JPH0228118A - Production of immunologically active peptide - Google Patents
Production of immunologically active peptideInfo
- Publication number
- JPH0228118A JPH0228118A JP63178447A JP17844788A JPH0228118A JP H0228118 A JPH0228118 A JP H0228118A JP 63178447 A JP63178447 A JP 63178447A JP 17844788 A JP17844788 A JP 17844788A JP H0228118 A JPH0228118 A JP H0228118A
- Authority
- JP
- Japan
- Prior art keywords
- active peptide
- immunologically active
- acid
- animal
- bone
- 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.)
- Pending
Links
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Landscapes
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Abstract
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は、免疫活性ペプチドの製造方法に関する。[Detailed description of the invention] [Industrial application field] The present invention relates to a method for producing an immunoactive peptide.
近年、社会の高齢化に伴い、加齢による各種骨関節疾患
に随伴した、いわゆる老化性骨粗鬆症(オステオポロー
ゼ)、すなわち骨萎縮を起こすことが知られている。In recent years, with the aging of society, it has been known that so-called senile osteoporosis (osteoporosis), that is, bone atrophy, is associated with various bone and joint diseases due to aging.
生骨髄カルシウムについて、老化性骨粗鬆症に対する有
効性が指摘されている(天井および均温、新薬と臨床、
↓ユ、1(1984))。しかしながら、カルシウムの
吸収、蓄積、代謝、排泄等の経路は、複雑であり生体内
では、これらが各種ホルモン、ペプチドにより調節をう
けていることが、示唆されている。It has been pointed out that raw bone marrow calcium is effective against senile osteoporosis (ceiling and isotherm, new drugs and clinical studies,
↓Yu, 1 (1984)). However, the pathways of absorption, accumulation, metabolism, excretion, etc. of calcium are complex, and it has been suggested that these are regulated in vivo by various hormones and peptides.
現在、多種多様の生理活性ペプチドが見出だされ、それ
らの細胞内カルシウム調節に対する作用がン主目されて
いる。At present, a wide variety of physiologically active peptides have been discovered, and their effects on intracellular calcium regulation are the focus of attention.
しかしながら、骨および骨髄中の生理活性ペプチドの存
在については、全く検討されておらず、現在なお不明の
ままである。However, the presence of bioactive peptides in bone and bone marrow has not been investigated at all and remains unclear at present.
天然補助食品の効果を検討するうえにもその存在の有無
の検索は、重要な基礎的な情報を提供する物と考える。We believe that searching for the presence or absence of natural supplements provides important basic information when examining the effects of natural supplements.
本発明は、骨および骨髄の微粉砕物中に含まれる各種免
疫活性ペプチド成分の製造をその目的とするものである
。The object of the present invention is to produce various immunoactive peptide components contained in finely ground bone and bone marrow.
即ち本発明は、鳥獣骨または骨髄を含有する粉砕物を酸
水溶液と接触させ、酸水溶液中に免疫活性ペプチドを抽
出することを特徴とする、免疫活性ペプチドの製造方法
をその要旨とするものである。That is, the gist of the present invention is a method for producing an immunoactive peptide, which is characterized by contacting a pulverized product containing bird and animal bones or bone marrow with an aqueous acid solution, and extracting the immunoactive peptide into the aqueous acid solution. be.
鳥獣骨または骨髄を含有する粉砕物における、鳥獣とし
ては、鶏、豚、猪、牛等を挙げることができる。Examples of the birds and animals in the crushed material containing bird and animal bones or bone marrow include chickens, pigs, boars, and cows.
鳥獣骨または骨髄を含有する粉砕物の製造方法としては
、たとえば、鳥獣の頭部と四肢を除去した鳥獣骨を、約
2°C以下の温度下で粗粉砕し、液体アンモニウム冷却
下で凍結し、粉砕したのち、脱窒素化し、マイクロ波お
よび熱風等により乾燥する方法等を挙げることができる
。As a method for producing a crushed product containing bird and animal bones or bone marrow, for example, bird and animal bones from which the head and limbs have been removed are coarsely ground at a temperature of about 2°C or less, and then frozen under liquid ammonium cooling. Examples include a method of pulverizing, denitrifying, and drying using microwaves, hot air, or the like.
また、食肉処理工場において抜管した牛骨を約20゛C
に冷却し、出口孔径6.5胴のチョンパーにより、牛骨
細片とし、これの約100部に対し55部の氷水を加え
、さらに出口孔径約2.5 mmのチゴッパーにより細
片化し、次いでこれの約155部に氷水約45部を加え
て摺動磨砕機(たとえばスーパーマスコロイダ−MKZ
81.5−40、増幸産業株式会社製造)を通過させ、
さらに磨砕機(プーラ−社製造)に、冷却下に通過させ
て粉砕する等の方法(特願昭55−67591号明細書
、実施例)を挙げることができる。In addition, extubated beef bones at a meat processing plant are heated to approximately 20°C.
The beef bones were cooled to 100 mL, cut into small pieces using a chomper with an outlet hole diameter of 6.5 mm, and 55 parts of ice water was added to about 100 parts of the beef bones. Approximately 45 parts of ice water is added to approximately 155 parts of this, and a sliding mill (for example, Super Mascolloid-MKZ) is used.
81.5-40, manufactured by Masuko Sangyo Co., Ltd.),
Further, a method of pulverizing the material by passing it through a grinder (manufactured by Puller Co., Ltd.) under cooling (Japanese Patent Application No. 55-67591, Examples) can be mentioned.
免疫活性ペプチドを抽出する酸水溶液としては、鉱酸水
溶液および有機酸水溶液を挙げることができる。Examples of the acid aqueous solution for extracting immunoactive peptides include mineral acid aqueous solutions and organic acid aqueous solutions.
鉱酸としては、塩酸、硫酸、燐酸等を挙げることができ
る。Examples of mineral acids include hydrochloric acid, sulfuric acid, and phosphoric acid.
有機酸としては、蟻酸、酢酸、プロピオン酸、酪酸等を
挙げることができる。これらの酸は、分岐していても差
支えない。Examples of organic acids include formic acid, acetic acid, propionic acid, butyric acid, and the like. These acids may be branched.
次に、実施例により本発明をさらに説明するが、本発明
は、かかる実施例に限定されるものではない。Next, the present invention will be further explained with reference to examples, but the present invention is not limited to these examples.
実施例1
成牛の頭部と四肢とを除去した新鮮な牛骨を2℃の温度
下で粗粉砕し、液体アンモニウム冷却下に凍結(約−1
00”C)粉砕したのち、マイクロ波および熱風(約9
0°C15分間)乾燥した。Example 1 Fresh beef bones from which the head and limbs of an adult cow were removed were coarsely ground at a temperature of 2°C, and frozen under liquid ammonium cooling (approximately -1
00”C) After crushing, microwave and hot air (approx.
Dry at 0°C for 15 minutes).
収量は乾燥重量比で約33%であった。得られた乾燥物
は、第1表に示す組成を有していた。The yield was about 33% on a dry weight basis. The obtained dried product had the composition shown in Table 1.
第 1 表
(重量%)
上記により得られた牛骨及び骨髄乾燥物1gに、0、I
N塩酸の10mff1を加え、polytron(Po
lyLron 社製造)にてホモジナイズした後、4
000rpmにて30分間遠心分離し、上滑を分離した
。沈渣は、0.IN塩酸の10+y/2を添加し、十分
撹拌したのち遠心分離し、その上清をさきの上清と合せ
、凍結乾燥し、これを塩酸抽出物とした。Table 1 (% by weight) 0, I
Add 10 mff1 of N-hydrochloric acid and
After homogenizing with lyLron Co., Ltd.),
The mixture was centrifuged at 000 rpm for 30 minutes, and the supernatant was separated. The sediment is 0. 10+y/2 of IN hydrochloric acid was added, thoroughly stirred, and then centrifuged. The supernatant was combined with the previous supernatant and freeze-dried to obtain a hydrochloric acid extract.
実施例2
ビーフインド(三菱化成食品株式会社販売、登録商標)
Igに、0.1M酢酸10m1を加え、polytro
nによりホモジナイズし、熱湯浴中にて10分間煮沸し
た。直ちに水冷した後氷酢酸を加え、IM濃度に調整し
た。実施例1と同様に遠心分離し、上清を分離した。沈
渣は、1M酢酸10mj2に懸濁し、再び同様に遠心分
離し、上清を分離した。これをさきの上清と合せ凍結乾
燥し、熱酢酸抽出物とした。Example 2 Beef India (Sold by Mitsubishi Kasei Foods Co., Ltd., registered trademark)
Add 10ml of 0.1M acetic acid to Ig, and
The mixture was homogenized by n and boiled for 10 minutes in a hot water bath. After immediately cooling with water, glacial acetic acid was added to adjust the concentration to IM. Centrifugation was performed in the same manner as in Example 1, and the supernatant was separated. The precipitate was suspended in 10 mj2 of 1M acetic acid, centrifuged again in the same manner, and the supernatant was separated. This was combined with the previous supernatant and lyophilized to obtain a hot acetic acid extract.
■)ラジオイムノアッセイ(RIA)法検索する活性ペ
プチドとして、
Vasoactive 1ntestinalPol
ypeptide (VIP)
Peptide hfstidineisoleuc
fne (PHI)
Pancreatic Po1ypeptide(p
p) ガストリン放出ペプチド(CARP)Meth
ionine−enkephalinArg−Gay−
Leu (Met−Enk8)ロイモルフィン
ガラニン
ソマトスフチン
epidermal growth
f a c t o r (EGF)
を選定した。■) Radioimmunoassay (RIA) method Vasoactive 1ntestinalPol is used as an active peptide to search.
ypeptide (VIP) Peptide hfstidineisoleuc
fne (PHI) Pancreatic polypeptide (p
p) Gastrin releasing peptide (CARP) Meth
ionine-enkephalinArg-Gay-
Leu (Met-Enk8) leumorphin galanin somatosfutin epidermal growth factor (EGF) was selected.
上記活性ペプチドのそれぞれに特異的なRIAの確立に
、それぞれ、抗ブタVIP血清R502、抗ブタPH1
(1−15)血清R8403、抗ヒトpp血清RO21
2、抗ブタGRP血清R6902、抗MeL−Enk8
血清RO171、抗ブタロイモルフイン血清ROO14
、抗ブタガラニン血清R1985、抗ソマトスタフチン
血清0AL272および抗ヒトEGF血清TKR102
をそれぞれ用いた。For establishment of RIA specific for each of the above active peptides, anti-pig VIP serum R502, anti-pig PH1, respectively, were used.
(1-15) Serum R8403, anti-human pp serum RO21
2. Anti-pig GRP serum R6902, anti-MeL-Enk8
Serum RO171, anti-porcine leumorphin serum ROO14
, anti-porcine galanin serum R1985, anti-somatostafutin serum 0AL272 and anti-human EGF serum TKR102.
were used respectively.
いずれの抗血清も、RIA系に用いた標準抗原に特異的
であり、既知の類似ペプチドとは交差反応しないことを
確認している。It has been confirmed that all antisera are specific to the standard antigen used in the RIA system and do not cross-react with known similar peptides.
2)標識抗原:
標識抗原は、標準抗原に対応するペプチドをクロラミン
T法により +!5iを導入し、5ephadexG
10カラム(IX30cm)または5EP−PAKC
1Bカートリッジ(WatersAssoctates
)により精製した。2) Labeled antigen: Labeled antigen is obtained by preparing a peptide corresponding to a standard antigen using the chloramine T method +! Introduced 5i and 5ephadexG
10 columns (IX30cm) or 5EP-PAKC
1B Cartridge (Waters Associates)
).
3)標準希釈液:
標準希釈液は、0.025M EDTA、0.14M
NaC1,0,14M 塩化ナトリウムおよび0
.5%ウシ血清アルブミンを含む0. OI M リ
ン酸緩衝液を用いた。3) Standard dilution solution: Standard dilution solution is 0.025M EDTA, 0.14M
NaCl 1,0,14M Sodium chloride and 0
.. 0.0 with 5% bovine serum albumin. OIM phosphate buffer was used.
なお、ソマトスタチンRIA系では、70mMEDTA
、0.1%ゼラチンを含む0.1 Mヒスチジン緩衝液
(pH6,0)を用いた。In addition, in the somatostatin RIA system, 70mMEDTA
, 0.1 M histidine buffer (pH 6,0) containing 0.1% gelatin was used.
4)RIA測定: tAは、二重測定で行い、ガラス試験管を用いた。4) RIA measurement: tA was performed in duplicate and using glass test tubes.
標準希釈液(0,4nfり、標準ペプチド(0,1m1
)および抗血清溶液(0,1nf)および標準抗原溶液
(5,000〜10.OOOcpm10.1 ml、
0.1 ml)をガラス試験管に分注、混和させた。Standard dilution solution (0.4nf), standard peptide (0.1ml
) and antiserum solution (0.1nf) and standard antigen solution (5,000-10.00cpm 10.1 ml,
0.1 ml) was dispensed into a glass test tube and mixed.
次いで、4 ’Cで8時間反応させた後、正常家兎血清
(100倍希釈、0.1nf抗家兎ガンマグロブリン血
清(20倍希釈、0.1mj2)、10%ポリエチレン
グリコール液(ポリエチレングリコール6000.0.
1nf)を順次添加、混和し、さらに4°Cに3時間反
応後、遠心分離(3,000rpm、30分、4’C)
l、、上清を吸引除去し、沈渣の放射活性をガンマカウ
ンタで測定した。Next, after reacting at 4'C for 8 hours, normal rabbit serum (100-fold dilution, 0.1nf anti-rabbit gamma globulin serum (20-fold dilution, 0.1mj2), 10% polyethylene glycol solution (polyethylene glycol 6000) .0.
1nf) was added in sequence, mixed, and further incubated at 4°C for 3 hours, followed by centrifugation (3,000 rpm, 30 minutes, 4'C).
l. The supernatant was removed by suction, and the radioactivity of the precipitate was measured using a gamma counter.
5)活性ペプチドの検索:
4)に記載した方法により、実施例1および2により得
られた牛骨及び骨髄粗抽出物の活性ペプチドを検索した
。5) Search for active peptides: The crude bovine bone and bone marrow extracts obtained in Examples 1 and 2 were searched for active peptides by the method described in 4).
実施例1および2により得られた牛骨及び骨髄粗抽出物
(0,1g)に標準希釈液(1,0mj2)を加え、十
分混和したのち不溶物を遠心分離(4゜000rpm、
30分、4゛C)にて除去した。得られた抽出液を倍々
または4倍希釈した希釈系列について測定を行った。The standard dilution solution (1.0 mj2) was added to the crude bovine bone and bone marrow extracts (0.1 g) obtained in Examples 1 and 2, and after mixing thoroughly, the insoluble matter was centrifuged (4°000 rpm,
It was removed for 30 minutes at 4°C. Measurements were performed on a dilution series in which the obtained extract was diluted twofold or fourfold.
活性ペプチド成分の含有量は、各RIA系における粗抽
出物の希釈曲線から評価し、希釈各点について、得られ
た免疫活性の平均値を牛骨及び骨髄乾燥物あたりに換算
し、測定値とした。なお、標準曲線ならびに希釈曲線は
IogiL−1og変換し、直線化した。The content of active peptide components is evaluated from the dilution curve of the crude extract in each RIA system, and the average value of the obtained immune activity for each dilution point is converted to the amount per dried bovine bone and bone marrow, and the measured value and did. In addition, the standard curve and dilution curve were converted into IogiL-1og and linearized.
6)結果: 実施例1で得られた塩酸抽出物のVIP、PP。6) Results: VIP, PP of the hydrochloric acid extract obtained in Example 1.
Met−Enk8およびPH1について、標準曲線なら
びに希釈曲線を作図し、第1図に示した。Standard curves and dilution curves were drawn for Met-Enk8 and PH1 and shown in FIG.
VIPとMet−Enk8とが、標準ペプチドと平行し
た。VIP and Met-Enk8 were paralleled with standard peptides.
実施例1で得られた塩酸抽出物(収率28%)中の免疫
活性を、第2表に示した。The immunoactivity in the hydrochloric acid extract (yield 28%) obtained in Example 1 is shown in Table 2.
第 2 表 p m o I / g乾燥物 実施例2で得られた酢酸抽出物のvrp、pp。Table 2 p m / g dry matter vrp, pp of the acetic acid extract obtained in Example 2.
Met−Enk8.EGF、GRPおよびロイモルフィ
ンについて標準曲線ならびに希釈曲線を作図し、第2図
に示した。Met-Enk8. Standard curves and dilution curves were constructed for EGF, GRP and leumorphin and are shown in FIG.
VIP、PP、Met−Enk8.GRPおよびロイモ
ルフインが標準ペプチドと平行した。VIP, PP, Met-Enk8. GRP and leumorphin were paralleled with standard peptides.
実施例2で得られた酢酸抽出物(収率24%)中の免疫
活性を、第3表に示した。The immunoactivity in the acetic acid extract (yield 24%) obtained in Example 2 is shown in Table 3.
第3表
哺乳動物などの動物組機とくに神経および消化管組織に
分布し存在するもので、生体の様々な調節機能を行って
いると考えられていることから、加齢による各種骨関節
疾患に随伴した、いわゆる老化性骨粗■症(オステオボ
ローゼ)の患者に、本発明の免疫活性ペプチタドを投与
することにより、生体の骨代謝機構に有効に作用するも
のと考えられる。Table 3: These substances are distributed and present in the tissues of animals such as mammals, especially the nerves and gastrointestinal tract, and are thought to perform various regulatory functions in the living body. It is believed that by administering the immunoactive peptide of the present invention to a patient suffering from so-called senile osteoporosis (osteoborosis), it will effectively act on the bone metabolic mechanism of the body.
第1図は、実施例1で得られた活性ペプチドの免疫活性
希釈曲線および標準曲線であり、第2図は、実施例2で
得られた活性ペプチドの免疫活性希釈曲線および標準曲
線である。
pmol/g乾燥物
〔発明の効果〕FIG. 1 shows the immunoreactivity dilution curve and standard curve of the active peptide obtained in Example 1, and FIG. 2 shows the immunoreactivity dilution curve and standard curve of the active peptide obtained in Example 2. pmol/g dry matter [Effects of the invention]
Claims (1)
接触させ、酸水溶液中に免疫活性ペプチドを抽出するこ
とを特徴とする免疫活性ペプチドの製造方法。(1) A method for producing an immunoactive peptide, which comprises contacting a pulverized product containing bird and animal bones or bone marrow with an aqueous acid solution, and extracting the immunoactive peptide into the aqueous acid solution.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP63178447A JPH0228118A (en) | 1988-07-18 | 1988-07-18 | Production of immunologically active peptide |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP63178447A JPH0228118A (en) | 1988-07-18 | 1988-07-18 | Production of immunologically active peptide |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH0228118A true JPH0228118A (en) | 1990-01-30 |
Family
ID=16048680
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP63178447A Pending JPH0228118A (en) | 1988-07-18 | 1988-07-18 | Production of immunologically active peptide |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH0228118A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107874062A (en) * | 2017-11-14 | 2018-04-06 | 王清秀 | FOS ox bone marrow peptide compound powder and its application |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS61268700A (en) * | 1985-05-24 | 1986-11-28 | Dai Ichi Pure Chem Co Ltd | Novel physiologically active peptide |
-
1988
- 1988-07-18 JP JP63178447A patent/JPH0228118A/en active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS61268700A (en) * | 1985-05-24 | 1986-11-28 | Dai Ichi Pure Chem Co Ltd | Novel physiologically active peptide |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107874062A (en) * | 2017-11-14 | 2018-04-06 | 王清秀 | FOS ox bone marrow peptide compound powder and its application |
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