JP2764276B2 - Functional novel peptides and their use - Google Patents
Functional novel peptides and their useInfo
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- JP2764276B2 JP2764276B2 JP63221280A JP22128088A JP2764276B2 JP 2764276 B2 JP2764276 B2 JP 2764276B2 JP 63221280 A JP63221280 A JP 63221280A JP 22128088 A JP22128088 A JP 22128088A JP 2764276 B2 JP2764276 B2 JP 2764276B2
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Description
【発明の詳細な説明】 （産業上の利用分野） 本発明は、機能性を有する新規ペプチド及びその利用
に関するものである。The present invention relates to a novel peptide having functionality and its use.
更に詳細には、本発明は、魚介類を自己消化処理と蛋
白分解酵素処理を同時に行って得られたもので、分子量
が500〜6,000の機能性ペプチドに関するものである。More specifically, the present invention relates to a functional peptide having a molecular weight of 500 to 6,000, which is obtained by simultaneously performing autolysis and proteolytic enzyme treatment on fish and shellfish.
更には、本発明は、この機能性ペプチドを有効成分と
してなる高脂血症治療及び予防剤、糖尿病治療及び予防
剤、又は、血圧降下及び血管拡張剤に関するものであ
る。Furthermore, the present invention relates to a therapeutic and preventive agent for hyperlipidemia, a therapeutic and preventive agent for diabetes, or a hypotensive and vasodilator comprising the functional peptide as an active ingredient.
（従来技術及び問題点） 一般に、魚介類を酸、アルカリ、蛋白分解酵素等で加
水分解して呈味性の加水分解物を得ることは普通に行わ
れている。(Prior Art and Problems) Generally, it is common practice to hydrolyze fish and shellfish with acids, alkalis, proteolytic enzymes and the like to obtain a tasty hydrolyzate.
また、本発明者らは、先に、魚介類をまず自己消化分
解し、次いで蛋白分解酵素による処理を行い、新規なペ
プタイドを得ることができたのである（特願昭60−2268
34）。Furthermore, the present inventors have firstly obtained a novel peptide by subjecting fish and shellfish to autolytic digestion and then to treatment with a proteolytic enzyme (Japanese Patent Application No. 60-2268).
34).
ここに得られた新規なペプタイドについて各種試験を
したところ機能性においてすぐれたものがみられたので
あるが、製造法において、蛋白分解酵素の添加時期にむ
つかしい点があること、また、加水分解後の精製処理に
工業的にかなり困難な点があるなどの問題点があったの
である。Various tests were carried out on the novel peptide obtained here, and although some were found to be excellent in functionality, it was difficult to add the proteolytic enzyme in the production method. There are problems such as that there are industrially difficult points in the purification treatment of phenol.
また、魚介類から得られる各種ペプタイドについて
は、牛乳から得られる多くの機能性ペプタイド（New Fo
od Industry Vol.29,No.4（1987）29〜43頁）と同じ様
に、新らしい機能性ペプタイドの出現が期待されるので
ある。For various peptides obtained from fish and shellfish, many functional peptides obtained from milk (New Fo
As in od Industry Vol. 29, No. 4 (1987), pp. 29-43), the emergence of new functional peptides is expected.
（課題を解決するための手段） 本発明者らは、よりすぐれた機能性ペプチドを求めて
鋭意研究した結果、魚介類を自己消化処理と蛋白分解酵
素処理を同時に行うことによって、分子量500〜6,000の
新規な機能性ペプチドを得ることに成功したのである。(Means for Solving the Problems) As a result of intensive research for a better functional peptide, the present inventors have conducted simultaneous autolysis and proteolytic enzyme treatment of fish and shellfish, thereby obtaining a molecular weight of 500 to 6,000. Has been successfully obtained.
本発明は、魚介類を、自己消化処理と蛋白分解酵素処
理を同時に行って得られたもので、分子量が500〜6,000
の機能性ペプチドに関するものである。The present invention is obtained by simultaneously performing autolysis and proteolytic enzyme treatment on fish and shellfish, and has a molecular weight of 500 to 6,000.
The present invention relates to a functional peptide of
また、本発明の機能性ペプチドは、高脂血症の治療及
び予防剤、糖尿病の治療及び予防剤、血圧降下及び血管
拡張剤、肥満症の治療及び予防剤、動脈硬化症の治療及
び予防剤のそれぞれの有効成分となるものである。In addition, the functional peptide of the present invention may be used as a therapeutic or preventive agent for hyperlipidemia, a therapeutic or preventive agent for diabetes, a hypotensive or vasodilator, a therapeutic or preventive agent for obesity, or a therapeutic or preventive agent for arteriosclerosis. Are the active ingredients.
本発明の機能性ペプチドは、魚介類を原料として製造
するものであるが、先ず、これを採肉機、デボーナー等
によって処理して魚肉質を分離する。原料は出来る限り
新鮮なものが好ましい。分離した魚肉は、10kg程度のす
り身に分割し、このまま次の処理に使用してもよいが、
−20〜−50℃、例えば−30℃、程度の冷気を吹き付けて
急速凍結し、−20〜−25℃に保存しておき、必要に応じ
てこれを適宜使用するようにしてもよい。The functional peptide of the present invention is produced using fish and shellfish as a raw material. First, the functional peptide is treated by a meat extractor, a deboner or the like to separate fish meat. The raw materials are preferably as fresh as possible. The separated fish meat may be divided into about 10 kg of surimi and used as it is for the next processing,
The mixture may be rapidly frozen by blowing cold air at about -20 to -50 ° C, for example, about -30 ° C, and stored at -20 to -25 ° C, which may be used as needed.
魚介類としては、イワシ、アジ、マグロ、カツオ、サ
ンマ、サバ等赤身魚；ヒラメ、タイ、キス、コノシロ、
タラ、ニシン、ブリ等白身魚；サメ、エイ等軟骨魚肉；
ワカサギ、コイ、イワナ、ヤマメ等淡水魚肉；アイザ
メ、アンコウ等深海魚肉のほか、エビ、カニ、タコ、ア
ミ類、各種貝類等も適宜使用できる。As the seafood, sardines, horse mackerel, tuna, bonito, saury, mackerel, and other red fish; flounder, Thailand, kiss, conoshiro,
White fish such as cod, herring and yellowtail; cartilage fish such as sharks and rays;
Freshwater fish such as smelt, carp, char, and yamame; deep-sea fish such as shark and anglerfish, as well as shrimp, crab, octopus, mysids, and various shellfish can be used as appropriate.
本発明においては、採肉した後、粉砕機等によって魚
介肉を粉砕し、加水すると同時に蛋白分解酵素を添加
し、酵素適温（使用酵素によって異なるが、20〜60℃程
度）にまで加温し、pHも適値（pH3〜９程度）に調整
し、ゆるやかに撹拌しつつ、自己消化処理と蛋白分解酵
素処理に同時に行わせ、２〜20時間、好ましくは３〜５
時間程度で処理を停止するために、処理液を10〜15分煮
沸する。In the present invention, after the meat has been minced, the fish meat is crushed by a crusher or the like, and the proteolytic enzyme is added at the same time as the water is added. The pH is adjusted to an appropriate value (about pH 3 to 9), and the digestion is carried out simultaneously with the autolysis treatment and the protease treatment with gentle stirring, for 2 to 20 hours, preferably 3 to 5 hours.
The treatment solution is boiled for 10 to 15 minutes to stop the treatment in about an hour.
自己消化酵素については、魚介類を酵素処理するまで
加熱しないで保持させる。Regarding autolyzing enzymes, fish and shellfish are kept without heating until they are treated with the enzymes.
また、蛋白分解酵素としては、蛋白質を分解し得る酵
素であればすべての酵素が単独で又は混合して使用し得
る。その起源は、動植物のほか微生物に求めることがで
き、ペプシン、レニン、トリプシン、キモトリプシン、
パパイン、ブロメレインのほか、細菌プロテアーゼ、糸
状菌プロテアーゼ、放線菌プロテアーゼ等も広く利用で
きる。これらの酵素は、通常、市販されているものが使
用されるが、未精製の酵素、酵素を含有した培養液、麺
といった固体又は液体の酵素含有物も、目的により必要
に応じて使用することができる。Further, as the proteolytic enzyme, any enzyme can be used alone or in combination as long as it is an enzyme capable of decomposing proteins. Its origin can be found in microorganisms as well as animals and plants, and pepsin, renin, trypsin, chymotrypsin,
In addition to papain and bromelain, bacterial proteases, filamentous fungal proteases, actinomycetes proteases and the like can be widely used. As these enzymes, those which are commercially available are generally used, but unpurified enzymes, culture solutions containing the enzymes, solid or liquid enzyme contents such as noodles may also be used as necessary according to the purpose. Can be.
蛋白分解酵素の量は、酵素の精製度によって相違し、
また、処理時間によっても相違するが、処理液の0.05〜
１％、好ましくは0.1％程度の使用で十分である。The amount of protease depends on the degree of purification of the enzyme,
In addition, although it differs depending on the processing time, 0.05 to
Use of 1%, preferably about 0.1% is sufficient.
酵素反応停止処理液はバイブロスクリーン等によって
濾過し、必要によりジェクター処理した後、シャープレ
ス遠心分離機等を用いて例えば10,000〜30,000rpmで遠
心分離する。The treatment liquid for stopping the enzyme reaction is filtered through a vibroscreen or the like, and if necessary, subjected to a Jector treatment, and then centrifuged at, for example, 10,000 to 30,000 rpm using a Sharples centrifuge.
これを減圧濃縮等によって濃縮し（30Bx程度にまで）
た後、再度遠心分離してペプチド原液を得る。This is concentrated by vacuum concentration etc. (to about 30Bx)
After that, centrifugation is again performed to obtain a peptide stock solution.
こうして得たペプチド原液は、活性炭濾過、珪藻土濾
過等によって精製し、そのまま機能性ペプチド原液とし
て使用することができる。The peptide stock solution thus obtained can be purified by activated carbon filtration, diatomaceous earth filtration, or the like, and used directly as a functional peptide stock solution.
ここに得られる機能性ペプチド原液は、デキストリン
を添加もしくは添加せずに、凍結乾燥したり、噴霧乾燥
したりして、粉末化することもできるものである。The functional peptide stock solution obtained here can also be freeze-dried or spray-dried, with or without the addition of dextrin, to be powdered.
本発明の機能性ペプチドは、文献未載で新規なもので
あり、ESP−２と命名された。The functional peptide of the present invention is a novel peptide which has not been published yet and is named ESP-2.
ESP−２の物理化学的性質は次のとおりである。 The physicochemical properties of ESP-2 are as follows.
1.元素分析値 C;55.22％、H;5.83％、N;7.31％、 O;31.64％ 2.分子量 セファデックスＧ−50カラムクロマトグラフィーによ
り分子量500〜6,000と認められる。1. Elemental analysis value C; 55.22%, H; 5.83%, N; 7.31%, O; 31.64% 2. Molecular weight The molecular weight is recognized to be 500 to 6,000 by Sephadex G-50 column chromatography.
ゲル濾過による分子量分布図は第１図に示される。 The molecular weight distribution diagram by gel filtration is shown in FIG.
3.融 点 145℃分解 4.比旋光度 ▲［α］20 D▼＝19゜ 5.UVスペクトル 第２図のとおり。3. Decomposition at 145 ° C 4. Specific rotation ▲ [α] 20 D ▼ = 19 ＝ 5. UV spectrum As shown in Fig. 2.
6.IRスペクトル 第３図のとおり。6. IR spectrum As shown in Fig. 3.
7.溶剤に対する溶解性 水、メタノール、DMSO等極性溶媒に可溶であるが、ク
ロロホルム、ヘキサン等非極性溶媒に不溶 8.呈色反応 ニンヒドリン反応 ＋ ビウレット反応 ＋ 銅−フォーリン反応 ＋ フェノール硫酸反応 − 9.塩基性、酸性、中性の区別 微酸性、pH6.21（10％溶液） 10.物質の色、形状 黄白色粉末（凍結乾燥品） 11.水分含量 3.75％（常圧乾燥法） 12.塩分 3.97％（Clとして電位差滴定法により測定） 13.全窒素及びアミノ酸態窒素 Ｔ−N:14.04％（ミクロケールダール法） アミノ酸態−N:2.22％（ホルモール法） 本発明の機能性ペプチドは、各種の生理活性機能を有
している。7. Solubility in solvents It is soluble in polar solvents such as water, methanol and DMSO, but insoluble in non-polar solvents such as chloroform and hexane. 8. Color reaction Ninhydrin reaction + Biuret reaction + Copper-Forin reaction + Phenol sulfate reaction- 9.Basic, acidic, neutral Slightly acidic, pH 6.21 (10% solution) 10.Color and shape of substance Yellow-white powder (lyophilized product) 11.Moisture content 3.75% (normal pressure drying method) 12 Salt content 3.97% (measured by potentiometric titration as Cl) 13. Total nitrogen and amino acid nitrogen TN: 14.04% (Miclocale Dahl method) Amino acid form -N: 2.22% (Formol method) Functional peptide of the present invention Has various physiologically active functions.
本発明の機能性ペプチドは、後記する試験例からも明
らかなように高脂血症の治療及び予防、糖尿病の治療及
び予防、血圧降下及び血管拡張、肥満症の治療及び予
防、動脈硬化症の治療及び予防にすぐれた効果を示すの
で、これら疾病の予防、治療用医薬、輸液ないし栄養食
品、健康食品として広範に使用することができる。The functional peptide of the present invention can be used for the treatment and prevention of hyperlipidemia, the treatment and prevention of diabetes, the lowering and vasodilation of blood pressure, the treatment and prevention of obesity, the treatment and prevention of atherosclerosis, Since it shows excellent effects in treatment and prevention, it can be widely used as a medicine for preventing and treating these diseases, infusion or nutritional food, and health food.
医薬として使用する場合には、経口又は非経口投与す
ることができる。経口投与の場合には、例えば常法にし
たがい、錠剤、顆粒剤、粉末剤、カプセル剤、散剤とす
ることができ、又、非経口投与の場合には、例えば注射
薬製剤、点滴剤、坐剤として使用することができる。When used as a medicament, it can be administered orally or parenterally. In the case of oral administration, for example, tablets, granules, powders, capsules, and powders can be prepared in the usual manner. In the case of parenteral administration, for example, injection preparations, drops, suppositories It can be used as an agent.
また、本発明の機能性ペプチドは、卵黄と併用して経
口投与した場合、きわめて顕著な薬効が奏される。Further, when the functional peptide of the present invention is orally administered in combination with egg yolk, extremely remarkable drug effect is exhibited.
卵黄としては、鶏卵のほか、ウズラ、アヒルその他鳥
類の卵が主として使用されるが、他の動物の卵も適宜使
用できる。卵黄としては、卵黄自体をそのまま使用して
もよいが、水や生理的食塩水等溶媒で稀釈して卵黄液と
して使用してもよく、稀釈率は適宜自由に定めればよ
い。経済性と有効性とを両立せしめるには、約10〜50％
卵黄液とするのが好都合である。As egg yolk, besides chicken eggs, quail, duck and other bird eggs are mainly used, but eggs of other animals can also be used as appropriate. As the yolk, the yolk itself may be used as it is, or may be diluted with a solvent such as water or physiological saline and used as a yolk liquid, and the dilution ratio may be freely determined as appropriate. Approximately 10-50% to achieve both economic efficiency and effectiveness
It is convenient to use egg yolk fluid.
投与する場合は、卵黄（液）に機能性ペプチドを５〜
50％程度溶解させて用いてもよいし、両者を単に混合し
て用いてもよいし、両者をカプセルに入れ体内で混合す
るようにしてもよい。このように卵黄を用いれば経口投
与しても所期の薬効を得ることができる。In the case of administration, the functional peptide should be added
It may be used by dissolving it by about 50%, or both may be simply mixed and used, or both may be put in a capsule and mixed in the body. As described above, the use of egg yolk can provide the desired drug effect even when administered orally.
本発明の機能性ペプチドは、天然起源でありしかも食
品でもあるために毒性が全くないか又は極めて低く、き
わめて安全である（LD50＞3,000mg/kg皮下、＞5,000mg/
kg経口：いずれもラット）。The functional peptides of the present invention have no or very low toxicity because they are both natural and food, and are very safe (LD 50 > 3,000 mg / kg subcutaneous,> 5,000 mg / kg
kg oral: rats).
本発明の機能性ペプチドは、その種類、投与方法、患
者の症状、年令等によって異なるが、約0.1〜6000mg/kg
/日であり、１日に１〜４回投与するのが好ましい。な
お、予防目的のために健常人が服用する場合には、投与
量、投与回数等に格別の制限はない。また必要ある場合
には、他の薬剤との併用も可能である。The functional peptide of the present invention varies depending on its type, administration method, patient condition, age, etc., but is about 0.1 to 6000 mg / kg.
/ Day, preferably 1 to 4 times a day. In addition, when taken by a healthy person for the purpose of prevention, there is no particular limitation on the dose, the number of times of administration, and the like. If necessary, it can be used in combination with other drugs.
以下、本発明を、試験例、製造例、調剤例により差に
詳細に説明する。Hereinafter, the present invention will be described in detail with reference to test examples, production examples, and preparation examples.
試験例１ 製造例によってイワシから調製した機能性ペプチドES
P−２を用い、ラットを次の３群に分けて血圧降下効果
を試験した。Test Example 1 Functional peptide ES prepared from sardines according to Production Example
Using P-2, the rats were divided into the following three groups to test the blood pressure lowering effect.
１）静脈注射群 ２）生理食塩水溶解経口投与群 ３）30％卵黄水溶液経口投与群。 1) Intravenous injection group 2) Oral administration group with physiological saline solution 3) Oral administration group of 30% yolk aqueous solution.
ラットは、Hos:SHR（高血圧自然発症ラット）10週令
を星野実験動物（株）から購入し、これを１週間予備飼
育したものを使用した。Rats used were 10-week-old Hos: SHR (spontaneously hypertensive rats) purchased from Hoshino Experimental Animal Co., Ltd., and preliminarily bred for 1 week.
投与は、次のようにして行い、投与後、非観血的尾動
脈血圧装置（（株）理研開発製PS−100）を用いて経時
的に血圧を測定し、表−１の結果を得た。The administration was performed as follows. After the administration, the blood pressure was measured over time using a non-invasive tail artery blood pressure device (PS-100 manufactured by Riken Co., Ltd.), and the results in Table 1 were obtained. Was.
静注投与:117mg/kgの投与量になるように、機能性ペ
プチドESP−２を生理食塩水に溶解したもの1ml/animal
をラットに投与した。Intravenous administration: 1 ml / animal obtained by dissolving the functional peptide ESP-2 in physiological saline to give a dose of 117 mg / kg
Was administered to rats.
経口投与：（イ）機能性ペプチドESP−２の投与量が400
0mg/kgになるようにイワシペプチドを生理食塩水に溶解
したもの6ml/animalを投与した。Oral administration: (a) 400 dose of functional peptide ESP-2
6 ml / animal was prepared by dissolving the sardine peptide in physiological saline so as to be 0 mg / kg.
（ロ）機能性ペプチドESP−２の投与量が4000mg/kgにな
るようにイワシペプチドを30％卵黄液に溶解したもの6m
l/animalを投与した。(B) The sardine peptide dissolved in 30% egg yolk solution so that the dose of the functional peptide ESP-2 becomes 4000 mg / kg 6 m
l / animal was administered.
表１から次のことが判る。 Table 1 shows the following.
１）静脈内への投与で明らかに血圧降下効果が認めら
れた。1) The intravenous administration clearly showed a blood pressure lowering effect.
２）機能性ペプチドESP−２を卵黄に溶解して経口投
与した場合、血圧は顕著に降下した。2) When the functional peptide ESP-2 was dissolved in egg yolk and administered orally, the blood pressure significantly decreased.
試験例２ 本試験例では機能性ペプチドESP−２を卵黄に溶解し
た系で、投与量と血圧降下との関係を、Nifedipineを陽
性対照試料とし、試験例１と同様にSHRラットを用いて
試験した。Test Example 2 In this test example, in a system in which the functional peptide ESP-2 was dissolved in egg yolk, the relationship between the dose and the decrease in blood pressure was tested using Nifedipine as a positive control sample using SHR rats as in Test Example 1. did.
所定量のイワシペプチドを30％卵黄液になるように卵
黄水に溶解して経口投与した。A predetermined amount of sardine peptide was dissolved in yolk water to give a 30% yolk solution and orally administered.
投与後、非観血的尾動脈血圧装置（（株）理研開発製
PS−100）を用いて経時的に血圧を測定した。測定は５
回繰返して行ない、最高最低値を棄却し３点の平均を測
定値とし、第４図の結果を得た。After administration, non-invasive tail artery blood pressure device (manufactured by Riken Co., Ltd.)
Blood pressure was measured over time using PS-100). Measurement is 5
The maximum and minimum values were rejected, and the average of the three points was used as the measured value to obtain the results shown in FIG.
第４図から次のことが判る。 The following can be seen from FIG.
１）投与量の増加とともに血圧降下量は確実に増大し
た。1) The blood pressure lowering amount definitely increased with the increase of the dose.
２）投与後、約３時間まで降下量は増大し、投与後６
時間経過後も、なお降圧効果は持続していた。2) After administration, the amount of fall increases up to about 3 hours, and
Even after the passage of time, the antihypertensive effect continued.
対照試料として用いたNifedipineの場合は、投口30分
後で降圧量46.3mmHgと、急速に降圧したが、投与後３時
間後には、14.9mmHgへと減少し、６時間後には６頭中４
頭は投与前の血圧値にまで上昇してしまった。In the case of Nifedipine, which was used as a control sample, the blood pressure decreased rapidly to 46.3 mmHg 30 minutes after the injection, but decreased to 14.9 mmHg 3 hours after administration, and 4 of 6 animals 6 hours later.
The head had risen to the pre-dose blood pressure value.
試験例３ 機能性ペプチドESP−２の2,4,8および16g/animalを各
々20mlの精製水に溶解し、カニューレを用いて、個体別
にウサギに経口的に投与した。ウサギの耳は剃毛して、
血管を観察しやすくしてから投薬し、耳血管の変化を観
察して血管拡張試験を行った。Test Example 3 Each of 2, 4, 8, and 16 g / animal of the functional peptide ESP-2 was dissolved in 20 ml of purified water, and orally administered to rabbits individually using a cannula. Rabbit ears are shaved,
The medicine was administered after the blood vessels were easily observed, and changes in the ear blood vessels were observed to perform a vasodilation test.
ウサギはStd:NZW（ニュージーランドホワイト種）、1
4〜15週令、体重4.0±0.5kgの雄を静岡県実験動物農業
協同組合より購入し、１週間予備飼育した後、健常な動
物を試験に供した。Rabbit is Std: NZW (New Zealand White), 1
Males aged 4 to 15 weeks and weighing 4.0 ± 0.5 kg were purchased from the Shizuoka Prefectural Experimental Animal Agriculture Cooperative, bred for one week, and then tested for healthy animals.
観察の時期は、投薬前、20,30,60および120分とし、
その都度ウサギの耳を観察したところ、2gの投与で30分
後にはかなりの血管拡張がみとめられた。The time of observation is 20, 30, 60 and 120 minutes before medication,
In each case, the ears of the rabbits were observed, and significant vasodilation was observed 30 minutes after administration of 2 g.
試験例４ 高脂血症及び動脈硬化に対する作用 体重約220gのWistar系雄性ラットを１群６匹とし、粉
末飼料（CA−１、日本クレア社製）にコレステロールお
よびコール酸をそれぞれ１および0.5％混合したものを1
4日間摂取させ、同時に機能性ペプチドESP−２を500お
よび1000mg/kg、比較対照薬クロフィブレートを200mg/k
gをそれぞれ金属製胃ゾンデを用いて経口投与した。投
与期間終了後、上述の正常ラットの場合と同様に血液お
よび肝臓を採取して脂質含有量を測定した。Test Example 4 Effects on hyperlipidemia and arteriosclerosis Wistar male rats weighing about 220 g were grouped into 6 rats, and cholesterol and cholic acid were added to powder feed (CA-1, manufactured by CLEA Japan) at 1 and 0.5%, respectively. Mix 1
4 days, at the same time 500 and 1000 mg / kg of functional peptide ESP-2 and 200 mg / k of clofibrate for comparison
g was orally administered using a metal gastric tube. After the administration period, blood and liver were collected and the lipid content was measured as in the case of the normal rats described above.
結果は表２に示される。 The results are shown in Table 2.
試験例５ 糖尿病に対する作用 体重約180gのWistar系雄性ラットに0.01Mクエン酸バ
ッファー（pH4.5）に溶解したストレプトゾシン（Upjoh
n社製）を45mg/kg静脈内投与し、投与１週間後に尾静脈
より採血し、血中グルコース値が270〜330mg/dlの範囲
内にある動物をSTZ糖尿病ラットとみなした。このSTZ糖
尿病ラットを１群６匹とし、粉末飼料（CA−１、日本ク
レア社製）にDAN−100を1.25、2.5または５％の割合に
混合したものを40g/匹/day7日間摂取させた。投与期間
終了後、上述の正常ラットの場合と同様に血液および肝
臓を採取してグルコース量及び脂質含有量を測定した。 Test Example 5 Effect on Diabetes Streptozocin (Upjoh) dissolved in 0.01 M citrate buffer (pH 4.5) was administered to male Wistar rats weighing about 180 g.
n company) was administered intravenously at 45 mg / kg, and one week after administration, blood was collected from the tail vein, and animals whose blood glucose level was in the range of 270 to 330 mg / dl were regarded as STZ diabetic rats. The STZ diabetic rats were grouped into 6 rats, and a mixture of DAN-100 at a ratio of 1.25, 2.5 or 5% in powder feed (CA-1, manufactured by CLEA Japan) was ingested at 40 g / animal / day for 7 days. . After the end of the administration period, blood and liver were collected and the amount of glucose and the amount of lipid were measured in the same manner as in the case of the normal rats described above.
結果は表３に示される。 The results are shown in Table 3.
試験例６ 肥満症に対する作用 体重約300gの雄性Zucker（遺伝性肥満）ラット（faf
a）を１群３〜４匹とし、粉末飼料（CA−１、日本クレ
ア社製）にESP−２を５または10％の割合に混合したも
のを20g/匹/6day3ヶ月間摂取させた。投与期間終了後、
血液および肝臓を採取して脂質含有量を測定した。 Test Example 6 Effect on obesity Male Zucker (hereditary obesity) rat weighing about 300 g (faf
a) was divided into 3 to 4 animals per group, and powdered feed (CA-1, manufactured by CLEA Japan) was mixed with ESP-2 at a ratio of 5 or 10% at 20 g / animal / 6 days for 3 months. After the end of the administration period,
Blood and liver were collected to determine lipid content.
結果は表４に示される。 The results are shown in Table 4.
製造例１ 新鮮イワシをデボーナーで処理して採肉し、得られた
イワシ肉身10kgを粉砕機で粉砕した後、等量の水及びデ
ナチーム（市販プロテアーゼ製剤商品名）10gを加え、
よく混合し、45℃に４時間保持して自己消化及び酵素分
解を行った。反応液をpH7に中和し、次いで15分間煮沸
して酵素を失活せしめた。 Production Example 1 Fresh sardines were treated with a deboner to extract meat, and 10 kg of the obtained sardine meat was pulverized with a pulverizer.
The mixture was mixed well and kept at 45 ° C. for 4 hours to perform autolysis and enzymatic degradation. The reaction was neutralized to pH 7, then boiled for 15 minutes to inactivate the enzyme.
これをバイブロスクリーン（150メッシュ）で濾過
し、濾液を5000rpmでジェクター処理した後、シャープ
レス遠心分離機で処理し（15,000rpm）、20Bxとなるま
で常温加熱濃縮し、そして再度シャープレス遠沈処理
（15,000rpm）した。This was filtered through a vibro screen (150 mesh), and the filtrate was subjected to a Jector treatment at 5000 rpm, followed by a Sharpless centrifugal separator (15,000 rpm), concentrated by heating at room temperature to 20 Bx, and again centrifuged by Sharpless. (15,000 rpm).
得られた清澄液を減圧濃縮機にて４倍に濃縮し、濃縮
液を活性炭濾過し、次いで珪藻土濾過し、わずか黄色を
帯び透明な機能性ペプチドESP−２の溶液を得た。The obtained clarified solution was concentrated four-fold with a vacuum concentrator, and the concentrated solution was filtered with activated carbon and then with diatomaceous earth to obtain a slightly yellowish and transparent solution of the functional peptide ESP-2.
製造例２ 製造例１で得た機能性ペプチドESP−２をそのまま凍
結乾燥し、黄白色の機能性ペプチドESP−２を得た。Production Example 2 The functional peptide ESP-2 obtained in Production Example 1 was freeze-dried to obtain a yellow-white functional peptide ESP-2.
製造例３ 製造例１で得た機能性ペプチドESP−２溶液に５％の
デキストリンを加え、噴霧乾燥して、機能性ペプチドES
P−２乾燥粉末を得た。Production Example 3 5% dextrin was added to the functional peptide ESP-2 solution obtained in Production Example 1 and spray-dried to give a functional peptide ES.
P-2 dry powder was obtained.
製造例４ 凍結イワシ10kgを粉砕し、等量の水及びペプシン（市
販）8gを添加して、よく混合し、45℃で３時間保持し、
自己消化及び酵素分解を行った。反応液をpH7に調整
し、15分間煮沸して酵素を失活せしめた。Production Example 4 10 kg of frozen sardines were crushed, and equal amounts of water and 8 g of pepsin (commercially available) were added, mixed well, and kept at 45 ° C. for 3 hours.
Autolysis and enzymatic degradation were performed. The reaction was adjusted to pH 7 and boiled for 15 minutes to inactivate the enzyme.
反応液を冷却後、遠心分離し、分離液を限外濾過によ
り５倍に濃縮し、濃縮液を活性炭濾過し、次いで珪藻土
濾過し、わずか黄色を帯び透明な機能性ペプチドESP−
２の溶液を得た。After cooling, the reaction solution was centrifuged, the separated solution was concentrated 5 times by ultrafiltration, the concentrated solution was filtered with activated carbon, and then filtered with diatomaceous earth to give a slightly yellow and transparent functional peptide ESP-.
Solution 2 was obtained.
調剤例１ （１）ESP−２の凍結乾燥粉末（製造例２） 10g （２）塩化ナトリウム 9g （３）クロロブタノール 5g （４）炭酸水素ナトリウム 1g 全成分を蒸留水1000mlに溶解し、褐色アンプル1000個
に1mlずつ分注し、窒素ガスで置換して封入し、注射剤
を製造した。Preparation Example 1 (1) Lyophilized powder of ESP-2 (Production Example 2) 10 g (2) 9 g of sodium chloride (3) 5 g of chlorobutanol (4) 1 g of sodium bicarbonate 1 ml was dispensed into 1000 pieces, replaced with nitrogen gas, and sealed to produce injections.
調剤例２ （１）ESP−２の噴霧乾燥粉末（製造例３） 10g （２）卵黄粉末 3g （３）乳糖 87g （４）コーンスターチ 29g （５）ステアリン酸マグネシウム 1g （１），（２），（３）及び17g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5gのコーンスターチと（５）と
を加え、得られた混合物を圧縮錠剤機で打錠し、錠剤10
00個を製造した。Preparation Example 2 (1) Spray-dried powder of ESP-2 (Production Example 3) 10 g (2) Egg yolk powder 3 g (3) Lactose 87 g (4) Corn starch 29 g (5) Magnesium stearate 1 g (1), (2), (3) and 17 g of corn starch were mixed, granulated together with a paste made from 7 g of corn starch, 5 g of corn starch and (5) were added to the granules, and the resulting mixture was compressed with a compression tablet machine. Ten
00 pieces were produced.
調剤例３ ESP−２の溶液（製造例４）20kg、卵黄30kg、グラニ
ュー糖10kgに水を加えて全量を100kgとし、これを良く
混合した。Formulation Example 3 Water was added to 20 kg of a solution of ESP-2 (Production Example 4), 30 kg of egg yolk, and 10 kg of granulated sugar to make the total amount 100 kg, and these were mixed well.
次いで90〜95℃で15〜30秒間殺菌処理し、品温を70℃
程度にまで急冷し、予じめ殺菌した褐色ビンに100gずつ
充填し、直ちに密栓し、ドリンク剤1000本を製造した。Next, sterilize at 90-95 ° C for 15-30 seconds, and raise the product temperature to 70 ° C.
The mixture was quenched to the extent, filled into brown bottles previously sterilized in an amount of 100 g each, immediately sealed, and 1,000 drinks were produced.
第１図は機能性ペプチドESP−２のゲル濾過による分子
量分布を示す図で、第２図はESP−２のUVスペクトルを
示す図で、第３図はESP−２のIRスペクトルを示す図
で、第４図はESP−２の降圧効果を図示したグラフであ
る。FIG. 1 shows the molecular weight distribution of the functional peptide ESP-2 by gel filtration, FIG. 2 shows the UV spectrum of ESP-2, and FIG. 3 shows the IR spectrum of ESP-2. FIG. 4 is a graph illustrating the step-down effect of ESP-2.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 ＦＩ Ａ６１Ｋ 38/00 ＡＤＮ Ｃ１２Ｐ 21/06 // Ｃ１２Ｐ 21/06 Ａ６１Ｋ 37/18 ＡＤＮ ────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. 6 Identification code FI A61K 38/00 ADN C12P 21/06 // C12P 21/06 A61K 37/18 ADN
Claims (6)
チドESP−２。 （１）分子量:500〜6000 （セファデックスＧ−50カラムクロマトグラフィーによ
る） （２）融点:145℃（分解） （３）比旋光度：▲［α］20 D▼＝19゜ （４）微酸性:pH6.21（10％溶液） （５）物質の色・形状：黄白色粉末 （６）塩分:3.97％（Clとして電位差滴定法により測
定） （７）全窒素及びアミノ態窒素 全窒素:14.04％（ミクロケールダール法） アミノ態窒素:2.22％（ホルモール法）1. A functional peptide ESP-2 having the following physicochemical properties. (1) Molecular weight: 500-6000 (by Sephadex G-50 column chromatography) (2) Melting point: 145 ° C. (decomposition) (3) Specific rotation: ▲ [α] 20 D ▼ = 19 ゜ (4) Fine Acidity: pH 6.21 (10% solution) (5) Color and shape of substance: yellow-white powder (6) Salt content: 3.97% (measured by potentiometric titration as Cl) (7) Total nitrogen and amino nitrogen Total nitrogen: 14.04% (Mic locale Dahl method) Amino nitrogen: 2.22% (Formol method)
有効成分とする高脂血症の治療及び予防の組成物。2. A composition for treating and preventing hyperlipidemia comprising the functional peptide ESP-2 according to claim 1 as an active ingredient.
有効成分とする糖尿病の治療及び予防の組成物。3. A composition for treating and preventing diabetes, comprising the functional peptide ESP-2 of claim 1 as an active ingredient.
有効成分とする血圧降下及び血管拡張の組成物。4. A composition for lowering blood pressure and vasodilation comprising the functional peptide ESP-2 according to claim 1 as an active ingredient.
有効成分とする肥満症の治療及び予防の組成物。5. A composition for treating and preventing obesity, comprising the functional peptide ESP-2 of claim 1 as an active ingredient.
有効成分とする動脈硬化症の治療及び予防の組成物。6. A composition for treating and preventing arteriosclerosis, comprising the functional peptide ESP-2 according to claim 1 as an active ingredient.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63221280A JP2764276B2 (en) | 1988-09-06 | 1988-09-06 | Functional novel peptides and their use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63221280A JP2764276B2 (en) | 1988-09-06 | 1988-09-06 | Functional novel peptides and their use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02154693A JPH02154693A (en) | 1990-06-14 |
| JP2764276B2 true JP2764276B2 (en) | 1998-06-11 |
Family
ID=16764309
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63221280A Expired - Fee Related JP2764276B2 (en) | 1988-09-06 | 1988-09-06 | Functional novel peptides and their use |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2764276B2 (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2683529B2 (en) * | 1994-02-08 | 1997-12-03 | 静次 片野 | Gel food |
| JP2805194B2 (en) * | 1996-03-22 | 1998-09-30 | 阪急共栄物産株式会社 | Peptide for increasing blood triglyceride concentration and blood triglyceride concentration increase inhibitor containing the peptide as an active ingredient |
| JP2000264845A (en) * | 1999-02-04 | 2000-09-26 | Nippon Synthetic Chem Ind Co Ltd:The | Hypocholesterolemic agent and its use |
| TWI225070B (en) | 1999-12-01 | 2004-12-11 | Food Industry Res & Dev Inst | Novel peptides used as angiotensin converting enzyme inhibitor and preparation process thereof |
| JP4862235B2 (en) * | 2001-02-27 | 2012-01-25 | Ｊｎｃ株式会社 | Blood sugar level rise inhibitor |
| NO322425B1 (en) * | 2003-07-04 | 2006-10-02 | Berge Biomed As | Use of a hydrolyzate of proteinaceous fish material for the preparation of a pharmaceutical agent for the treatment and / or prevention of pathologically high levels of tracylglycerols, hypercholesterolemia, hyperhomocysteinemia, or pathologically low levels of beta-oxidation in an animal or human. |
| EP1824501B1 (en) * | 2004-11-15 | 2009-08-19 | Mg Pharma Inc. | Protein hydrolysate with antidiabetic effect |
| KR100763938B1 (en) * | 2004-12-30 | 2007-11-28 | 부경대학교 산학협력단 | Hydrolysates from Alaska pollack frame |
| JP5589176B2 (en) * | 2008-06-13 | 2014-09-17 | 国立大学法人佐賀大学 | New peptide degradation product |
| JP6207863B2 (en) * | 2013-04-09 | 2017-10-04 | 仙味エキス株式会社 | Allergy resistance acquisition agent |
| WO2015056619A1 (en) * | 2013-10-16 | 2015-04-23 | 日本水産株式会社 | Peptide or acid addition salt thereof, food and beverage, and composition for preventing diabetes and the like |
| JP6251427B2 (en) * | 2017-02-03 | 2017-12-20 | 仙味エキス株式会社 | Food composition for acquiring fish allergy resistance |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6287058A (en) * | 1985-10-14 | 1987-04-21 | Osajima Kazuharu | Novel peptide |
-
1988
- 1988-09-06 JP JP63221280A patent/JP2764276B2/en not_active Expired - Fee Related
Also Published As
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| JPH02154693A (en) | 1990-06-14 |
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