時間インキュベートした。反応
停止後この溶液をＯＤＳ−カラム（Cosmosil5C18-AR,
4.6 × 150mm）を用い、０．１％トリフロロ酢酸を含む
アセトニトリルの直線的濃度勾配によるＨＰＬＣによっ
て精製した。アセトニトリル34％付近に溶出する画分を
凍結乾燥したところ約10mgの純品の配列番号５のペプチ
ドを得た。Production Example 7 Preparation of Peptide of SEQ ID NO: 5 by Enzyme The peptide of SEQ ID NO: 5 can also be obtained by enzymatic hydrolysis of the peptide of SEQ ID NO: 1. The enzyme used is a metalloendopeptidase that specifically cleaves the peptide bond of Asn-Lys contained in the peptide of SEQ ID NO: 1,
Use asparaginyl endopeptidase or the like. An example of the preparation method is shown below, but the enzyme used is not limited to this example. SEQ ID NO: 1 peptide 20 mg
In addition, 500U of metalloendopeptidase (manufactured by Seikagaku Corporation)
100 mM glycine-NaOH buffer (pH = 10.0)
2 ml was added and incubated at 70 ° C. for 5 hours. After stopping the reaction, this solution was used for ODS-column (Cosmosil5C 18 -AR,
4.6 x 150 mm) and purified by HPLC with a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid. The fraction eluted near 34% acetonitrile was freeze-dried to obtain about 10 mg of the pure peptide of SEQ ID NO: 5.
【００１７】製造例８ 化学合成法（Fmoc）による配列
番号６のペプチドの調製 置換率 0.52meq／ｇの Fmoc-Val-樹脂 0.4ｇをＳＡＭ２
ペプチド合成装置（バイオサーチ社）の反応容器にセッ
トし、２と同様な方法で合成し相当する８残基ペプチド
−樹脂を得た。以下製造例３と同様にして、約 100mgの
純品のMet-Ile-Thr-Leu-Ala-Ile-Pro-Val を得た。Production Example 8 Preparation of peptide of SEQ ID NO: 6 by chemical synthesis method (Fmoc) 0.4 g of Fmoc-Val-resin having a substitution rate of 0.52 meq / g was added to SAM2.
It was set in a reaction vessel of a peptide synthesizer (Biosearch) and synthesized in the same manner as in 2 to obtain a corresponding 8-residue peptide-resin. Thereafter, in the same manner as in Production Example 3, about 100 mg of pure Met-Ile-Thr-Leu-Ala-Ile-Pro-Val was obtained.
【００１８】製造例９ 化学合成法（Fmoc）による配列
番号７のペプチドの調製 置換率 0.71meq／ｇの Fmoc-Pro-樹脂 0.3ｇをＳＡＭ２
ペプチド合成装置（バイオサーチ社）の反応容器にセッ
トし、２と同様な方法で合成し相当する７残基ペプチド
−樹脂を得た。以下製造例３と同様にして、約 100mgの
純品のMet-Ile-Thr-Leu-Ala-Ile-Pro を得た。Production Example 9 Preparation of peptide of SEQ ID NO: 7 by chemical synthesis method (Fmoc) 0.3 g of Fmoc-Pro-resin having a substitution rate of 0.71 meq / g was added to SAM2.
It was set in a reaction vessel of a peptide synthesizer (Biosearch) and synthesized in the same manner as in 2 to obtain a corresponding 7-residue peptide-resin. Thereafter, in the same manner as in Production Example 3, about 100 mg of pure Met-Ile-Thr-Leu-Ala-Ile-Pro was obtained.
【００１９】製造例10 化学合成法（Fmoc）による配列
番号８のペプチドの調製 置換率 0.55meq／ｇの Fmoc-Ile-樹脂 0.4ｇをＳＡＭ２
ペプチド合成装置（バイオサーチ社）の反応容器にセッ
トし、２と同様な方法で合成し相当する６残基ペプチド
−樹脂を得た。以下製造例３と同様にして、約 100mgの
純品のMet-Ile-Thr-Leu-Ala-Ile を得た。Production Example 10 Preparation of peptide of SEQ ID NO: 8 by chemical synthesis method (Fmoc) 0.4 g of Fmoc-Ile-resin having a substitution rate of 0.55 meq / g was added to SAM2.
It was set in a reaction vessel of a peptide synthesizer (Biosearch) and synthesized in the same manner as in 2 to obtain a corresponding 6-residue peptide-resin. Thereafter, in the same manner as in Production Example 3, about 100 mg of pure Met-Ile-Thr-Leu-Ala-Ile was obtained.
【００２０】製造例11 化学合成法（Fmoc）による配列
番号９のペプチドの調製 置換率 0.70meq／ｇの Fmoc-Ala-樹脂 0.3ｇをＳＡＭ２
ペプチド合成装置（バイオサーチ社）の反応容器にセッ
トし、２と同様な方法で合成し相当する５残基ペプチド
−樹脂を得た。以下製造例３と同様にして、約50mgの純
品のMet-Ile-Thr-Leu-Ala を得た。Production Example 11 Preparation of peptide of SEQ ID NO: 9 by chemical synthesis method (Fmoc) 0.3 g of Fmoc-Ala-resin having a substitution rate of 0.70 meq / g was added to SAM2.
It was set in a reaction vessel of a peptide synthesizer (Biosearch) and synthesized in the same manner as in 2 to obtain a corresponding 5-residue peptide-resin. Thereafter, in the same manner as in Production Example 3, about 50 mg of pure Met-Ile-Thr-Leu-Ala was obtained.
【００２１】製造例12 化学合成法（Fmoc）による配列
番号10のペプチドの調製 置換率 0.58meq／ｇの Fmoc-Leu-樹脂 0.3ｇをＳＡＭ２
ペプチド合成装置（バイオサーチ社）の反応容器にセッ
トし、２と同様な方法で合成し相当する４残基ペプチド
−樹脂を得た。以下製造例３と同様にして、約50mgの純
品のMet-Ile-Thr-Leu を得た。Production Example 12 Preparation of peptide of SEQ ID NO: 10 by chemical synthesis method (Fmoc) 0.3 g of Fmoc-Leu-resin having a substitution rate of 0.58 meq / g was added to SAM2.
It was set in a reaction vessel of a peptide synthesizer (Biosearch) and synthesized in the same manner as in 2 to obtain a corresponding 4-residue peptide-resin. Thereafter, in the same manner as in Production Example 3, about 50 mg of pure Met-Ile-Thr-Leu was obtained.
【００２２】配列番号１〜10までのペプチドのアミノ酸
組成、ＨＰＬＣの溶出位置、及び薄層クロマトグラフィ
ーのRf値を表−２にまとめて示した。尚、配列番号１の
天然品（製造例１）及び合成品（製造例２）は、それら
の結果から同一品であることが判明した。従って、以下
では合成品を用いて検討を行っている。Table 2 shows the amino acid compositions of the peptides of SEQ ID NOS: 1 to 10, the elution position of HPLC, and the Rf value of thin layer chromatography. The natural product (Production Example 1) and the synthetic product (Production Example 2) of SEQ ID NO: 1 were found to be the same product from the results. Therefore, in the following, studies are conducted using synthetic products.
【００２３】 上記表中、ＭはMet 、ＩはIle 、ＴはThr 、ＬはLeu 、
ＡはAla 、ＰはPro 、ＶはVal 、ＤはAsp 、ＫはLys 、
ＧはGly 、ＲはArg を示す。 *1 ６Ｎ−ＨＣｌ 110℃の加水分解後の値である。従っ
て、ペプチド組成のAsnはAsp として検出される。 *2 ＯＤＳ−カラム（Cosmosil 5C18-AR, 4.6×150mm
）を用いて、アセトニトリルの直線的濃度勾配により
測定している。 *3 メルク製のキーゼルゲルプレートを用い、室温にて
展開した。展開溶媒の組成は、ブタノール：酢酸：ピリ
ジン：水＝15:3:10:12である。[0023] In the above table, M is Met, I is Ile, T is Thr, L is Leu,
A is Ala, P is Pro, V is Val, D is Asp, K is Lys,
G represents Gly and R represents Arg. * 1 The value after hydrolysis with 6N-HCl at 110 ° C. Therefore, the peptide composition Asn is detected as Asp. * 2 ODS-column (Cosmosil 5C 18 -AR, 4.6 x 150 mm
) Is used for measurement with a linear concentration gradient of acetonitrile. * 3 It was developed at room temperature using a Kiesel gel plate made by Merck. The composition of the developing solvent is butanol: acetic acid: pyridine: water = 15: 3: 10: 12.
【００２４】[0024]
【試験例】以下、試験例により本発明ペプチドの薬理活
性を示す。 試験例１ ファゴサイトーシス活性の測定 試験方法： ヒト末梢血にリン酸緩衝液を含む生理食塩
水（ＰＢＳ）を加え、1000rpm−５分の遠心分離で血球
を洗浄したのち、ＰＢＳにて４×106 個／mlの血球の懸
濁液を調製した。この溶液 100μl を採り、ＰＢＳに溶
解させたペプチド溶液10μl を加え37℃で10分インキュ
ベートを行い、ついでヒト末梢血でオブソニル化した４
×108 個／mlの蛍光標識ラテックスビーズ液10μl を加
え、さらに５分インキュベートを行った。ＥＤＴＡを含
むＰＢＳで反応を停止させ、遠心により血球を分離し、
これに塩化アンモニウム溶血剤を加えて溶血させ白血球
を得た。これをＥＴＤＡを含むＰＢＳに懸濁後、フロー
サイトメトリーにて測定した。[Test Example] The pharmacological activity of the peptide of the present invention will be shown below by a test example. Test Example 1 Measurement of phagocytosis activity Test method: Physiological saline (PBS) containing phosphate buffer was added to human peripheral blood, and blood cells were washed by centrifugation at 1000 rpm for 5 minutes, and then 4 × with PBS. A suspension of 10 6 cells / ml was prepared. 100 μl of this solution was taken, 10 μl of the peptide solution dissolved in PBS was added, the mixture was incubated at 37 ° C. for 10 minutes, and then obsonated with human peripheral blood.
10 µl of a fluorescent labeled latex bead solution of × 10 8 cells / ml was added, and the mixture was further incubated for 5 minutes. Stop the reaction with PBS containing EDTA, separate the blood cells by centrifugation,
An ammonium chloride hemolyzing agent was added to this to cause hemolysis, and white blood cells were obtained. This was suspended in PBS containing ETDA and then measured by flow cytometry.
【００２５】結果： 数値はコントロール（ＰＢＳ）に対する倍率で示した。Results: Numerical values are shown as magnifications relative to the control (PBS).
【００２６】試験例２ 活性酸素産出量の測定 試験方法： 成人ヒト末梢血をＰＢＳにて洗浄した後、
赤血球を溶血させＨＥＰＥＳ−生理食塩緩衝液に溶かし
２×106 ／mlの好中球浮遊液を作製した。この液 125μ
l にＨＥＰＥＳ−生理食塩緩衝液 355μl 、５mMチトク
ロム−Ｃ溶液10μl 、及び10μl のペプチド水溶液を加
え、37℃で15分インキュベートした。15分後氷中にて急
冷することによりインキュベートを停止させた。この溶
液を遠心分離し、上澄み液の 550nm及び 468nmの吸光度
から活性酸素産出量を計算した。活性酸素産出量は下記
の計算式によって算出した。 活性酸素産出量＝（Ａ1 −Ａ2 ）×ｋ ただし式中、 Ａ1 は波長550nm による吸光度 Ａ2 は波長468nm による吸光度 ｋ は係数（測定に１cmセルを用いた場合の係数は95で
ある。）Test Example 2 Measurement of Active Oxygen Production Test Method: After washing adult human peripheral blood with PBS,
Erythrocytes were hemolyzed and dissolved in HEPES-physiological saline buffer to prepare a 2 × 10 6 / ml neutrophil suspension. This liquid 125μ
355 μl of HEPES-physiological saline buffer, 10 μl of 5 mM cytochrome-C solution, and 10 μl of an aqueous peptide solution were added to 1 l, and the mixture was incubated at 37 ° C. for 15 minutes. After 15 minutes, the incubation was stopped by quenching in ice. This solution was centrifuged, and active oxygen production was calculated from the absorbance of the supernatant at 550 nm and 468 nm. The amount of active oxygen produced was calculated by the following formula. Active oxygen production = (A 1 −A 2 ) × k where A 1 is the absorbance at wavelength 550 nm A 2 is the absorbance at wavelength 468 nm k is the coefficient (the coefficient when using a 1 cm cell for measurement is 95) .)
【００２７】結果： ペプチド濃度は30μM で、数値はコントロール（水）に
対する倍率で示した。Results: The peptide concentration was 30 μM, and the numerical value was shown as the ratio to the control (water).
【図１】酵素加水分解物をＤＥＡＥ−セルロースカラム
にロードして得られた活性画分を、ＯＤＳカラムによる
ＨＰＬＣにより溶離させた時の、該成分の吸光度を測定
したチャートである。FIG. 1 is a chart in which the absorbance of the component was measured when the active fraction obtained by loading the enzymatic hydrolyzate on a DEAE-cellulose column was eluted by HPLC with an ODS column.
【図２】図１のＯＤＳカラムによるＨＰＬＣの活性画分
を、フェネチルカラムによるＨＰＬＣにより溶離させた
時の、該成分の吸光度を測定したチャートである。FIG. 2 is a chart in which the absorbance of the component was measured when the active fraction of HPLC by the ODS column of FIG. 1 was eluted by HPLC by the phenethyl column.
【図３】図２のフェネチルカラムによるＨＰＬＣの活性
画分を、中性ＯＤＳカラムにより溶離させた時の、該成
分の吸光度を測定したチャートである。FIG. 3 is a chart in which the absorbance of the component when the active fraction of HPLC by the phenethyl column of FIG. 2 is eluted by the neutral ODS column.
フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｃ０７Ｋ 7/08 8318−4Ｈ Ｃ１２Ｐ 21/06 9282−4Ｂ Continuation of front page (51) Int.Cl. 6 Identification code Office reference number FI Technical display area C07K 7/08 8318-4H C12P 21/06 9282-4B
Claims (4)
理活性を有するペプチド。 配列番号１：Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro-Gly-Arg 配列番号２：Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro-Gly 配列番号３：Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro 配列番号４：Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys 配列番号５：Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn 配列番号６：Met-Ile-Thr-Leu-Ala-Ile-Pro-Val 配列番号７：Met-Ile-Thr-Leu-Ala-Ile-Pro 配列番号８：Met-Ile-Thr-Leu-Ala-Ile 配列番号９：Met-Ile-Thr-Leu-Ala 配列番号10：Met-Ile-Thr-Leu 1. A peptide having the physiological activity of SEQ ID NOs: 1 to 10 represented by the following formula. SEQ ID NO: 1: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro-Gly-Arg SEQ ID NO: 2: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn -Lys-Pro-Gly SEQ ID NO: 3: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro SEQ ID NO: 4: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val -Asn-Lys SEQ ID NO: 5: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn SEQ ID NO: 6: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val SEQ ID NO: 7: Met- Ile-Thr-Leu-Ala-Ile-Pro SEQ ID NO: 8: Met-Ile-Thr-Leu-Ala-Ile SEQ ID NO: 9: Met-Ile-Thr-Leu-Ala SEQ ID NO: 10: Met-Ile-Thr-Leu
されるペプチド及びその医薬上許容される塩よりなる群
から選択される１種又は２種以上を有効成分とする免疫
系賦活作用を有する医薬組成物。2. An immune system activating action which comprises, as an active ingredient, one or more selected from the group consisting of the peptides represented by SEQ ID NOS: 1 to 10 and pharmaceutically acceptable salts thereof according to claim 1. A pharmaceutical composition having.
進作用である請求項２記載の医薬組成物。3. The pharmaceutical composition according to claim 2, wherein the immune system activating action is a phagocytosis promoting action.
である、請求項１記載の配列番号１ないし８のペプチド
及びその医薬上許容される塩の１種又は２種以上を有効
成分とする請求項２記載の医薬組成物。4. An active ingredient comprising one or two or more of the peptides of SEQ ID NOs: 1 to 8 and pharmaceutically acceptable salts thereof, wherein the immune system activating action is an active oxygen production promoting action. The pharmaceutical composition according to claim 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6037707A JP2673659B2 (en) | 1994-02-11 | 1994-02-11 | peptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6037707A JP2673659B2 (en) | 1994-02-11 | 1994-02-11 | peptide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH07224093A true JPH07224093A (en) | 1995-08-22 |
| JP2673659B2 JP2673659B2 (en) | 1997-11-05 |
Family
ID=12505004
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP6037707A Expired - Lifetime JP2673659B2 (en) | 1994-02-11 | 1994-02-11 | peptide |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2673659B2 (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0873374A (en) * | 1994-09-05 | 1996-03-19 | Honen Corp | Medicinal composition having promoting action on tnf secretion |
| JP2002502368A (en) * | 1997-05-15 | 2002-01-22 | エラン コーポレーション ピーエルシー | Peptides that enhance the transport of active substances across tissues, compositions and methods of use |
| EP3117831A1 (en) * | 2015-07-16 | 2017-01-18 | Nuritas Limited | Peptides for use in promoting transport of glucose into skeletal muscle |
| WO2018101477A1 (en) * | 2016-12-01 | 2018-06-07 | 国立大学法人埼玉大学 | Endocytosis enhancer for drug delivery systems |
| CN111848735A (en) * | 2020-07-09 | 2020-10-30 | 中国科学院华南植物园 | Immunoregulation active peptide and preparation method and application thereof |
| US10905734B2 (en) | 2015-07-16 | 2021-02-02 | Nuritas Limited | Growth promoting peptides and uses thereof |
| US11253456B2 (en) | 2015-07-16 | 2022-02-22 | Nuritas Limited | Anti-inflammatory peptides, and uses thereof |
-
1994
- 1994-02-11 JP JP6037707A patent/JP2673659B2/en not_active Expired - Lifetime
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0873374A (en) * | 1994-09-05 | 1996-03-19 | Honen Corp | Medicinal composition having promoting action on tnf secretion |
| JP2002502368A (en) * | 1997-05-15 | 2002-01-22 | エラン コーポレーション ピーエルシー | Peptides that enhance the transport of active substances across tissues, compositions and methods of use |
| EP3932418A3 (en) * | 2015-07-16 | 2022-04-20 | Nuritas Limited | Peptides for use in promoting transport of glucose |
| US10905734B2 (en) | 2015-07-16 | 2021-02-02 | Nuritas Limited | Growth promoting peptides and uses thereof |
| WO2017009491A1 (en) * | 2015-07-16 | 2017-01-19 | Nuritas Limited | Peptides for use in promoting transport of glucose |
| US11779531B2 (en) | 2015-07-16 | 2023-10-10 | Nuritas Limited | Anti-inflammatory peptides, and uses thereof |
| CN108135963A (en) * | 2015-07-16 | 2018-06-08 | 努里塔斯有限公司 | For promoting the peptide of glucose transport |
| US11707500B2 (en) | 2015-07-16 | 2023-07-25 | Nuritas Limited | Growth promoting peptides, and uses thereof |
| EP3117831A1 (en) * | 2015-07-16 | 2017-01-18 | Nuritas Limited | Peptides for use in promoting transport of glucose into skeletal muscle |
| WO2017009487A1 (en) * | 2015-07-16 | 2017-01-19 | Nuritas Limited | Topical compositions |
| US10925922B2 (en) | 2015-07-16 | 2021-02-23 | Nuritas Limited | Growth promoting peptides, and uses thereof |
| US11253456B2 (en) | 2015-07-16 | 2022-02-22 | Nuritas Limited | Anti-inflammatory peptides, and uses thereof |
| US11510987B2 (en) | 2016-12-01 | 2022-11-29 | Saitama University | Endocytosis enhancer for drug delivery system |
| JPWO2018101477A1 (en) * | 2016-12-01 | 2019-10-24 | 国立大学法人埼玉大学 | Endocytosis enhancer for drug delivery system |
| WO2018101477A1 (en) * | 2016-12-01 | 2018-06-07 | 国立大学法人埼玉大学 | Endocytosis enhancer for drug delivery systems |
| CN111848735A (en) * | 2020-07-09 | 2020-10-30 | 中国科学院华南植物园 | Immunoregulation active peptide and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2673659B2 (en) | 1997-11-05 |
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