JPS6043333B2 - Method for producing anti-inflammatory active substances - Google Patents
Method for producing anti-inflammatory active substancesInfo
- Publication number
- JPS6043333B2 JPS6043333B2 JP51099385A JP9938576A JPS6043333B2 JP S6043333 B2 JPS6043333 B2 JP S6043333B2 JP 51099385 A JP51099385 A JP 51099385A JP 9938576 A JP9938576 A JP 9938576A JP S6043333 B2 JPS6043333 B2 JP S6043333B2
- Authority
- JP
- Japan
- Prior art keywords
- inflammatory
- substance
- inflammatory active
- active substance
- ultrafiltration
- 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.)
- Expired
Links
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
【発明の詳細な説明】
本発明は、炎症性疾患の治療に用いられる抗炎症活性物
質の製法に関するものである。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing anti-inflammatory active substances for use in the treatment of inflammatory diseases.
生体の有する自己防御機能の一つとして、炎症に対し治
療作用を示す自己物質が存在するとの仮説が提出されて
いる。A hypothesis has been proposed that as one of the self-defense functions of living organisms, there are self-substances that exhibit therapeutic effects against inflammation.
例えばこの物質は血漿中に存在して分子量数百の低分子
物質であるとされている(特開昭50−82214)が
、その物質の精製は充分にはなされておらず、物理化学
的性質についても未だ明らかではない。本発明者等は、
啼乳動物の体液より、かゝる低分子抗炎症物質とは異な
る分子量の抗炎症物質前駆体(分子量1000〜100
00)を分離し、これを高分子化することにより、抗炎
症活性物質に変え、−その精製を容易ならしめることに
成功し、本発明を完成した。For example, this substance exists in blood plasma and is said to be a low-molecular substance with a molecular weight of several hundred (Japanese Patent Application Laid-open No. 82214-1982), but the substance has not been sufficiently purified and its physicochemical properties is still not clear. The inventors,
Anti-inflammatory substance precursors with molecular weights different from such low-molecular-weight anti-inflammatory substances (molecular weight 1000 to 100
The present invention has been completed by separating 00) and converting it into an anti-inflammatory active substance by polymerizing it, thereby facilitating its purification.
本発明によれば、抗炎症活性物質は啼乳動物の血清、血
漿、炎症性滲出液、乳汁、唾液などの体液を、ます限外
ろ過して高分子の蛋白質、糖質を除いた低分子の限外ろ
過液を取り、そのカルシウム濃度を低下させることによ
り同分画中の低分子抗炎症活性物質前駆体を高分子化さ
せ、再び限外濾過によりろ過されない高分子化した抗炎
症活性物質を分画し、次いでゲル濾過クロマトグラフィ
ー処理により活性分画を取り、更に弱塩基性イオン交換
クロマトグラフィー、ゲルろ過クロマトグラフィーおよ
び弱酸性イオン交換クロマトグラ’フィーを行うことに
より、高度に精製された電気泳動的に単一な物質として
得ることができる。According to the present invention, the anti-inflammatory active substance is a low molecule obtained by ultrafiltering body fluids such as serum, plasma, inflammatory exudate, milk, and saliva of mammals to remove high molecular weight proteins and carbohydrates. By taking the ultrafiltrate and reducing its calcium concentration, the low-molecular anti-inflammatory active substance precursor in the same fraction is polymerized, and the polymerized anti-inflammatory active substance that is not filtered by ultrafiltration is recovered. The active fraction was obtained by gel filtration chromatography, and then subjected to weakly basic ion-exchange chromatography, gel filtration chromatography, and weakly acidic ion-exchange chromatography. It can be obtained electrophoretically as a single substance.
この物質は糖質を主成分とし一部アミノ酸を含む糖蛋白
と考えられ、カラゲニン浮腫に対し静脈内投与で強力な
抗炎症活性を示す。又、ラットのCMC(カルボキシメ
チル−セルロース)嚢法を用いた生体内の白血球遁走試
験では、静脈内投与により強い遁走阻害活性を示し、生
体外のポイデン・チャンバー(B()ydenCham
ber)を用いた遁走試験でも同物質の共存により白血
球遁走を著しく阻止する。本発明を実施するに当つて、
出発原料としては、啼乳動物の体液例えば人、牛、豚、
羊、ラット、モルモツトなどの血液、ラットの実験的炎
症部位への滲出液、又は分泌液として人、牛、馬あるい
は豚などの乳汁、唾液等が用いられるが、多量の原料入
手の点を考慮すると、牛、馬、豚等大動物家畜の血液及
び乳汁が最適である。This substance is thought to be a glycoprotein mainly composed of carbohydrates and some amino acids, and exhibits strong anti-inflammatory activity against carrageenan edema when administered intravenously. In addition, in an in vivo leukocyte fugue test using the CMC (carboxymethyl cellulose) sac method in rats, intravenous administration showed a strong fugetaxis inhibitory activity;
Even in a fugue test using the same substance, white blood cell fugue is significantly inhibited by the coexistence of the same substance. In carrying out the present invention,
Starting materials include body fluids of mammals such as humans, cows, pigs, etc.
Blood from sheep, rats, guinea pigs, etc., exudate from experimentally inflamed sites in rats, or secretions from humans, cows, horses, or pigs, such as milk or saliva, are used, but consideration should be given to the availability of large quantities of raw materials. Blood and milk from large animals such as cows, horses, and pigs are then optimal.
以下に分画、精製法について各段階毎に説明を加える。Each step of the fractionation and purification method will be explained below.
〔第1段階〕原料は、まず低温遠心分離操作により血球
、脂質等を除き、次いで高分子蛋白等を除く目的で限外
泊過を行う。[First step] The raw material is first subjected to low-temperature centrifugation to remove blood cells, lipids, etc., and then subjected to ultrafiltration in order to remove high-molecular proteins, etc.
以下の操作はできるかぎり4℃付近の低温室にて行う。
限外ろ過には例えば、アミコン社(AmicOnCO.
)製ダイアフロー装置を用い、フィルターとしてはUM
−10等分子量1万以下のものを通過させるものを使用
すると有利である。又、同じくアミコン社製のフオロー
・ファイバー装置(DC−2型、HIDP−10)を用
いると大量の原料処理が短時間にでき更に好適である。
例えば、血清を限外沖過する場合、まず細菌繁殖を防止
する意味で、1%ナトリウム・アザイド(NaN3)を
1110喀量加え、透析、希釈等の操作を加えることな
く直ちに限外沖過器にかける。この過程で、透析や希釈
を行い液のカルシウム濃度が低下すると(約5TrLg
%以下)、抗炎症物質前駆体の一部は高分子化し、限外
枦過器より流出せず収量が低下する。又、限外枦過は内
容物濃度上昇による高粘度のための時間と共に速度が低
下するので、原料の112〜1B容量が沖過された時点
で終了するのが好適である。沖過された低分子分画は凍
結により保存することができる。〔第■段階〕
この限外p過された低分子分画中の抗炎症活性物質前駆
体について、共存するカルシウム・イオンの低下による
高分子化操作を行なう。The following operations are performed in a cold room at around 4°C as much as possible.
For ultrafiltration, for example, Amicon CO.
) diaflow device was used, and the filter was UM.
It is advantageous to use a material that allows the passage of -10 equivalent molecular weights of 10,000 or less. Further, it is more preferable to use a follow fiber device (Model DC-2, HIDP-10) also manufactured by Amicon, as it can process a large amount of raw material in a short time.
For example, when ultrafiltrating serum, first add 1110 molar volume of 1% sodium azide (NaN3) to prevent bacterial propagation, and immediately place it in the ultrafilter without performing any operations such as dialysis or dilution. Put it on. During this process, when the calcium concentration of the solution decreases due to dialysis or dilution (approximately 5TrLg
% or less), some of the anti-inflammatory substance precursors become polymerized and do not flow out of the ultrafilter, resulting in a reduced yield. In addition, since the speed of ultrafiltration decreases with time due to high viscosity due to increase in content concentration, it is preferable to finish when 112 to 1B capacity of the raw material has been passed. The filtered low molecular weight fraction can be preserved by freezing. [Step (II)] The anti-inflammatory active substance precursor in the ultrapolar-filtered low-molecular fraction is subjected to a polymerization operation by reducing coexisting calcium ions.
即ち、水等による希釈、透析又はカルシウムを除くよう
なキレート剤例えばEDTA(エチレンジアミン四酢酸
)、GBDTA(グリコールエチレンジアミン四−酢酸
)等の添加により高分子化が起り、再度の限外沖過(U
M−1蒔)で?液中に出なくなる。高分子化はカルシウ
ムイオン濃度5m9%で充分に起り、血清(カルシウム
を約10m9%含む)を例にとれば、その限外P過液を
水で2倍に希釈するのみ−てよい。但し、高分子化を確
実にする意味で3〜4倍希釈を用いる方が有効である。
この低カルシウム化した分画を限外淵過(UM−10,
.UM−2等のフィルター)し、今度は枦過されずに残
つた高分子分画を採取する。この低カルシウム化による
高分子化過程と、それに続く限外ろ過濃縮過程で目的の
抗炎症活性物質を著しく精製することができる。本発明
の特徴は一つはこの過程にある。〔第■段階〕抗炎症活
性物質を含む第■段階の濃縮液をゲル洒過クロマトグラ
フィーにかける。That is, polymerization occurs by dilution with water, dialysis, or addition of a chelating agent that removes calcium, such as EDTA (ethylenediaminetetraacetic acid), GBDTA (glycolethylenediaminetetraacetic acid), etc.
M-1 Maki)? It will not come out in the liquid. Polymerization sufficiently occurs at a calcium ion concentration of 5m9%, and taking serum (containing about 10m9% calcium) as an example, it is only necessary to dilute the ultra-P filtrate twice with water. However, in order to ensure polymerization, it is more effective to use 3- to 4-fold dilution.
This low-calcification fraction was subjected to ultrafiltration (UM-10,
.. filter (such as UM-2), and collect the remaining high molecular fraction without being filtered. The target anti-inflammatory active substance can be significantly purified through this polymerization process by low calcium conversion and the subsequent ultrafiltration concentration process. One of the features of the present invention lies in this process. [Step II] The concentrated solution from Step II containing the anti-inflammatory active substance is subjected to gel permeation chromatography.
ゲル沖過には、セスアデツクスG−75等(Sepha
dexlPharmaciaCO.)の架橋テキストラ
ンが有効に用”いられる。又、展開溶液としては、0.
9%の食塩水等の生理的条件に近い塩溶液を用いられる
。クロマト・カラムよりの溶離液は一定量づつ分取し、
その275mμ附近の紫外線吸収を目安にクロマトグラ
ムを書くと、抗炎症活性物質は第1のピーク附近に存在
し、ほとんど間隙容積(VOidvOIume)直後に
溶出することが分かる。この第1ピーク及びその後部に
流出する裾野の部分を広く採取し、UM−2等のフィル
ターで限外淵過して活性物質を濃縮する。〔第■段階〕
第■段階の濃縮液を弱塩基性イオン交換体例えばDEA
E−セルロース(ジエチルアミノエチル−セルロース、
SefvaCO.製)などを用いるイオン交換クロマト
グラフィーにかける。Sepha Dex G-75 etc. (Sepha
dexlPharmaciaCO. ) can be effectively used. Also, as a developing solution, 0.
A salt solution close to physiological conditions, such as 9% saline, can be used. The eluent from the chromatography column is collected in fixed amounts,
When a chromatogram is drawn using the ultraviolet absorption around 275 mμ as a guide, it can be seen that the anti-inflammatory active substance exists near the first peak and elutes almost immediately after the interstitial volume (VOidvOIume). This first peak and the tail portion flowing to the rear thereof are broadly sampled and subjected to ultrafiltration using a filter such as UM-2 to concentrate the active substance. [Step ■] The concentrated liquid from Step ■ is treated with a weakly basic ion exchanger such as DEA.
E-cellulose (diethylaminoethyl cellulose,
SefvaCO. The sample is subjected to ion exchange chromatography using a commercially available product such as
このイオン交換体をあらかじめ低イオン強度の弱酸性緩
衝液例えば0.05M,.PH5.8の酢酸緩衝液など
で緩衝化しておき、そのクロマトカラム上へ、第■段階
の濃縮液を吸着させる。この場合、濃縮後の塩濃度を0
.1M食塩水になるように調整しておく。吸着させた後
、0.2Mの酢酸緩衝液(PH5.8)を充分に流し2
757T1.μ紫外部吸収を指標として溶離するものを
できるかぎり除いた後、1Mの同緩衝液に換え濃縮溶離
する分画を集める。この分画に抗炎症活性物質が存在し
ている。〔第■段階〕
第■?階の1M溶離分画をゲル淵過する。This ion exchanger is preliminarily mixed with a weakly acidic buffer of low ionic strength, for example, 0.05M, . The column is buffered with an acetate buffer having a pH of 5.8 or the like, and the concentrated solution of the second stage is adsorbed onto the chromatography column. In this case, the salt concentration after concentration is 0.
.. Adjust the solution to 1M saline. After adsorption, 0.2M acetate buffer (PH5.8) was thoroughly poured.
757T1. After removing as much of the eluting material as possible using μ ultraviolet absorption as an indicator, the buffer is exchanged with the same 1M buffer and the fractions to be concentrated and eluted are collected. Anti-inflammatory active substances are present in this fraction. [Stage ■] Stage ■? The 1M elution fraction of 100% is gel-filtered.
ゲル沖過は、第■段階よりも更に架橋度のあらい網目構
造の大きなもの例えばセフアデツクスG−200などが
有効である。ゲル淵過クロマトグラフィーは第■段階と
同様に行い、抗炎症活性物質が流出する第1ピークを集
める。限外枦過(UM−10、■−2等)で濃縮後、水
にて充分に透析して脱塩し、透析内液は凍結乾燥し低温
保存する。第■段階までの精製により抗炎症活性物質は
極めて高度に純化されるが、更にもう一段階精製過程を
おいてもよい。即ち、弱酸性イオン交換体例えばCM−
セフアデツクス(カルボキシメチルーセフアデツクス)
などのカラムを通す第■段階である。〔第■段階〕
第■段階での第1ピークを水で透析したものをCM−セ
フアデツクスを用いるイオン交換クロマトグラフィーに
かける。As for gel filtering, it is effective to use gels with a higher degree of cross-linking and a larger network structure than those used in the second stage, such as Sephadex G-200. Gel filtration chromatography is performed in the same manner as in step (2), and the first peak from which the anti-inflammatory active substance flows is collected. After concentration by ultrafiltration (UM-10, ■-2, etc.), it is thoroughly dialyzed against water to desalt it, and the dialyzed solution is freeze-dried and stored at low temperature. Although the anti-inflammatory active substance is purified to a very high degree through the purification up to step (2), one more step of purification may be performed. That is, weakly acidic ion exchangers such as CM-
Cephadex (carboxymethyl-cephadex)
This is the second stage where the liquid is passed through a column such as [Step (1)] The first peak obtained in the step (2) is dialyzed against water and subjected to ion exchange chromatography using CM-Sephadex.
CM−セフアデツクスはあらかじめ低濃度の緩衝液例え
ば0.02M酢酸緩衝液(PH5.8)て緩衝化してお
き、活性分画を上部にのせた後0.02r!4の同緩衝
液を流して非吸着溶離してくるものを集める。この中に
抗炎症活性物質が含まれており、限外?過で濃縮後水で
充分透析し凍結乾燥することにより、目的の活性物質を
得ることができる。次に実施例をあげて本発明の方法を
更に具体的に説明する。CM-Sephadex is buffered in advance with a low-concentration buffer such as 0.02M acetate buffer (PH5.8), and after placing the active fraction on top, 0.02r. 4. Flow the same buffer solution in step 4 and collect the non-adsorbed elute. This contains anti-inflammatory active substances, and is it limitless? The desired active substance can be obtained by concentrating with filtration, thoroughly dialyzing with water, and freeze-drying. Next, the method of the present invention will be explained in more detail with reference to Examples.
実施例1
牛血液より抗炎症活性物質の分画、精製
牛血液180eを静置して凝固させ、5℃前後の低温放
置て血清を充分に分離させた後血餅を除去する。Example 1 Fractionation and Purification of Anti-inflammatory Active Substances from Bovine Blood Bovine blood 180e was allowed to stand still to coagulate, left to stand at a low temperature of around 5°C to sufficiently separate serum, and then blood clots were removed.
血餅を除去した血清分画中の血球は遠心分離にて除く。
この場合、ドラバール型連続遠心機例えばバーチエス(
Barces)遠心機などを用い、回転数を5000r
.p.mで行えば能率よく透明な血清が約60′得られ
る。血清に殺菌剤としてナトリウム・アザイドを最終0
.01%になるように加え、限外淵過操作を行う。アミ
コン製ハイフローセル型p過機(又はバイオエンジニア
リング社製.ダイアフィルターMC−6型機)を用い、
フィルターは直径150T0t(7)UM−10を使用
し、窒素−ガス加圧下(4k91cf1)に行う。UM
−10フィルターを通過するものは分子量約1万以下の
ものであり、約48′が得られた。この淵液を水で4倍
に希J釈する。この希釈過程て低分子の抗炎症活性物質
前駆体は高分子化し、次の限外淵過ではフィルターを通
過しなくなる。この段階では、約200eと処理量が多
いので、アミコン製フオロー・ファイバー(DC−2型
、HIDP−10)を用いると能率が・よい。この様に
して通過するものを除き、通過しない高分子物質を濃縮
して30m1にまでする。この場合小型ダイアフロー限
外ろ過機を用い濃縮すると具合がよい。この濃縮した分
画をゲル淵過操作にかける。Blood cells in the serum fraction from which blood clots have been removed are removed by centrifugation.
In this case, a Dravall type continuous centrifuge, such as Birches (
Barces) Using a centrifuge, etc., increase the rotation speed to 5000 r.
.. p. If carried out at m, approximately 60' of clear serum can be obtained efficiently. Add sodium azide to the serum as a bactericidal agent.
.. 0.01%, and perform ultrafiltration operation. Using Amicon's high-flow cell type p-filtration machine (or Bio Engineering Co., Ltd.'s Diafilter MC-6 model machine),
A filter with a diameter of 150T0t (7) UM-10 is used, and the test is carried out under nitrogen gas pressure (4k91cf1). UM
What passed through the -10 filter had a molecular weight of about 10,000 or less, and a molecular weight of about 48' was obtained. Dilute this fluid by 4 times with water. During this dilution process, the low-molecular anti-inflammatory active substance precursor becomes a polymer and no longer passes through the filter during the next ultrafiltration. At this stage, the throughput is large, about 200 e, so it is efficient to use Amicon's follow fiber (DC-2 type, HIDP-10). In this way, those that pass are removed, and the polymer substances that do not pass are concentrated to a volume of 30 ml. In this case, it is convenient to concentrate using a small diaflow ultrafilter. This concentrated fraction is subjected to gel filtration.
まず、セフアデツクス(Sephadex)G−75を
0.9%食塩水に懸濁し、5×95cmのクロマトカラ
ム、(Excelカラム、SB−501硝英KK)につ
める。この上部に濃縮液30m1をのせ、展開は0.9
%食塩水で行い、2757n,μの紫外部吸収を指標に
して溶離してくる各フラクシヨンを分取する。抗炎症活
性物質は間隙容積(VOidVOlLlnle)に接し
てすぐ出てくる第1ピークに存在する。この第1ピーク
部ノ分を合併し、限外枦過(フィルターUM−10)で
濃縮し、約30mtにする。濃縮液は水にて1晩透析し
、次いで弱塩基性DEM−セルロースを用いるイオン交
換カラムクロマトグラフィーにかける。DEAE−セル
ロースはあらかじめPH5.8の0.01M酢酸緩衝液
にて緩衝化しておき、26×40C7Ttのカラム(E
xcel)に充填しておく。この上に上記濃縮液をおき
吸着させた後、0.2M..PH=5.8の酢酸緩衝液
にて溶離するものをよく洗い出し、2757T1,Pの
吸収で溶離するものが無くなつたのを確認後、1Mの同
緩衝液に変える。この1M緩衝液により活性物質は濃縮
されながらカラムにより溶離してくる。次に、セフアデ
ツクスG−200を用いゲル枦過を行う。First, Sephadex G-75 was suspended in 0.9% saline and loaded into a 5 x 95 cm chromatography column (Excel column, SB-501 Nitrous KK). Place 30ml of concentrated liquid on top of this, and the development is 0.9
% saline solution, and each eluting fraction is fractionated using the ultraviolet absorption of 2757n, μ as an index. The anti-inflammatory active substance is present in the first peak immediately adjacent to the interstitial volume (VOidVOlLlnle). The first peak fractions are combined and concentrated by ultrafiltration (filter UM-10) to approximately 30 mt. The concentrate is dialyzed overnight against water and then subjected to ion exchange column chromatography using weakly basic DEM-cellulose. DEAE-cellulose was buffered in advance with 0.01M acetate buffer at pH 5.8, and then placed on a 26×40C7Tt column (E
xcel). After placing the above concentrated solution on top of this and adsorbing it, 0.2M. .. Thoroughly wash out the eluting material with an acetate buffer of pH=5.8, and after confirming that there is no more eluting material due to absorption of 2757T1,P, change to the same buffer of 1M. The active substance is concentrated by this 1M buffer and eluted through the column. Next, gel filtration is performed using Sephadex G-200.
先のG−75の場合と同様、0.9%食塩水にてG−2
00をカラム(4×95C!rl)につめ、上部にDE
AE−セルロースクロマトグラフィーによる1M溶離部
(約20m1)をのせ、0.9%の食塩水にて展関する
。この場合も活性物質は間隙容積のすぐ後の第1ピーク
に存在する。この第1ピーク部分を限外淵過(UM−1
0又はUM−2)で濃縮し、水にて充分透析後、凍結乾
燥する。この段階で26m9の活性分質が得られた。こ
の物質はかなり精製され強い抗炎症活性を示す。又、次
のステップとして弱酸性のCM−セフアデツクスを用い
るイオン交換クロマトグラフィーを行うと更に精製され
るが、一部抗炎症活は低下する場合もある。凍結乾燥標
品又はセフアデツクスG−200クロマトグラフィー処
理の第1ピークを0.01M酢酸緩衝液(PH5.8)
にて透析後、CM−セフアデツクスカラム(2.6×4
0cr!t)にのせ同緩衝液に食塩を加えるグラージエ
ント(Gradint)の系でクロマトグラフィーを行
う。クロマト分画で食塩濃度0.02〜0.2Mの範囲
で溶離してきた部分を採取し、限外淵過で濃縮(UM−
10又はUM−2)し水で透析後、凍結乾燥すると、1
0m9の活性標品が得られた。この物質の各種性質を以
下列記する。G-2 in 0.9% saline as in the case of G-75 above.
Pack 00 into a column (4x95C!rl) and add DE to the top.
A 1M eluate (approximately 20 ml) from AE-cellulose chromatography was loaded and expanded with 0.9% saline. In this case too, the active substance is present in the first peak immediately after the interstitial volume. This first peak portion was filtered through ultrafiltration (UM-1
0 or UM-2), thoroughly dialyzed against water, and freeze-dried. At this stage, 26m9 of active substance was obtained. This substance is highly purified and exhibits strong anti-inflammatory activity. In addition, as a next step, ion exchange chromatography using weakly acidic CM-Sephadex is performed for further purification, but the anti-inflammatory activity may be partially reduced. The first peak of the freeze-dried sample or Sephadex G-200 chromatography was added to 0.01M acetate buffer (PH5.8).
After dialysis using a CM-Sephadex column (2.6 x 4
0 cr! Chromatography is performed using a gradient system in which sodium chloride is added to the same buffer solution. In the chromatographic fractionation, the portion eluted in the salt concentration range of 0.02 to 0.2M was collected and concentrated by ultrafiltration (UM-
10 or UM-2) and lyophilized after dialysis with water, 1
0 m9 of active preparation was obtained. Various properties of this substance are listed below.
凍結乾燥標品は白色粉末であり、水に溶解するとPH=
4.3(17y191m1)を呈する。The freeze-dried sample is a white powder, and when dissolved in water, the pH =
4.3 (17y191m1).
このものの紫外部吸収スペクトルは第1図のごとく28
0wLμ附近に肩をもつ。又、赤外吸収スペクトルは第
2図に示すとおりである。分子量はゲル枦過法によると
、20万以上と考えられる。元素分析値は下記のようで
ある。The ultraviolet absorption spectrum of this product is 28 as shown in Figure 1.
It has a shoulder near 0wLμ. Moreover, the infrared absorption spectrum is as shown in FIG. The molecular weight is estimated to be 200,000 or more according to the gel filtration method. The elemental analysis values are as follows.
又、このものの組成は下記のようである。The composition of this product is as follows.
糖の分析によると、グルコース、ガラクトース、フコー
スが多く、微量ではあるがラムノース、マンノースも含
んでいる。According to sugar analysis, it contains a lot of glucose, galactose, and fucose, and also contains trace amounts of rhamnose and mannose.
アミノ酸分析では、アスパラギン酸、グルタミン酸、グ
ルタミン、セリン、グリシン、アラニン、バリン、ロイ
シンが存在する。Amino acid analysis reveals aspartic acid, glutamic acid, glutamine, serine, glycine, alanine, valine, and leucine.
なお、シアル基、グルクロン酸、ガラクトサミンは認め
られない。Note that sialic groups, glucuronic acid, and galactosamine are not recognized.
抗カラゲニン浮腫作用 ウインター
(Winter)の方法(1)で行つたラットの抗カラ
ゲニン浮腫作用で、この凍結乾燥標品溶液をカラゲニン
投与3吟前に静脈内に注射しておくと、3時間後の判定
時に足浮腫の著しい抑制が見られる。Anti-carrageenan edema effect The anti-carrageenan edema effect in rats was carried out using Winter's method (1).If this freeze-dried sample solution was intravenously injected 3 hours before carrageenan administration, Significant suppression of foot edema is seen at the time of evaluation.
下.に示す表の抑制率より、50%抑制量を求めるとD
勅=0.88μy/BOdyとなる。なお、プロナーゼ
(PrOnase)にて、当物質の蛋白部分を分解して
も、抗カラゲニン活性に影響は与えない。under. From the suppression rate shown in the table, the 50% suppression amount is calculated as D.
= 0.88μy/BOdy. Note that even if the protein portion of this substance is decomposed with pronase (PrOnase), the anti-carrageenan activity is not affected.
CMC嚢法(3)を用いた白血球遊走阻止作用:ラツト
の背部にCMC(カルボキシメチル セルロース)を皮
下投与しその局所に遊走してくる白血球数を測定するC
MC嚢法を用いる。Inhibition of leukocyte migration using the CMC bag method (3): CMC (carboxymethyl cellulose) is subcutaneously administered to the back of rats and the number of leukocytes migrating to that area is measured.
The MC capsule method is used.
標品溶液は、CMC投与時及び3時間後に2回静注する
。6時間目の局所白血球数からその遊走阻害をみると著
しい抑制がみとめられる。The standard solution is injected intravenously twice, at the time of CMC administration and 3 hours later. When looking at the local leukocyte count at 6 hours, a remarkable inhibition of migration was observed.
表に示すように、0.5μFllbOdyを2回投与す
るのみで、白血球遊走を72%阻害している。(3)飯
塚義夫、他;第24回日本薬理学要旨集、305頁(1
967)石川浩、他;薬誌、?1472(1968)ボ
イデン●チャンバー(BOydenChamber)法
(4)での白血球遊走阻止作用:マイクロボア・フィル
ターで2室に分けられたBOydenChem?rの上
室にモルモツト白血球〔腹腔内の多形核白血球(PMN
−1euc0cyte)〕を、下室にその遊走因子(モ
ルモツト血清+チモーザン(ZymOsan))を入れ
白血球がフィルター内を因子側に移動するのを測定する
。As shown in the table, leukocyte migration was inhibited by 72% with just two administrations of 0.5 μFllbOdy. (3) Yoshio Iizuka et al.; 24th Japanese Pharmacology Abstracts, p. 305 (1
967) Hiroshi Ishikawa, et al.; Pharmaceutical Journal, ? 1472 (1968) Leukocyte migration inhibition effect using the BOyden Chamber method (4): BOydenChem? divided into two chambers with a microbore filter? Guinea pig leukocytes in the upper chamber of r [polymorphonuclear leukocytes (PMNs in the peritoneal cavity)
-1euc0cyte)], its migratory factor (guinea pig serum + thymosan (ZymOsan)) is placed in the lower chamber, and the movement of leukocytes within the filter toward the factor is measured.
標品を遊走因子側に入れた場合は白血球遊走にあまり影
響ないが、標品が白血球側に存在すると(4.4pfI
m1)、その白血球遊走を著しく抑制(86%)する。
この抑制作用は白血球に対する標品の細胞毒性によるも
のではなく、遊走抑制である。(4)S.BOyden
;J.Exptl.Med.、川−、453(1962
)実施例2
ラットの実験的炎症局所への滲出液を原料とする方法ラ
ットの背部に無菌的にスポンジ(例えば、ポリウレタン
製のモルトブレンなどが利用できる)を移殖し4日後に
これを取り出す。When the preparation is placed on the migration factor side, it does not have much effect on leukocyte migration, but when the preparation is placed on the leukocyte side (4.4pfI
m1), significantly inhibits leukocyte migration (86%).
This inhibitory effect is not due to the cytotoxicity of the preparation to leukocytes, but is due to migration inhibition. (4) S. Boyden
;J. Exptl. Med. , Kawa-, 453 (1962
) Example 2 A method using exudate from a rat's experimentally inflamed area as a raw material A sponge (for example, polyurethane maltbren can be used) is aseptically implanted on the back of a rat and removed after 4 days.
普通、直径3鑞、厚さ1C77!の円径スポンジなら2
〜3個を1匹に移殖することができ、スポンジ中の滲出
液は10nt程度は採取可能である。この滲出液をプー
ルし原料とする。滲出液を遠心分離し(1万R.p.m
l2紛)混入している血球を除去後、限外t過操作に入
る。以下は実施例1の方法と同じである。このようにし
て精製された抗炎症活性物質も牛血中の物質とほぼ同じ
性質を示す。。実施例3
牛乳を原料とする方法
新鮮牛乳を低温(イ)〜5℃)で遠心分離(1方R.p
.ml3扮)し、浮上する脂質を除去後、再び遠心操作
を行つて充分に脂質をとり除く。Normally, diameter 3, thickness 1C77! For a sponge with a diameter of 2
~3 pieces can be transferred to one animal, and about 10 nt of exudate from the sponge can be collected. This exudate is pooled and used as raw material. The exudate was centrifuged (10,000 R.p.m.
12) After removing the contaminated blood cells, enter the ultra-thigh temperature operation. The following procedure is the same as the method of Example 1. The anti-inflammatory active substance purified in this way also exhibits almost the same properties as the substance in bovine blood. . Example 3 Method using milk as raw material Fresh milk was centrifuged (one way R.p.
.. After removing floating lipids, perform centrifugation again to thoroughly remove lipids.
脱脂した牛乳は限外ろ過操作(UM−10など)により
分子量1万以下のものを採取する。牛乳が血清と違う点
は、含まれるカルシウム濃度が何倍も高いことであり、
低カルシウム化による抗炎症物質前駆体を高分子化させ
るには、多量の水による希釈が必要となる。しかるに容
量が増加すると次の操作”に都合がわるいため透析操作
で脱カルシウムをした方が得策である。即ち、限外p過
により取出した分子量1万以下の分画を透析チューブに
入れ、水に対して充分透析する。透析内液をアミコン製
フェロー・ファイバーにて通過するものを除き、内部に
残る高分子のものを濃縮後、セフアデツクスG−75を
用いるクロマトグラフィーにかける。以下は実施例1と
同じに行う。このようにして取出した物質も牛血清中の
抗炎症性物質とほぼ同じ性質を示す。Defatted milk with a molecular weight of 10,000 or less is collected by ultrafiltration (UM-10, etc.). The difference between milk and serum is that it contains many times more calcium.
Dilution with a large amount of water is required to polymerize anti-inflammatory substance precursors due to low calcium content. However, if the volume increases, it will be difficult to carry out the next operation, so it is better to remove calcium by dialysis.In other words, put the fraction with a molecular weight of 10,000 or less extracted by ultrapolar filtration into a dialysis tube, and add water to the dialysis tube. The dialyzed fluid is thoroughly dialyzed against the dialysis fluid, except for the one that passes through Amicon's Ferro fiber, and the remaining polymers are concentrated, and then subjected to chromatography using Sephadex G-75.The following is an example. The procedure is carried out in the same manner as in 1. The substance thus extracted also exhibits almost the same properties as the anti-inflammatory substance in bovine serum.
第1図は実施例1による牛血清より分画、精製した抗炎
症活性物質の紫外部吸収スペクトルを示したもので、2
mgの標品を3m1の水にとかして測定した。Figure 1 shows the ultraviolet absorption spectrum of the anti-inflammatory active substance fractionated and purified from bovine serum according to Example 1.
Measurements were made by dissolving mg of the standard in 3 ml of water.
Claims (1)
濃度低下による高分子化操作を施した後、再び限外濾過
を行なつて濾過されない高分子物質を分画し、更に得ら
れた高分子分画を精製することを特徴とする抗炎症活性
物質の製法。1 After ultrafiltrating mammalian body fluids and subjecting the filtrate to polymerization by lowering the calcium concentration, ultrafiltration is performed again to fractionate the unfiltered polymer substances, and the resulting polymer A method for producing an anti-inflammatory active substance, which comprises purifying a molecular fraction.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP51099385A JPS6043333B2 (en) | 1976-08-20 | 1976-08-20 | Method for producing anti-inflammatory active substances |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP51099385A JPS6043333B2 (en) | 1976-08-20 | 1976-08-20 | Method for producing anti-inflammatory active substances |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS5326312A JPS5326312A (en) | 1978-03-11 |
JPS6043333B2 true JPS6043333B2 (en) | 1985-09-27 |
Family
ID=14246031
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP51099385A Expired JPS6043333B2 (en) | 1976-08-20 | 1976-08-20 | Method for producing anti-inflammatory active substances |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS6043333B2 (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4284623A (en) * | 1979-11-09 | 1981-08-18 | Beck Lee R | Method of treating inflammation using bovine milk |
DE3219248A1 (en) * | 1982-05-21 | 1983-11-24 | Solco Basel AG, Birsfelden | METHOD FOR OBTAINING CELL-BREATHING ACTIVE INGREDIENTS FROM CALF BLOOD |
US4956349A (en) * | 1983-10-27 | 1990-09-11 | Stolle Research & Development Corporation | Anti-inflammatory factor, method of isolation, and use |
US5980953A (en) * | 1994-10-03 | 1999-11-09 | Stolle Milk Biologics, Inc. | Anti-inflammatory factor, method of isolation, and use |
-
1976
- 1976-08-20 JP JP51099385A patent/JPS6043333B2/en not_active Expired
Also Published As
Publication number | Publication date |
---|---|
JPS5326312A (en) | 1978-03-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Cheung et al. | Compartmental distribution of complement activation products in artificial kidneys | |
CA1120468A (en) | Hgi-glycoprotein capable of stimulating proliferation and differentiation of human granulocyte, process for preparing same and leukopenia curative containing same | |
CA1142175A (en) | Fractionation of protein mixtures by salting out followed by dialysis treatment | |
CA1112166A (en) | Method of preparing and intravenously administrable immune globulin preparation containing antibodies and preparations produced according to this method | |
CH626256A5 (en) | Device for the continuous, extracorporal separation of specific substances from blood | |
US4137307A (en) | Process for preparing haptoglobin aqueous solution using strong anion exchanger | |
JPS60193470A (en) | Abdominal membrane diagnotis and composition used therein | |
NURMOHAMED et al. | Long-term Efficacy and Safety of a Low Molecular Weight Heparin in Chronic Hemodialysis Patients A Comparison with Standard Heparin | |
JPS59137417A (en) | Preparation of colonization stimulation factor and kallikrein originated from human urine | |
JPH11507033A (en) | Method for purifying thrombopoietin | |
Scharschmidt et al. | Removing substances from blood by affinity chromatography: II. Removing bilirubin from the blood of jaundiced rats by hemoperfusion over albumin-conjugated agarose beads | |
US4061735A (en) | Haptoglobin in aqueous solution and process for preparing the same | |
JPS6043333B2 (en) | Method for producing anti-inflammatory active substances | |
US4148886A (en) | Polypeptide possessing thymic activity | |
US4608253A (en) | Process for removing immune complex in blood by use of the immobilized pepsin | |
US4770877A (en) | Isolation of a high molecular weight aortic endothelial cell growth inhibitor | |
JPH0153650B2 (en) | ||
Brunner et al. | Preparative isolation of middle molecular weight fractions from the hemofiltrate of patients with chronic uremia | |
Demus et al. | Identification of water-insoluble membrane proteins by immunoelectrophoresis in a solubilizing urea-triton solvent | |
US4610815A (en) | Process for producing and obtaining anaphylatoxin- and cocytotaxin-containing leucotaxine preparations and of anaphylatoxin and cocytotaxin proteins in molecularly homogeneous, biologically active form | |
JPS5930686B2 (en) | Method for producing peptides with immunoregulatory effects | |
JP2985158B2 (en) | Recovery method of highly active lactenin fraction | |
JPH02502640A (en) | Purification of GM-CSF | |
JPH0352480B2 (en) | ||
Ayala et al. | Immunochemical characterization of the abnormal paraprotein in a case of scleromyxoedema |