JPH0433764B2 - - Google Patents
Info
- Publication number
- JPH0433764B2 JPH0433764B2 JP57137208A JP13720882A JPH0433764B2 JP H0433764 B2 JPH0433764 B2 JP H0433764B2 JP 57137208 A JP57137208 A JP 57137208A JP 13720882 A JP13720882 A JP 13720882A JP H0433764 B2 JPH0433764 B2 JP H0433764B2
- Authority
- JP
- Japan
- Prior art keywords
- family
- reaction
- antitumor
- shellfish
- fraction
- 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 - Lifetime
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Description
本発明はイタヤガイ科、リユウテンサザエ科、
ミミガイ科及びマルスダレガイ科に属する貝類の
煮汁の乾燥粉末又は濃縮物から得た水溶性含糖蛋
白質からなる抗腫瘍性物質とその製造法に関す
る。
従来の抗腫瘍性物質は大部分が低分子化合物で
あり、癌細胞に対する直接細胞毒性が主な作用で
あり、宿主である生体に対する一般毒性が強く、
生体の受ける障害が大きいという問題がある。そ
のため分子量が比較的高く毒性の低い、しかも免
疫賦活作用を持つた抗腫瘍性物質である多糖類な
どを植物、微生物等から抽出して粗粉末の形で使
用しているが、抗癌スペクトルが狭く臨床的な利
用に限界がある。
貝類から抗腫瘍性物質が得られることは特公昭
57−8088号、特開昭54−41314号及び同54−41315
号公報に報告されている。これらの特許公報に開
示されている抗腫瘍性物質の製造は、(1)生の貝類
を原料とし、これをそのままもしくは加熱して貝
殻を除去し、(2)肝臓部分を除去し、(3)得られた除
肝臓肉を低温条件下において水、塩水溶液等の水
性溶媒中においてブレンダー等で細断、磨砕し、
あるいはさらに超音波処理等の物理的衝撃を加え
均質化して抽出を行ない、抗腫瘍性物質が常温以
下の冷水に可溶化し易いようにし、(4)生成混合物
から遠心分離等により細断された肉等の水不溶性
物質を除去し、(5)このようにして得られた抽出液
を透析、限外過、ゲル過、カラムクロマトグ
ラフイー等公知の蛋白質性物質分離精製手段で精
製して目的とする抗腫瘍性物質を得るものであ
る。貝類から得られるこの抗腫瘍性物質は抗癌ス
ペクトルが広く、その薬効も強い点で注目に値す
る。しかしながら上記した製造法では、食用とし
て利用価値のある高価な貝類の肉部分を原料とし
これを細断、分解してしまわなければならないう
えに、有効成分の抽出操作が煩雑であり、処理し
た細断肉片の廃棄にも問題がある等工業的に不利
であり、活性物質を多量に確保するには難点があ
つた。
本発明者等はある範囲内の貝類についてそれか
ら得られる抗腫瘍性物質の抗癌スペクトルが広い
ことに着目してこれら貝類から抗腫瘍性物質を工
業的有利に得るべく研究を積み重ねた結果、従来
廃物として棄てられていたこれら貝類の煮汁か
ら、すなわち従来食用としてこれら貝類を蒸煮も
しくは加熱加工する際副生し、河川を汚染する有
機成分を多量に含むためその廃棄に多大の費用を
要していた貝類の煮汁から抗腫瘍性物質が得られ
ることを見出し、さらにかかる抗腫瘍性物質は塩
基性陰イオン交換体に吸着される分子量10000〜
300000の範囲内の水溶性含糖蛋白質であることを
発見して本発明を完成した。
したがつて本発明の主たる目的はある特定範囲
内の貝類の煮汁から抗腫瘍性物質を取得する方法
を提供することにある。本発明の他の目的はこれ
ら貝類の煮汁から効率よく新規な抗腫瘍性物質を
取得する方法を提供するにある。本発明のさらに
他の目的は新規な抗腫瘍性物質を提供することに
ある。
すなわち本発明の要旨は、イタヤガイ科、リユ
ウテンサザエ科、ミミガイ科及びマルスダレガイ
科に属する貝類の可食部分を熱水性溶媒で蒸煮も
しくは加熱加工する際に得られる煮汁の乾燥粉末
もしくは濃縮物を原料とし、これを塩基性陰イオ
ン交換体を使用するイオン交換クロマトグラフイ
ー及びゲル過ゲルを用いるゲル過に付して分
子量10000〜300000の範囲内の水溶性含糖蛋白質
からなる抗腫瘍性物質を分取することを特徴とす
る抗腫瘍性物質の製造法にあり、またかく得られ
る水溶性含糖蛋白質性物質、すなわち次の特性：
帯微褐白色ないしは白色の固状；
水に易溶性でメタノール、エタノール、アセ
トンなどの有機溶媒に不溶；
両性電解質；
KBrペレツトによる赤外線吸収スペクトル
で3500〜3300cm-1、1650cm-1、1540cm-1及び
1400cm-1附近に主ピークを示す；
ビウレツト反応、キサントプロテイン反応、
フエノール試薬による反応が陽性；
アンスロン硫酸反応、フエノール硫酸反応が
陽性でシステイン硫酸反応が偽陽性；
紫外線吸収スペクトルにおいてλH2O nax278nmに
吸収をもつ；
分子量範囲として10000〜300000のものを含
む；
明確な融点を示さず、235℃以上で分解しは
じめる；
加水分解物中のアミノ酸としてアスパラギン
酸、ハイドロプロリン、スレオニン、セリン、
グルタミン酸、プロリン、グリシン、アラニ
ン、システイン、バリン、メチオニン、イソロ
イシン、ロイシン、チロシン、フエニルアラニ
ン、リジン、アルギニン、ヒスチジン、ハイド
ロキシリジンが認められる；
xi 加水分解物中の糖としてフルクトース、マン
ノース、フコース、イノシトール、ガラクトー
スが認められる；
を有する含糖蛋白質性物質からなる抗腫瘍剤にあ
る。
上記特性において、分子量はゲル過クロマト
グラフイーにより測定したものであり、呈色反応
は阿南功一、紺野邦夫、田村善蔵、松橋通生、松
木重一郎編、丸善発行（1976）「基礎生化学実験
法５」の化学的測定法に従つて行なつたものであ
り、アミノ酸測定のための加水分解は6N−HC
を用い、105〜110℃、24時間の条件で実施したも
のであり、糖測定のための加水分解は2N−HC
を用い、80〜90℃、10時間の条件で実施したもの
である。
本発明の抗腫瘍性物質は前記したごとく水溶性
含糖蛋白質であり、その精製度により元素分析
値、構成物質の含有量等はある程度変動するので
上記においては特定しなかつたが、代表的な特定
の物質として後記実施例５において得られる粉末
標品4fについてのこれらの測定値をつぎに示す。
元素分析：Ｃ＝43.2％、Ｈ＝6.5％、Ｎ＝10.1％、
灰分＝１％、Ｐ＝0.12％、Ｓ＝0.1％
比施光度：〔α〕20 D−15.7（ｃ＝0.5％、水中）
蛋白質含有量：ローリーフオーリー法によるフエ
ノール試薬による発色の結果、牛胎児血清アルブ
ミン換算の蛋白質含有量は60.5％であつた。
糖含有量：フエノール硫酸法によつて測定したグ
ルコース換算の糖含有量は19.9％であつた。
核酸含量：デイツシユ反応によるジフエニルアミ
ン法で測定したアデニール酸換算の核酸含量は
0.8％以下であつた。
本標品4fの紫外線吸収スペクトル及び赤外線吸
収スペクトルをそれぞれ第４図及び第５図に示
す。
以上のような本発明の抗腫瘍性物質の諸特性に
ついて、貝類から得た抗腫瘍性物質について文献
を調べたが、特公昭57−8088号、特開昭54−
41314号及び同54−41315号公報、さらにJ.
NationalCancerInstitute，60、No.６（1978）1499
−1500に記載の佐々木らの報告する性質と比べて
も同一性は認められず、全く新規な物質である。
以下本明細書において、本発明に関して使用す
る用語“貝類”はイタヤガイ科、リユウテンサザ
エ科、ミミガイ科及びマルスダレガイ科に属する
貝類を意味するものとする。
添付図面の第１図、第２図はホタテ貝の煮汁か
ら得られる水溶液を塩基性陰イオン交換体を使用
してイオン交換クロマトグラフイー処理して陰イ
オン交換体に吸着された物質を塩化ナトリウム水
溶液で溶出した場合の溶出パターンの結果を示
し、横軸は溶出液の画分ナンバーを表わし、白丸
は各画分の糖の含有量の指標、すなわちフエノー
ル硫酸法で発色させ、その490nm波長の吸光度
（光学密度）を、黒丸は各画分の蛋白質の含有量
の指標、すなわちロウリー（Lowry）法で発色
させ、その750nm波長の吸光度（光学密度）を表
わす。第３図は上記のようにしてイオン交換クロ
マトグラフイーから溶出分離した第１図のＣ画分
を分子量が1500〜100000の中に分画範囲を持つゲ
ル過用ゲルでゲル過した場合の結果を示し、
横軸は溶出液の画分ナンバーを表わし、白丸及び
黒丸は第１図、第２図と同じくそれぞれ各画分の
糖含有量及び蛋白質含有量の指標を表わす。第４
図、第５図は第３図にようにゲル過に付し、そ
の後さらに分子量300000を超える画分をゲル過
により除去した後に得られる分子量10000〜
300000の水溶性含糖蛋白質物質の紫外線吸収スペ
クトル図及び赤外線吸収スペクトル図をそれぞれ
表わす。
本発明で原料とする貝類の煮汁は、生又は加熱
した貝類の可食部分を熱水性溶媒液又は蒸気で蒸
煮もしくは加熱加工して食用貝肉を得る際に副生
する煮汁又は液汁を含包する。貝類を蒸煮もしく
は加熱加工する加熱媒体として使用し得る熱水性
溶媒は熱水蒸気、熱水でも、他の熱水性溶媒蒸気
あるいは熱水性溶媒液でもよい。
前記定義したとおり、本発明においては広くア
ワビ（Haliotisdiscus）の属するミミガイ科
（Haliotidas）の貝類、チヨウセンサザエ
（Marmarostoma argyrostoma）、タツマキサザ
エ（Tarbo Petholatus reevsi）、サザエ
（Batillus cornutus）の属するリユウテンサザエ
科（Turbinidae）の貝類、イタヤガイ（Pecten
albicans），ホタテガイ（Patinopecten
yessoensis）の属するイタヤガイ科
（Pectinidae）、アサリ（Tapes philippinarum），
ハマグリ（Meretrix lusoria）、チヨウセンハマ
グリ（Meretrixlamarcki DESHYES）の属する
マルスダレガイ科（Veneridae）の貝類を原料と
して使用し得るものであるが、以下においては特
に量的に入手しやすい、
ホタテガイ（Patinopecten yessoensis）
サザエ（Batillus cornutus）
アワビ（Haliotis discus）
ハマグリ（Meretrix lusoria）
について詳しく説明する。
本発明で原料として使用する煮汁を得る工程は
貝類を熱水性溶媒で蒸煮もしくは加熱加工してそ
の可食部分を採取する工程に該当し、この目的の
ために従来慣用の任意の方法で行なうことができ
る。勿論、本発明においては、煮汁を利用してこ
れから抗腫瘍剤有効成分、すなわち分子量10000
〜300000の範囲内の水溶性含糖蛋白質性抗腫瘍性
物質（以下抗腫瘍性物質又は単に有効成分とい
う）を採取するものであるから、貝類の可食部分
を採取する本来の目的を損なうことなく、しかも
抗腫瘍性物質を効果的に取得できるように該蒸煮
もしくは加熱加工工程を制御することが重要であ
る。よつて以下ではかゝる本発明の目的を好まし
く達成し得る熱水性溶媒による蒸煮もしくは加熱
加工工程の実施態様について煮汁の採取を中心と
して説明する。
貝類を熱水性溶媒で蒸煮、加熱加工して可食部
分を採取しかつ有効成分を含有する煮汁を得る蒸
煮加熱加工工程の操作は、熱水性溶媒蒸気又は熱
水性溶媒液を加熱媒体兼抽出溶媒として使用し、
貝類に熱水性溶媒蒸気及び／又は熱水性溶媒液を
接触させて煮汁を得る操作を一回又は二回以上行
なう方法が採用される。貝類と熱水性溶媒との接
触方法は、貝類に熱水性溶媒蒸気を直接当てる方
法、貝類に熱水性溶媒液を直接注ぎかける方法、
あるいは貝類を熱水性溶媒液中に浸漬して加熱す
る方法等がある。熱水性溶媒蒸気と熱水性溶媒液
は一方だけを使用しても、両者を同時に使用して
もよい。本発明の目的のためには、処理する貝類
の殻や肝臓は除いても除かなくてもよく、また貝
の肉部分は細断したり、磨砕、破砕あるいは粉砕
したりする必要がないので、蒸煮加熱処理後は全
量食用に供することができる。蒸煮加熱の温度条
件は約50〜120℃、特に60〜120℃が好ましい。煮
汁を得る操作は一回だけでも、三回以上行なつて
もよいが、通常は二回に分けて行なうのが好まし
く、又特に２回目は0.2〜20％の食塩を含む熱水
を用いるのが好ましい。一回だけ行なう場合には
生の貝に熱水性溶媒蒸気を直接吹きつける方法、
生の貝類に熱水性溶媒液を直接注ぎかける方法、
あるいは生の貝類を熱水性溶媒液中に浸漬して加
熱する方法により有効成分をいわゆる一番煮汁と
して得る方法が採用でき、二回に分けて行なう場
合には上記の方法で一番煮汁を採取した後、加熱
した貝類を食塩を含む熱水性溶媒液中に浸漬して
加熱する方法により有効成分をいわゆる二番煮汁
として得る方法が好ましい。一回だけ処理する場
合の好ましい処理条件を第１表にまとめて示す。
また二回に分けて処理を行なう場合の好ましい処
理条件を第２表にまとめて示す。上記した方法の
うち蒸煮加熱処理を二回に分けて行なうのが好ま
しく、その場合第一回の処理を熱水性溶媒蒸気を
使用し有効成分を一番煮汁として採取し、次いで
加熱した貝肉を食塩を含む熱水性溶媒液と接触さ
せて有効成分を二番煮汁として採取する方法は熱
水性溶媒の使用量が少なく抗腫瘍性物質の抽出効
果も良く、目的とする煮汁の濃縮が簡単であるこ
と、加熱効率がよいこと、設備が簡単でよいこと
から最も好ましい方法である。
The present invention is directed to the family Asperidae, the family Ornatidae,
The present invention relates to an anti-tumor substance consisting of a water-soluble glycoprotein obtained from a dried powder or concentrate of the broth of shellfish belonging to the family Ophthalidae and the family Ophthalmidae, and to a method for producing the same. Most conventional antitumor substances are low-molecular compounds, and their main effect is direct cytotoxicity on cancer cells, with strong general toxicity toward the host organism.
There is a problem in that the damage to living organisms is large. Therefore, polysaccharides, which are antitumor substances with relatively high molecular weight and low toxicity and have immunostimulatory effects, are extracted from plants, microorganisms, etc. and used in the form of coarse powder, but the anticancer spectrum is low. It has limited clinical use. The fact that anti-tumor substances can be obtained from shellfish was reported by Tokukosho.
No. 57-8088, Japanese Patent Application Publication No. 54-41314 and No. 54-41315
It is reported in the No. The production of antitumor substances disclosed in these patent publications involves (1) using raw shellfish as a raw material, removing the shell as it is or heating it, (2) removing the liver part, and (3) ) The obtained liver-free meat is shredded and ground with a blender etc. in an aqueous solvent such as water or a salt solution under low temperature conditions,
Alternatively, the antitumor substance is further homogenized and extracted by applying physical impact such as ultrasonication, so that it is easily solubilized in cold water below room temperature, and (4) the resulting mixture is shredded by centrifugation, etc. Water-insoluble substances such as meat are removed, and (5) the extract obtained in this way is purified using known proteinaceous substance separation and purification methods such as dialysis, ultrafiltration, gel filtration, and column chromatography. The purpose is to obtain an antitumor substance. This antitumor substance obtained from shellfish is notable for its broad anticancer spectrum and strong medicinal efficacy. However, in the above-mentioned production method, the meat of expensive shellfish that has edible value is used as raw material, which must be shredded and decomposed, and the extraction of the active ingredient is complicated, and the processed It is industrially disadvantageous, as there is a problem in disposing of the cut pieces, and it is difficult to secure a large amount of the active substance. The present inventors focused on the broad anticancer spectrum of antitumor substances obtained from shellfish within a certain range, and as a result of repeated research in order to industrially advantageously obtain antitumor substances from these shellfish, The broth from these shellfish, which was previously discarded as waste, is a by-product when these shellfish are steamed or heat-processed for food, and it costs a lot of money to dispose of because it contains a large amount of organic components that pollute rivers. It was discovered that an anti-tumor substance can be obtained from the broth of clams, and furthermore, the anti-tumor substance has a molecular weight of 10,000 to 10,000 which can be adsorbed on a basic anion exchanger.
The present invention was completed by discovering that it is a water-soluble glycoprotein within the range of 300,000. Therefore, the main object of the present invention is to provide a method for obtaining antitumor substances from shellfish broth within a certain range. Another object of the present invention is to provide a method for efficiently obtaining novel antitumor substances from the broth of these shellfish. Yet another object of the present invention is to provide a novel antitumor substance. In other words, the gist of the present invention is to use a dried powder or concentrate of the broth obtained when the edible parts of shellfish belonging to the family Polygonidae, the family Cercotidae, the family Ophthalidae, and the family Mutinidae are steamed or heat-processed in a hydrothermal solvent. The raw material is subjected to ion exchange chromatography using a basic anion exchanger and gel filtration using a gel permeation gel to obtain an antitumor substance consisting of a water-soluble glycoprotein with a molecular weight within the range of 10,000 to 300,000. A method for producing an antitumor substance, which is characterized by fractionating a substance, and the water-soluble glycoprotein substance thus obtained has the following characteristics: A slightly brownish-white or white solid; Easily soluble in water Insoluble in organic solvents such as methanol, ethanol, and acetone; Ampholyte; Infrared absorption spectrum with KBr pellets shows 3500 to 3300 cm -1 , 1650 cm -1 , 1540 cm -1 and
Shows main peak around 1400 cm -1 ; Biuretz reaction, xanthoprotein reaction,
Reaction with phenol reagent is positive; Anthrone sulfuric acid reaction and phenol sulfuric acid reaction are positive, cysteine sulfuric acid reaction is false positive; Ultraviolet absorption spectrum has absorption at λ H2O nax 278 nm; Includes molecular weight range of 10,000 to 300,000; Clear It has no melting point and begins to decompose above 235°C; amino acids in the hydrolyzate include aspartic acid, hydroproline, threonine, serine,
Glutamic acid, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, arginine, histidine, and hydroxylidine are recognized; xi Sugars in the hydrolyzate include fructose, mannose, fucose, Inositol and galactose are found in antitumor agents consisting of glycoprotein-containing substances. In the above properties, the molecular weight is measured by gel permeation chromatography, and the color reaction is based on Koichi Anan, Kunio Konno, Zenzo Tamura, Michio Matsuhashi, Shigeichiro Matsuki (eds.), published by Maruzen (1976), Basic Biochemistry. The experiment was carried out according to the chemical measurement method described in ``Experimental Method 5'', and the hydrolysis for amino acid measurement was performed using 6N-HC.
The hydrolysis for sugar measurement was carried out using 2N-HC at 105-110℃ for 24 hours.
The experiment was carried out at 80 to 90°C for 10 hours. As mentioned above, the antitumor substance of the present invention is a water-soluble glycoprotein, and its elemental analysis value, content of constituent substances, etc. vary to some extent depending on its degree of purification, so it was not specified above, but representative These measured values for powder specimen 4f obtained as a specific substance in Example 5 below are shown below. Elemental analysis: C = 43.2%, H = 6.5%, N = 10.1%,
Ash content = 1%, P = 0.12%, S = 0.1% Specific light intensity: [α] 20 D -15.7 (c = 0.5%, in water) Protein content: As a result of color development with a phenol reagent by the Lowry-Folley method, cattle The protein content calculated as fetal serum albumin was 60.5%. Sugar content: The sugar content measured by the phenol sulfuric acid method was 19.9% in terms of glucose. Nucleic acid content: The nucleic acid content in terms of adenylate acid measured by the diphenylamine method using a date-shock reaction is
It was less than 0.8%. The ultraviolet absorption spectrum and infrared absorption spectrum of this specimen 4f are shown in Figures 4 and 5, respectively. Regarding the characteristics of the antitumor substance of the present invention as described above, we investigated the literature regarding antitumor substances obtained from shellfish, and found that
41314 and 54-41315, as well as J.
National Cancer Institute, 60 , No. 6 (1978) 1499
Even when compared with the properties reported by Sasaki et al. described in 1500, no identity is found, and it is a completely new substance. Hereinafter, in the present specification, the term "shellfish" used in connection with the present invention shall mean shellfish belonging to the families of the family Apocalypidae, the family Aperaceidae, the family Ophthalidae, and the family Muthidae. Figures 1 and 2 of the attached drawings show that an aqueous solution obtained from scallop broth is subjected to ion exchange chromatography using a basic anion exchanger, and the substances adsorbed on the anion exchanger are separated by sodium chloride. The results of the elution pattern when eluted with an aqueous solution are shown. The horizontal axis represents the fraction number of the eluate, and the white circles are indicators of the sugar content of each fraction. The black circles represent the absorbance (optical density) at a wavelength of 750 nm, which is an indicator of the protein content of each fraction, which is developed using the Lowry method. Figure 3 shows the results when fraction C in Figure 1, which was eluted and separated from ion exchange chromatography as described above, was subjected to gel filtration using a gel filtration gel with a molecular weight fractionation range of 1,500 to 100,000. shows,
The horizontal axis represents the fraction number of the eluate, and the white circles and black circles represent indicators of the sugar content and protein content of each fraction, respectively, as in FIGS. 1 and 2. Fourth
Figure 5 shows a molecular weight of 10,000 to 10,000 obtained after gel filtration as shown in Figure 3, and then further removal of the fraction with a molecular weight of over 300,000 by gel filtration.
3 shows the ultraviolet absorption spectrum and infrared absorption spectrum of 300,000 water-soluble glycoprotein substances, respectively. The broth of shellfish that is used as a raw material in the present invention includes broth or sap that is a by-product when obtaining edible shellfish meat by steaming or heat-processing the edible parts of raw or heated shellfish in a hydrothermal solvent solution or steam. wrap The hydrothermal solvent that can be used as a heating medium for steaming or heat-processing shellfish may be hot steam, hot water, or other hydrothermal solvent vapor or hydrothermal solvent liquid. As defined above, in the present invention, shellfishes of the family Haliotidae, to which Haliotisdiscus belongs, Marmarostoma argyrostoma, Tarbo Petholatus reevsi, and Batillus cornutus, to which Pecten, a shellfish of the family Turbinidae.
albicans), scallop (Patinopecten)
Yessoensis) belongs to Pectinidae, clams (Tapes philippinarum),
Shellfish of the family Veneridae, which includes clams (Meretrix lusoria) and Japanese clams (Meretrix lamarcki DESHYES), can be used as raw materials, but the following are particularly easy to obtain in quantity: scallops (Patinopecten yessoensis), turban shells (Batillus cornutus) Abalone (Haliotis discus) Clam (Meretrix lusoria) will be explained in detail. The process of obtaining the broth used as a raw material in the present invention corresponds to the process of steaming or heating shellfish in a hydrothermal solvent and collecting the edible parts thereof, and for this purpose, any conventional method can be used. be able to. Of course, in the present invention, the active ingredient of the antitumor agent, that is, the molecular weight of
Since the purpose is to collect water-soluble glycoprotein antitumor substances within the range of ~300,000 (hereinafter referred to as antitumor substances or simply active ingredients), it defeats the original purpose of collecting the edible parts of shellfish. It is important to control the steaming or heating process so that the antitumor substance can be effectively obtained. Therefore, embodiments of the steaming or heating processing step using a hydrothermal solvent that can preferably achieve the object of the present invention will be described below, focusing on the collection of broth. The operation of the steaming and heating processing process involves steaming and heating shellfish in a hydrothermal solvent to collect edible parts and obtain a broth containing active ingredients. Used as both extraction solvent and
A method is employed in which the operation of bringing shellfish into contact with a hydrothermal solvent vapor and/or a hydrothermal solvent liquid to obtain a broth is performed once or twice or more. Methods for contacting shellfish with a hydrothermal solvent include direct application of hydrothermal solvent vapor to shellfish, direct pouring of a hydrothermal solvent solution onto shellfish,
Alternatively, there is a method in which shellfish are immersed in a hydrothermal solvent solution and heated. Only one of the hydrothermal solvent vapor and the hydrothermal solvent liquid may be used, or both may be used simultaneously. For the purposes of the present invention, the shells and liver of the shellfish to be processed may or may not be removed, and the flesh of the shellfish need not be shredded, ground, crushed or crushed. Therefore, the entire amount can be eaten after steaming and heat treatment. The temperature condition for steaming and heating is preferably about 50 to 120°C, particularly 60 to 120°C. The operation to obtain the broth may be carried out once or three or more times, but it is usually preferable to carry out the process in two parts, and especially for the second time, it is recommended to use hot water containing 0.2 to 20% salt. is preferred. For one-time use, direct spraying of hot water solvent vapor onto raw shellfish;
A method of directly pouring a hydrothermal solvent solution onto raw shellfish,
Alternatively, a method can be adopted in which raw shellfish are immersed in a hydrothermal solvent solution and heated to obtain the active ingredients as so-called Ichiban broth.If the process is to be carried out in two batches, the above-mentioned method can be used to obtain the Ichiban broth. A preferred method is to obtain the active ingredients as a so-called second broth by immersing the heated shellfish in a hot aqueous solvent solution containing salt and heating the shellfish after collection. Table 1 summarizes the preferable treatment conditions when the treatment is carried out only once.
In addition, preferred treatment conditions when the treatment is carried out in two steps are summarized in Table 2. Of the above methods, it is preferable to carry out the steaming and heat treatment in two steps; in that case, the first step is to use hot aqueous solvent vapor to collect the active ingredients as the boiling liquid, and then the heated shellfish meat is heated. The method of contacting with a hydrothermal solvent solution containing salt and collecting the active ingredients as a second broth requires less amount of hydrothermal solvent, has a good extraction effect on antitumor substances, and allows the desired concentration of the broth. This is the most preferred method because it is simple, has good heating efficiency, and requires simple equipment.
【表】【table】
【表】
貝類から有効成分を効果的に採取するには前述
のごとく熱水性溶媒による蒸煮加熱処理の温度条
件に留意すべきであり、好ましい蒸煮加熱温度は
約50〜120℃である。低温すぎると有効成分が効
率良く採取できないし、高温すぎると有効成分が
破壊される。処理時の系の圧力は減圧、常圧、加
圧のいずれでもよい。貝類と熱水性溶媒との接触
時間は低温条件下では長くし、高温条件下では短
かくてよい。処理する貝類に対する熱水性溶媒の
使用量は、少なすぎると有効成分の採取効率が悪
くなり、多すぎるとその後の濃縮操作が煩雑にな
る。蒸煮加熱加工工程で使用する水性溶媒の例と
しては水、食塩水、海水、その他各種水溶性塩類
の水溶液、緩衝液（塩など添加物質の濃度は約
0.5〜20重量％程度が好ましい）、水溶性溶剤、例
えばエタノールのような低級アルコール類の水溶
液（溶剤濃度は約１〜50重量％程度が好ましい）
などがあり、好ましい水性溶媒は水又は食塩水で
ある。
こうして得られた一番煮汁、二番煮汁等の煮汁
中にはいずれも有効成分が含まれるので、このま
ま、あるいは約30〜100℃程度の温度で加熱濃縮
したり、透析や限外過処理により脱塩したり、
あるいは熱風搬送型乾燥法、凍結乾燥法等により
粉末にして抗腫瘍剤として使用してもよい。熱風
搬送型乾燥法により煮汁から有効成分を粉末とし
て得る場合、乾燥器への熱風入口温度は約200〜
350℃、特に約250〜310℃が好ましく、熱風出口
温度は約80〜170℃、特に約100〜150℃が好まし
く、接触（滞留）時間は約５〜80秒、特に５〜50
秒が好ましい。このような条件下では有効成分が
破壊されることはない。
本発明においては、かく得られる貝類煮汁の乾
燥粉末又は濃縮物から抗腫瘍性物質を分離、採取
して抗腫瘍剤として使用しようとするものである
が、本発明者らは工業的に多量生産に使用し得る
抗腫瘍性物質の分離、精製法について種々検討し
た結果、最も効率的に抗腫瘍性を損なうことなく
しかも経済的に精製できる方法として本発明の方
法を完成した。分子量10000〜300000の範囲内の
水溶性含糖蛋白質からなる本発明の抗腫瘍性物質
を製造する好ましい方法は、貝類の煮汁の乾燥粉
末または濃縮物を水性液とし、この水溶液を塩基
性陰イオン交換体を使用するイオン交換クロマト
グラフイーおよびゲル過用ゲルを用いるゲル
過に付して塩基性陰イオン交換体に吸着される分
子量10000〜300000の範囲の画分を取得すること
からなる。イオン交換クロマトグラフイーによる
処理は分子量10000〜300000の画分を分離取得す
るゲル過処理の後で行なってもよいが、ゲル
過処理の前に行なうのが特に好ましい。イオン交
換クロマトグラフイーによる処理において塩基性
陰イオン交換体に吸着された水溶性含糖蛋白質は
約0.07〜0.4モル濃度、好適には約0.1〜0.38モル
濃度、最適には約0.1〜0.3モル濃度の塩化ナトリ
ウムを含有する水溶液を溶出剤として用いること
が望ましい。またイオン交換クロマトグラフイー
及びゲル過処理に先立つて貝類の煮汁から得ら
れる水溶液から不溶解部を除去しておくことが望
ましく、また無機塩も除去しておかないとイオン
交換クロマトグラフイーやゲル過処理が円滑に
行われない。以下これら工程の詳細について述べ
る。また得られた物質の抗腫瘍活性の検定方法は
特記しない限り次の方法で行なつた。
抗腫瘍活性の検定方法
６週令のマウス（ICR系雌）腹腔内にサルコー
マ（Sarcoma）180腫瘍細胞を接種し、１週間後
に増殖した腫瘍細胞を腹水と共に抜き取り、この
細胞４×106個を他の６週令マウス（ICR系雌）
のそけい部皮下に移植する。１週間後固形腫瘍が
形成されていることを確認して、検定試料を所定
の濃度となるように生理食塩水に溶解してその腫
瘍内もしくはその周囲に移植後、５，７，９日目
に隔日３回投与する。腫瘍移植５週間後の固形腫
瘍を摘出し、その重量を試料の代りに生理食塩水
を投与した対照区の場合と比較する。中間経過に
ついては固形腫瘍の直径を測定することによつて
調べた。得られた結果は次の式によつて表示し
た。
腫瘍阻止率（％）
＝（１−薬剤投与区平均腫瘍重量／対照区平均腫瘍
重量）×100
完全治癒＝完全治癒マウス匹数／総マウス匹数
目的物質の分離方法につき詳述すると、貝類の
煮汁から得られる原料水溶液からまず不溶解部を
例えば遠心分離、過あるいは傾斜法により除去
し、後脱塩する。脱塩は例えば無機塩など低分子
化合物を分画できるゲル過用ゲルを用いるゲル
過処理により行なつても、原料水溶液をセルロ
ースチユーブに入れ蒸留水を流して透析により行
なつてもよい。例えば貝類煮汁の乾燥粉末又は濃
縮物を乾燥固形重量として約10〜25重量％になる
ように、０〜0.15モル／の塩化ナトリウムを含
有するPH７〜7.5の0.01〜0.1モル／、好ましく
は0.01〜0.5モル／のリン酸緩衝液よりなる溶
出剤中に溶解し、遠心分離機で不溶性物質を沈澱
物として除去し、得られる溶液を分子量が約500
〜5000の分画範囲を持つゲル過用ゲル（例えば
フアルマシア フアイン ケミカルズ
（Pharmacia Fine Chemicals）社製の登録商標
セフアテツクス（Sephadex）Ｇ−25。これはテ
キストランをエピクロロヒドリンで三次元的に架
橋して得られたゲルである。）を充填したカラム
に注入し、同じ緩衝液を溶出剤としてゲル過ク
ロマトグラフイーを行ない、溶出液の電気伝導度
が上昇する前の画分を取得する。この操作により
塩化ナトリウムなど無機塩は高分子量の有機物質
を主とする画分から分離することができる。この
操作において高分子の有機物と無機塩の画分を分
けるのに電気伝導度の差を見て判定したが、この
ような分離を確認する手段としてはその他いかな
る検出器を用いてもよい。
こうして得られる比較的高分子の有機物を含有
する溶出画分は排除限界分子量（分画できる上限
値）が充分大きい、例えば約500000から1000000
の塩基性陰イオン交換ゲル、好適には解離基とし
てジエチルアミノエチル基あるいはアミノエチル
基を有する陰イオン交換ゲル（例えばフアルマシ
ア フアイン ケミカルズ社製の登録商標DEAE
−セフアロース（Sepharose）CL−6B。これは
アガロースを２，３−ジブロモプロパノールを用
いて三次元架橋したものにジエチルアミノエチル
基をエーテル結合で結合したもので、対イオンと
してクロルイオンを持ち、排除限界分子量は約１
×106，総交換容量は15±2meq／100mlゲルであ
る。）を充填したカラムに注入し、吸着されない
物質（主として多糖類と溶出剤の塩化ナトリウム
とリン酸緩衝液の塩濃度で脱離する低イオン親和
性の物質からなる。）を素通りさせて取り除き、
吸着性物質をイオン交換体上に吸着させる。溶出
剤の塩濃度で吸着する物質を充分に吸着したイオ
ン交換カラムに0.07〜0.4モル濃度、好適には0.1
〜0.38モル濃度、最適には約0.1〜0.3モル濃度の
塩化ナトリウムを含有するPH7.0〜7.5の0.01〜0.1
モル濃度のリン酸緩衝液からなる溶出剤を徐々に
流し込んで溶出液を紫外線検出器などのモニター
を使用してこのイオン濃度で脱離溶出してくる画
分を採取する。
このイオン交換クロマトグラフイー処理におけ
る目的物を溶出する塩濃度の決定は次の実験結果
に基づいて行なつた。
ホタテ貝煮汁の乾燥粉末を原料とし、セフアデ
ツクスG25ゲル過分離で得られた比較的高分子
の有機物画分の凍結乾燥粉末5gをPH7.0の
0.01M／りん酸緩衝液20mlに溶解した試料液を
0.01M／りん酸緩衝液で平衡化させたDEAE−
セフアローズCL−6Bゲルカラムに添加する。溶
出剤に塩化ナトリウムの連続的濃度勾配をつけて
クロマトグラフイーを行うと第１図に示す溶出パ
ターンが得られ、これを塩化ナトリウム濃度がそ
れぞれ０，０〜0.1，0.1〜0.25，0.25〜0.38モル／
で溶出してくるＡ，Ｂ，Ｃ，Ｄの４画分に分画
し、凍結乾燥して粉末標品Ｄ−Ａ，Ｄ−Ｂ，Ｄ−
Ｃ，Ｄ−Ｄを得た。これらの画分の抗腫瘍活性は
サルコーマ180腫瘍細胞をマウスの右そけい部の
皮下に移植し、固形腫瘍の発生したICR系マウス
の腫瘍内局所投与での腫瘍阻止率で測定した結果
を下表に示す。[Table] In order to effectively extract active ingredients from shellfish, attention should be paid to the temperature conditions of steaming and heat treatment using a hydrothermal solvent as described above, and the preferred steaming and heating temperature is about 50 to 120°C. If the temperature is too low, the active ingredients cannot be collected efficiently, and if the temperature is too high, the active ingredients will be destroyed. The pressure of the system during treatment may be reduced pressure, normal pressure, or increased pressure. The contact time between the shellfish and the hydrothermal solvent may be longer under low temperature conditions and shorter under high temperature conditions. If the amount of hydrothermal solvent used for the shellfish to be treated is too small, the efficiency of collecting the active ingredient will be poor, and if it is too large, the subsequent concentration operation will be complicated. Examples of aqueous solvents used in the steaming and heating processing process include water, saline, seawater, aqueous solutions of various other water-soluble salts, and buffer solutions (concentrations of additives such as salts are approx.
(preferably about 0.5 to 20% by weight), a water-soluble solvent, such as an aqueous solution of a lower alcohol such as ethanol (the solvent concentration is preferably about 1 to 50% by weight)
etc., and the preferred aqueous solvent is water or saline. The broths obtained in this way, such as the first broth and the second broth, all contain active ingredients, so they can be used as is, concentrated by heating at a temperature of about 30 to 100 degrees Celsius, or subjected to dialysis or ultrafilter treatment. Desalt or
Alternatively, it may be made into powder by hot air conveyance drying method, freeze-drying method, etc. and used as an antitumor agent. When obtaining the active ingredient from the broth as a powder using the hot air conveyance drying method, the hot air inlet temperature to the dryer is approximately 200~200℃.
350°C, especially about 250-310°C is preferred, the hot air outlet temperature is preferably about 80-170°C, especially about 100-150°C, and the contact (residence) time is about 5-80 seconds, especially about 5-50°C.
Seconds are preferred. The active ingredients are not destroyed under these conditions. In the present invention, the antitumor substance is separated and collected from the dried powder or concentrate of the shellfish broth obtained in this way and is intended to be used as an antitumor agent. As a result of various studies on separation and purification methods for antitumor substances that can be used in the present invention, the method of the present invention was completed as the most efficient and economical purification method without impairing antitumor properties. A preferred method for producing the antitumor substance of the present invention comprising a water-soluble glycoprotein with a molecular weight in the range of 10,000 to 300,000 is to prepare a dry powder or concentrate of shellfish broth as an aqueous solution, and add a basic anion to the aqueous solution. It consists of ion exchange chromatography using an exchanger and gel filtration using a gel for gel filtration to obtain a fraction with a molecular weight in the range of 10,000 to 300,000 that is adsorbed on a basic anion exchanger. Although the treatment by ion exchange chromatography may be carried out after the gel filtration treatment to separate and obtain a fraction with a molecular weight of 10,000 to 300,000, it is particularly preferably carried out before the gel filtration treatment. The water-soluble glycoprotein adsorbed onto the basic anion exchanger during treatment by ion exchange chromatography has a concentration of about 0.07 to 0.4 molar, preferably about 0.1 to 0.38 molar, optimally about 0.1 to 0.3 molar. It is preferable to use an aqueous solution containing sodium chloride of 50% as the eluent. In addition, it is desirable to remove insoluble parts from the aqueous solution obtained from shellfish broth prior to ion exchange chromatography and gel filtration treatment. Over-processing is not carried out smoothly. Details of these steps will be described below. The antitumor activity of the obtained substances was assayed as follows unless otherwise specified. Method for assaying antitumor activity: Sarcoma 180 tumor cells were intraperitoneally inoculated into a 6-week-old mouse (ICR female), and after 1 week, the proliferated tumor cells were removed together with ascites, and 4 x 10 6 of these cells were collected. Other 6-week-old mice (ICR female)
Transplant subcutaneously to the groin area. After one week, it was confirmed that a solid tumor had formed, and the test sample was dissolved in physiological saline to a predetermined concentration and transplanted into or around the tumor, and then on days 5, 7, and 9. Administer 3 times every other day. Solid tumors are excised 5 weeks after tumor implantation, and their weights are compared with those of a control group in which physiological saline was administered instead of the sample. The interim course was investigated by measuring the diameter of solid tumors. The obtained results were expressed using the following formula. Tumor inhibition rate (%) = (1 - drug administration group average tumor weight / control group average tumor weight) × 100 Complete cure = number of completely cured mice / total number of mice To explain in detail the method for separating the target substance, First, undissolved parts are removed from the raw material aqueous solution obtained from the broth by, for example, centrifugation, filtration or decanting, and then desalted. Desalting may be carried out, for example, by gel filtration treatment using a gel filtration gel capable of fractionating low-molecular compounds such as inorganic salts, or by dialysis by placing the aqueous raw material solution in a cellulose tube and flowing distilled water through it. For example, the dried powder or concentrate of shellfish broth has a pH of 7 to 7.5 and contains 0 to 0.15 mol of sodium chloride, preferably 0.01 to 0.1 mol/, preferably 0.01 to 0.1 mol/containing sodium chloride of about 10 to 25% by dry solid weight. It is dissolved in an eluent consisting of 0.5 mol/phosphate buffer, and the insoluble substances are removed as a precipitate using a centrifuge, and the resulting solution has a molecular weight of about 500.
A gel-filtration gel with a fractionation range of ) is injected into a column packed with gel, and gel perchromatography is performed using the same buffer as the eluent to obtain the fraction before the electrical conductivity of the eluate increases. By this operation, inorganic salts such as sodium chloride can be separated from the fraction mainly consisting of high molecular weight organic substances. In this operation, the difference in electrical conductivity was used to separate the macromolecular organic matter and inorganic salt fractions, but any other detector may be used to confirm such separation. The elution fraction containing relatively high-molecular organic substances obtained in this way has a sufficiently large exclusion limit molecular weight (upper limit that can be fractionated), for example, about 500,000 to 1,000,000.
a basic anion exchange gel, preferably an anion exchange gel having a diethylaminoethyl group or an aminoethyl group as a dissociative group (e.g. DEAE, a registered trademark manufactured by Pharmacia Fine Chemicals)
-Sepharose CL-6B. This is agarose three-dimensionally cross-linked using 2,3-dibromopropanol and a diethylaminoethyl group bonded to it through an ether bond.It has a chloride ion as a counterion and has an exclusion limit molecular weight of approximately 1.
×10 6 , total exchange volume is 15±2meq/100ml gel. ) is injected into a column packed with the solution, and substances that are not adsorbed (mainly consisting of polysaccharides and substances with low ionic affinity that are desorbed by the salt concentration of the eluent sodium chloride and phosphate buffer) are removed by passing through.
The adsorptive substance is adsorbed onto the ion exchanger. The salt concentration of the eluent is 0.07 to 0.4 molar, preferably 0.1, to the ion exchange column that has sufficiently adsorbed the substance to be adsorbed.
0.01-0.1 at PH7.0-7.5 containing ~0.38 molar, optimally about 0.1-0.3 molar sodium chloride
An eluent consisting of a phosphate buffer solution with a molar concentration is gradually poured in, and a monitor such as an ultraviolet detector is used to collect the fraction that is desorbed and eluted at this ion concentration. The salt concentration at which the target substance was eluted in this ion exchange chromatography treatment was determined based on the following experimental results. Using dry powder of scallop broth as raw material, 5g of freeze-dried powder of relatively high molecular weight organic matter fraction obtained by Sephadex G25 gel permeation was added to a pH of 7.0.
Sample solution dissolved in 20ml of 0.01M phosphate buffer
DEAE− equilibrated with 0.01M/phosphate buffer
Add to Sepharose CL-6B gel column. When chromatography is performed using a continuous concentration gradient of sodium chloride as the eluent, the elution pattern shown in Figure 1 is obtained, with the sodium chloride concentration being 0, 0 to 0.1, 0.1 to 0.25, and 0.25 to 0.38, respectively. Mol/
It is fractionated into four fractions, A, B, C, and D, which are eluted by
C, DD were obtained. The antitumor activity of these fractions was determined by subcutaneously implanting Sarcoma 180 tumor cells into the right groin of mice, and measuring the tumor inhibition rate by local administration within the tumor of ICR mice that developed solid tumors. Shown in the table.
【表】
塩化ナトリウム濃度が0.1〜0.25M／付近の
Ｄ−Ｃ画分は腫瘍阻止率88.5％、腫瘍完全退消数
（完全治癒）４匹中２匹と高い抗腫瘍活性を示し
ている。
そこで上記と同じ原料をDEAE−セフアローズ
CL−6Bのカラムに通し、塩化ナトリウム濃度を
段階的に0.07M／，0.25M／，そして
0.35M／と変化させて溶出した精製溶出パター
ンを第２図に示す。第２図に示すように0.25M／
塩化ナトリウム緩衝液での溶出画分をピークの
ない前画分、糖のピークがある画分、蛋白質のピ
ークがある画分、後画分のＡ，Ｂ，Ｃ，Ｄ画分に
分け、凍結乾燥してそれぞれDS−Ａ，DS−Ｂ，
DS−Ｃ，DS−Ｄの４画分に分け、前述の検定法
に従つて抗腫瘍性を検討すると、次表の通りDS
−Ａ，DS−Ｂ，DS−Ｃの３画分に活性をもつ物
質が多く含まれている。[Table] The DC fraction with a sodium chloride concentration of around 0.1 to 0.25 M shows high antitumor activity, with a tumor inhibition rate of 88.5% and complete tumor regression (complete cure) in 2 out of 4 animals. Therefore, the same raw materials as above were used as DEAE-Sepharose.
Pass it through a CL-6B column and increase the sodium chloride concentration stepwise to 0.07M/, 0.25M/, and
Fig. 2 shows the purified elution pattern eluted at a concentration of 0.35M. 0.25M/as shown in Figure 2
Divide the elution fraction with sodium chloride buffer into a pre-fraction with no peak, a fraction with a sugar peak, a fraction with a protein peak, and post-fractions A, B, C, and D, and freeze. After drying, DS-A, DS-B,
When divided into four fractions, DS-C and DS-D, and examined for antitumor activity according to the assay method described above, DS
The three fractions -A, DS-B, and DS-C contain many active substances.
【表】
上記実験からDEAE−セフアローズのカラムに
吸着された画分を0.07〜0.4Mの塩化ナトリウム
を含有する溶出剤で溶出すると目的物質が効果的
に回収できることがわかる。
イオン交換クロマトグラフイー処理により得ら
れた目的物含有溶液はゲル過処理に付し目的と
する分子量10000〜300000の成分を分離するが、
それに先立ち脱塩濃縮する。脱塩はゲル過によ
り行なつても、透析により行なつてもよい。脱
塩、濃縮は、例えば本溶出画分を40℃以下（好ま
しくは20℃以下）の温度で溶出量の約1/5〜1/6に
減圧下で濃縮し、これを再び分子量が500〜5000
の内の分画範囲にある適当なゲル過用ゲル（例
えばセフアデツクスＧ−25）中に導き、蒸留水を
溶出剤として、この溶出液の電気伝導度を測定し
て脱塩処理を行なう。溶出液は再び減圧下で40℃
以下（好ましくは20℃以下）の温度で溶出液の1/
５〜1/6に濃縮し、そのあとは凍結乾燥で粉末化し
た。この段階における原料の煮汁粉末又は濃縮物
の粉末化したものに対する収率は約1/60〜1/100
であつた。
こうして精製した目的物含有混合物の精製度を
更にあげるためには分子量が1500〜100000の中に
分画範囲を持つゲル過用ゲル（例えばフアルマ
シア フアイン ケミカルズ社製の登録商標セフ
アデツクスＧ−75。これはデキストランをエピク
ロロヒドリンで三次元的に架橋したもの）を用い
て行なうゲル過処理が大量処理としての流速の
確保ができるのみならず、処理中の原因不明によ
る抗腫瘍性成分の失活もなく効果的であることを
見出し本発明方法に組み入れた。この操作例を述
べると、まずイオン交換ゲルから脱着し、脱塩、
粉末化した画分（前記Ｄ−Ｃ相当品）1gをPH7.0
〜7.5の０〜0.1M／リン酸緩衝液５mlに溶解
し、セフアデツクスＧ−75を充填し、PH7.0〜7.5
の０〜0.01M／リン酸緩衝液で平衡化したゲル
過カラムに注入し、PH7.0〜7.5の０〜0.1M／
リン酸緩衝液を溶出剤としてゲル過を行なう。
この溶出パターンの一例は第３図に示したもので
あり、蛋白質と糖の小さいピークのある前画分、
糖のピークがなく蛋白質をかなり含有する中画
分、蛋白質と糖の大きなピークのある後画分のそ
れぞれＡ，Ｂ，Ｃの３画分に分け、これらをそれ
ぞれ透析、凍結乾燥してＧ−Ａ，Ｇ−Ｂ，Ｇ−Ｃ
の粉末試料を得た。
この標品の抗腫瘍性活性を調べた結果を次表に
示す。[Table] The above experiment shows that the target substance can be effectively recovered when the fraction adsorbed on the DEAE-Sepharose column is eluted with an eluent containing 0.07 to 0.4M sodium chloride. The target substance-containing solution obtained by ion-exchange chromatography treatment is subjected to gel filtration treatment to separate the target component with a molecular weight of 10,000 to 300,000.
Prior to that, it is desalted and concentrated. Desalting may be performed by gel filtration or dialysis. For desalting and concentration, for example, the main elution fraction is concentrated under reduced pressure at a temperature of 40°C or lower (preferably 20°C or lower) to about 1/5 to 1/6 of the elution amount, and then concentrated again to a fraction with a molecular weight of 500 to 1/6. 5000
The desalting treatment is carried out by introducing the eluate into a suitable gel-passing gel (for example, Sephadex G-25) that has a fractionation range within the above range, and measuring the electrical conductivity of the eluate using distilled water as an eluent. The eluate was heated again at 40°C under reduced pressure.
1/1 of the eluate at a temperature below (preferably below 20°C)
It was concentrated to 5 to 1/6, and then lyophilized to powder. The yield at this stage is approximately 1/60 to 1/100 of the raw material powdered broth or concentrate powder.
It was hot. In order to further increase the degree of purification of the target product-containing mixture thus purified, it is necessary to use a gel filtration gel with a fractionation range of molecular weight between 1,500 and 100,000 (for example, Sephadex G-75, a registered trademark manufactured by Pharmacia Fine Chemicals Co., Ltd.). Gel filtration treatment using dextran (three-dimensionally cross-linked with epichlorohydrin) not only ensures a high flow rate for large-scale treatment, but also prevents the deactivation of antitumor components due to unknown reasons during treatment. It was found that this method was effective without any problems, and was incorporated into the method of the present invention. To describe an example of this operation, first, it is desorbed from the ion exchange gel, desalted,
1g of the powdered fraction (equivalent to D-C above) at pH 7.0
~7.5 dissolved in 5 ml of 0-0.1M/phosphate buffer, filled with Sephadex G-75, pH7.0-7.5
Inject into a gel filtration column equilibrated with 0-0.01M/phosphate buffer at pH 7.0-7.5.
Perform gel filtration using phosphate buffer as an eluent.
An example of this elution pattern is shown in Figure 3, where the pre-fraction with small protein and sugar peaks,
The middle fraction, which has no sugar peak and contains a considerable amount of protein, and the rear fraction, which has large protein and sugar peaks, are divided into three fractions, A, B, and C. These are each dialyzed and freeze-dried to obtain G- A, G-B, G-C
A powder sample was obtained. The results of examining the antitumor activity of this preparation are shown in the table below.
【表】
この結果Ｇ−Ａの画分が他のＧ−Ｂ，Ｇ−Ｃの
画分に比べて腫瘍阻止率も高く、またこの画分の
収率が原料に対し約1/50の21mgであることから
精製効率も良く、精製法に取入れた。尚、このＧ
−Ａの画分のおおよその下限の分子量を調べるた
めにフアルマシア フアイン ケミカルズ社製の
セフアデツクスＧ−50を用いてトリプトフアン、
ビタミンB12、チトクロームＣ、オブアルブミン
などを標準物質として用いてゲル過により本物
質の分子量を測定したところ、Ｇ−Ａ画分は分子
量として約10000以上のものが含まれていた。
得られたＧ−Ａ画分の精製を更に進めるため、
Ｇ−Ａ画分相当品1gを少量の０〜0.1M／のPH
7.0〜7.5のリン酸緩衝液に溶かし、PH7.0〜7.5の
０〜0.1M／のリン酸緩衝液で平衡化してある
分子量が10000〜2000000の蛋白質性物質などを分
画範囲として持つゲル過用ゲル（例えばフアル
マシア フアイン ケミカルズ社製の登録商標セ
フアクリル（Sephacryl）Ｓ−400。これはアリ
ルデキストランをＮ，N′−メチレンビスアクリ
ルアミドで架橋したもの。）を充填したカラムで
ゲル過を行ない、その後透析し、凍結乾燥して
粉末化した。その結果分子量300000以上の画分Ｓ
−Ａ，分子量が300000〜150000の画分Ｓ−Ｂ及び
分子量が150000以下の画分Ｓ−Ｃが得られた。
（各画分の分離は別に本ゲル過用ゲルを用いて
分子量測定用標準物質の溶出位置から求めた検量
線を基準にして行なつた。）得られた試料Ｓ−Ａ，
Ｓ−Ｂ，Ｓ−Ｃの抗腫瘍活性をサルコーマ180結
節型抗腫瘍に大して調べたところ、次表に示すと
おりであつた。[Table] As a result, the G-A fraction has a higher tumor inhibition rate than the other G-B and G-C fractions, and the yield of this fraction is 21 mg, about 1/50 of the raw material. Because of this, the purification efficiency was good, so we incorporated it into the purification method. Furthermore, this G
- In order to determine the approximate lower limit molecular weight of the fraction A, tryptophan,
When the molecular weight of this substance was measured by gel filtration using vitamin B 12 , cytochrome C, ovalbumin, etc. as standard substances, it was found that the GA fraction contained a molecular weight of about 10,000 or more. In order to further purify the obtained G-A fraction,
Add 1g of G-A fraction equivalent to a small amount of PH of 0 to 0.1M/
A gel filter that has a fractionation range of proteinaceous substances with a molecular weight of 10,000 to 2,000,000, which is dissolved in a phosphate buffer with a pH of 7.0 to 7.5 and equilibrated with a 0 to 0.1M phosphate buffer with a pH of 7.0 to 7.5. Gel filtration is performed in a column packed with a commercially available gel (for example, Sephacryl S-400, a registered trademark manufactured by Pharmacia Fine Chemicals, Inc., which is allyl dextran cross-linked with N,N'-methylenebisacrylamide), and then It was dialyzed and lyophilized to powder. As a result, the fraction S with a molecular weight of 300,000 or more
-A, a fraction SB with a molecular weight of 300,000 to 150,000, and a fraction S-C with a molecular weight of 150,000 or less were obtained.
(Separation of each fraction was separately performed using this gel for gel filtration, based on a calibration curve determined from the elution position of the standard substance for molecular weight measurement.) The obtained sample S-A,
The antitumor activity of S-B and S-C was investigated against Sarcoma 180 nodular antitumor, and the results were as shown in the following table.
【表】
本実験結果は分子量300000以上の画分は抗腫瘍
活性が低いこと及び300000〜150000の画分と
150000以下の画分との間には有意差は認められな
いことを示している。
本発明の水溶性含糖蛋白質からなる抗腫瘍性物
質細胞毒性もなく広い抗腫瘍スペクトルを示し、
腫瘍に対し異なつた部位からの投与（静注，腹腔
投与、皮下投与、腫瘍内投与）によつても著しい
腫瘍縮退効果を示すきわめてすぐれた抗腫瘍剤と
なり得るものである。
さらに本発明に従う抗腫瘍性物質は抗腫瘍剤と
して他の抗腫瘍剤と併用することもできる。免疫
学的効果の増強をもたらすような併用は特に効果
的である。
また本発明に従う抗腫瘍性物質の製造方法によ
ると、従来は廃棄されていた貝類煮汁から抗腫瘍
剤として有効な成分が効率よく分離される。本発
明による貝類煮汁の取得方法及び煮汁からの有効
成分の採取方法は非常に簡単であり、特に工業的
生産に適する方法である。本発明方法と特公昭57
−8088号、特開昭54−41314号及び同54−41315号
公報に記載の製造方法との相違点について述べる
と、これら従来方法では貝の肉部分を原料とし、
このものから肝臓を除去しなければならないし、
低温条件下に保って貝肉を粉砕し、超音波処理等
で均質化して冷水で抽出し、大量の固形物（粉砕
肉）を遠心分離して除去するなど煩雑な多くの操
作を必要とするし、水抽出物が目的物以外に多く
の他の物質を含有するためその分離に手間がかか
るという難点がある。これに対し、本発明方法で
は貝の肉部分は粉砕しないので煮汁との分離が極
めて簡単であるし、蒸煮加熱処理後の肉部分は食
用資源として本来の目的に使用でき、従来方法で
は不要物として廃棄されていた廃物の煮汁を利用
したもので、原料面からも大巾のコスト低下がで
きるうえに、この煮汁は不純物、特に分子量の高
い不純物の含有量が少なく、その分離精製工程も
簡単化できる効果がある。従来、抗腫瘍性物質を
海洋生物から抽出、分離する際にはできる限り低
温で処理するのが普通であり、加熱などの方法は
有効成分を変質させる可能性があるので高温条件
下での処理は通常行なわれていなかつた。本発明
方法で使用する貝類煮汁は高温条件下で処理され
たものであり、この高温処理によつても薬効が損
なわれずかつ煮汁中に有効成分が高収率で得られ
たことは驚くべきことであつた。かくして本発明
によれば、食用として価値のある貝肉部を損傷す
ることなくまた食用としての味覚、外観等の商品
価値を損なうことなく、簡単な処理操作によつて
貴重な抗腫瘍性成分を同時に得ることができる。
さらに従来食用を目的として肉部を加工していた
際産業用排水としては好ましくない河川を汚染す
る有機成分を含有したまま廃棄されていた排水中
の有機成分を減少させることができるので、環境
衛生の面でも利点がある。すなわち本発明方法は
経済的に有利であるばかりでなく、環境保全の見
地からも従来方法より優れた方法である。
以下実施例により本発明をさらに説明する。
実施例 １
生ホタテ貝に対し0.10重量倍の量の105〜110℃
の熱スチームを生ホタテ貝に直接吹き付け、10分
間90〜100℃に蒸煮することにより有効成分が凝
縮水に溶けた一番煮汁を得た。原料としたホタテ
貝にはその重量の約0.1倍量のフジ貝が付着して
いるものであつた。一番煮汁は７時間で90℃から
50℃に徐冷した後、熱風入口温度280℃、出口温
度125℃の噴霧乾燥器により滞留時間45秒で瞬間
的に乾燥して有効成分を粉末標品（1a）として
得た。この粉末の収率は貝に対して0.27重量％で
あつた。
一番煮汁を取得した後の加熱ホタテ貝から貝柱
部分のみを取り出しこれを原料とした。沸騰して
いる食塩水（塩濃度10重量％）450Kgの中へ貝柱
50Kgを浸漬した。浸漬により液温は70℃になるが
20分間加熱して再度沸騰させ、この時点で貝柱を
食塩水から引き揚げた。得られた煮汁中で上記と
同様にして貝柱50Kgずつをさらに３回処理した。
こうして得られた二番煮汁を15時間で90℃から40
℃に徐冷した後、一番煮汁の乾燥と同じ条件で噴
霧乾燥器により有効成分を粉末として得た。この
粉末標品（1b）の収率は貝に対して0.20重量％で
あつた。
一番煮汁から得た粉末２重量部と二番煮汁から
得た粉末１重量部とを混合して混合粉末の標品
（1e）を調合した。
実施例 ２
実施例１と同様の方法で得た混合粉末（1c）
600gにその５重量倍の蒸留水を加えた懸濁液と
し、この懸濁液を10000G，５℃で20分間遠心分
離して沈澱物を除き、上清液３を得た。この上
清液をゲル過用ゲルセフアデツクスＧ−25を膨
潤させて充填したのちPH7.5の0.01M／のリン
酸緩衝液に0.07M／の塩化ナトリウムを加えた
溶出剤で平衡化したカラムヘチヤージして同じ組
成の溶出剤で溶出させ、溶出液の電気伝導度が塩
などの溶出によつて上昇しはじめる前の画分を採
り、これを更に約１容の先に示したと同様の溶
出剤で平衡化したDEAE−セフアローズCL−6B
カラム中を通し、素通りする画分を捨て、充分に
素通り画分がなくなつたことを紫外線モニターで
確認したのち、溶出剤を0.01M／のPH7.5のリ
ン酸緩衝液に0.25M／の食塩を加えた溶出剤に
替えて同じカラムへ導入し、これによつて溶出す
る画分を取得した。この画分を５℃の条件で２昼
夜透析して脱塩し、凍結乾燥して7.2gの粉末標品
（2d）を得た。
本標品の抗腫瘍性をサルコーマ180肉腫結節型
に対して調べたところ、400mg／Kg×３回（腫瘍
移植後５，７，９日目に腫瘍内投与）の投与で腫
瘍阻止率68％であつた。
また粉末標品（2d）は繊維肉腫Meth／Ａフイ
ブロサルコーマを接種したBALB／ｃ系のマウ
スに対して400mg／Kg×６回（腫瘍移植後３，
５，７，９，11日目に腫瘍内投与）の投与量で腫
瘍阻止率100％、完全治癒６匹中６匹の成績を示
した。
実施例 ３
実施例２と同様の方法で得た粉末標品（2d）
30gを0.1MのPH7.5のリン酸緩衝液150mlに溶かし
て高さ90cm、直径14cmのカラムにセフアデツクス
G75を充填し0.1M／のPH7.5のリン酸緩衝液の
溶出剤で平衡化したカラムヘチヤージし、PH7.5
のリン酸緩衝液で溶出させた。この条件で溶出し
た液の内、排除限界分子量から約10000の分子量
までの溶出画分を得て、２昼夜の透析により脱塩
したのち粉末標品（3e）0.8gを得た。この標品の
抗腫瘍性をサルコーマ180肉腫結節型に対して調
べたところ、40mg／Kg×３回の投与で腫瘍阻止
率80.5％であつた。
実施例 ４
実施例３と同様にして得た粉末標品（3e）相当
品1.0gを10mlの0.01M／のPH7.5のリン酸緩衝液
に溶解し、内径2.5cm、高さ50cmのカラムへセフ
アクリルＳ−400を充填し、PH7.5の0.01M／の
リン酸緩衝液で充分に平衡化したカラムにチヤー
ジし、ゲル過クロマトグラフイーを行なつた。
そして検量線から溶出画分を取る位置を逆算し、
溶出する溶出液を分子量が300000以上の画分Ｓ−
Ａ，分子量が300000〜150000の画分Ｓ−Ｂ及び分
子量が150000以下の画分Ｓ−Ｃに分画した。本実
験で得られた各画分は２昼夜蒸留水で透析し、凍
結乾燥してＳ−Ａが0.18g、Ｓ−Ｂが0.35g、Ｓ−
Ｃが0.30g各々得られた。この標品Ｓ−Ａ、Ｓ−
Ｂ、Ｓ−Ｃの抗腫瘍性をサルコーマ180肉腫結節
型に対して調べたところ、本文中にも示したよう
に、Ｓ−ＢとＳ−Ｃの画分が強い抗腫瘍性を示し
たが、Ｓ−Ａの分子量300000以上の画分は強い抗
腫瘍性活性のものを含まないことが判つた。
実施例 ５
実施例４で強い抗腫瘍活性を示したＳ−ＢとＳ
−Ｃをこれらが得られた比率でＳ−Ｂ画分210mg
とＳ−Ｃ画分180mgを混合し、蒸留水に溶解し、
凍結乾燥した。かくして粉末標品（4f）を0.39g
得た。
本文中に示した本発明の水溶性含糖蛋白質から
なる抗腫瘍性物質の理化学的諸性質は本粉末標品
（4f）を使用して測定したものである。
実施例 ６
実施例５で得た粉末標品（4f）のサルコーマ
180肉腫結節型に対する抗腫瘍効果を次の表に示
す。[Table] The results of this experiment show that the fraction with a molecular weight of 300,000 or more has low antitumor activity, and that the fraction with a molecular weight of 300,000 to 150,000 has low antitumor activity.
This shows that no significant difference was observed between the fractions below 150,000. The antitumor substance consisting of the water-soluble glycoprotein of the present invention exhibits a wide antitumor spectrum without cytotoxicity,
It can be an extremely excellent antitumor agent that exhibits remarkable tumor regression effects even when administered to tumors from different sites (intravenous injection, intraperitoneal administration, subcutaneous administration, intratumoral administration). Furthermore, the antitumor substance according to the present invention can be used in combination with other antitumor agents as an antitumor agent. Combinations that result in enhanced immunological effects are particularly effective. Furthermore, according to the method for producing an antitumor substance according to the present invention, components effective as an antitumor agent can be efficiently separated from shellfish broth, which has conventionally been discarded. The method of obtaining shellfish broth and the method of collecting active ingredients from the broth according to the present invention is very simple and is particularly suitable for industrial production. The method of the present invention and the Special Publication Act of 1977
-8088, JP-A No. 54-41314, and JP-A No. 54-41315, these conventional methods use shellfish meat as the raw material,
I have to remove the liver from this thing,
Many complicated operations are required, such as keeping the shellfish meat under low-temperature conditions, pulverizing it, homogenizing it with ultrasonication, extracting it with cold water, and removing a large amount of solid matter (pulverized meat) by centrifugation. However, since the aqueous extract contains many other substances in addition to the target substance, it is difficult to separate it. In contrast, in the method of the present invention, the meat part of the shellfish is not crushed, so it is extremely easy to separate it from the broth, and the meat part after steaming and heat treatment can be used for its original purpose as an edible resource. This method uses boiled liquid from waste materials that had been discarded as raw materials, and in addition to significantly reducing costs from the raw material standpoint, this broth contains fewer impurities, especially impurities with high molecular weight, and the separation and purification process is simple. It has the effect of making it more effective. Conventionally, when extracting and separating antitumor substances from marine organisms, it has been common practice to process them at the lowest possible temperature, and since methods such as heating may alter the active ingredients, processing under high temperature conditions is not recommended. was not normally practiced. The shellfish broth used in the method of the present invention was processed under high temperature conditions, and it is surprising that the medicinal efficacy was not impaired even by this high temperature treatment and that the active ingredients were obtained in high yield in the broth. It was hot. Thus, according to the present invention, valuable anti-tumor components can be extracted through simple processing operations without damaging the edible shellfish meat, and without impairing the commercial value of the edible taste, appearance, etc. can be obtained at the same time.
Furthermore, it is possible to reduce the organic components in wastewater, which was previously disposed of when meat was processed for edible purposes and still contained organic components that contaminate rivers, which is undesirable as industrial wastewater. There are also advantages in terms of That is, the method of the present invention is not only economically advantageous, but also superior to conventional methods from the standpoint of environmental conservation. The present invention will be further explained below with reference to Examples. Example 1 105-110℃ in an amount 0.10 times the weight of raw scallops
Hot steam was directly sprayed onto the raw scallops and the raw scallops were steamed at 90-100°C for 10 minutes to obtain a broth in which the active ingredients were dissolved in the condensed water. The scallop used as a raw material had Fuji shellfish attached to it in an amount approximately 0.1 times its weight. Ichiban soup is boiled from 90℃ in 7 hours.
After slowly cooling to 50°C, it was instantaneously dried in a spray dryer with a hot air inlet temperature of 280°C and an outlet temperature of 125°C for a residence time of 45 seconds to obtain the active ingredient as a powder specimen (1a). The yield of this powder was 0.27% by weight based on the shellfish. Only the scallop part was taken out from the heated scallop after obtaining the first broth, and this was used as the raw material. Scallops into 450 kg of boiling salt water (salt concentration 10% by weight)
50Kg was immersed. The temperature of the liquid becomes 70℃ due to immersion.
Heat again for 20 minutes to boil, at which point the scallops were removed from the brine. In the resulting broth, 50 kg of scallops were further treated three times in the same manner as above.
The second broth obtained in this way was heated to 40℃ from 90℃ in 15 hours.
After slowly cooling to ℃, the active ingredient was obtained as a powder using a spray dryer under the same conditions as for drying the first broth. The yield of this powder specimen (1b) was 0.20% by weight based on the shellfish. A mixed powder specimen (1e) was prepared by mixing 2 parts by weight of the powder obtained from the first broth and 1 part by weight of the powder obtained from the second broth. Example 2 Mixed powder (1c) obtained in the same manner as Example 1
A suspension was prepared by adding 5 times the weight of distilled water to 600 g, and this suspension was centrifuged at 10,000 G and 5° C. for 20 minutes to remove the precipitate to obtain supernatant liquid 3. This supernatant was swollen and filled with GelSephadex G-25 for gel filtration, and then equilibrated with an eluent containing 0.07M sodium chloride added to 0.01M phosphate buffer at pH 7.5. Charge the column and elute with an eluent of the same composition, collect the fraction before the electrical conductivity of the eluate begins to rise due to the elution of salts, and add about 1 volume of this to the elution similar to that shown above. DEAE-Sepharose CL-6B equilibrated with agent
After passing through the column and discarding the fraction that passes through the column, and confirming with an ultraviolet monitor that there is no sufficient fraction that passes through the column, add the eluent to 0.25M/0.01M/pH 7.5 phosphate buffer. The eluent was replaced with an eluent containing sodium chloride and introduced into the same column, thereby obtaining a fraction to be eluted. This fraction was desalted by dialysis for 2 days and nights at 5°C, and lyophilized to obtain 7.2 g of powder sample (2d). When the antitumor properties of this preparation were investigated against Sarcoma 180 sarcoma nodular type, tumor inhibition rate was 68% with administration of 400 mg/Kg x 3 times (intratumoral administration on days 5, 7, and 9 after tumor implantation). It was hot. In addition, the powder preparation (2d) was administered at 400 mg/Kg x 6 times (3 times after tumor implantation) to BALB/c mice inoculated with fibrosarcoma Meth/A fibrosarcoma.
Intratumoral administration on days 5, 7, 9, and 11) showed a tumor inhibition rate of 100%, with 6 out of 6 animals completely cured. Example 3 Powder specimen (2d) obtained in the same manner as Example 2
Dissolve 30g in 150ml of 0.1M PH7.5 phosphate buffer and transfer to a column with a height of 90cm and a diameter of 14cm.
Charge the column packed with G75 and equilibrated with eluent of 0.1M phosphate buffer at PH7.5.
It was eluted with phosphate buffer. Of the liquid eluted under these conditions, an eluted fraction with a molecular weight from the exclusion limit molecular weight to approximately 10,000 was obtained, and after desalting by dialysis for two days and nights, 0.8 g of powder sample (3e) was obtained. When the antitumor properties of this preparation were investigated against Sarcoma 180 nodular type, the tumor inhibition rate was 80.5% after administration of 40 mg/Kg x 3 times. Example 4 1.0 g of the powder sample (3e) obtained in the same manner as in Example 3 was dissolved in 10 ml of 0.01M PH7.5 phosphate buffer, and a column with an inner diameter of 2.5 cm and a height of 50 cm was prepared. The column was charged with Hecephacryl S-400 and sufficiently equilibrated with 0.01M phosphate buffer at pH 7.5, and gel permeation chromatography was performed.
Then, calculate back the position from which to take the elution fraction from the calibration curve.
The eluate to be eluted is divided into a fraction S- with a molecular weight of 300,000 or more.
A, the fraction was fractionated into a fraction S-B with a molecular weight of 300,000 to 150,000 and a fraction S-C with a molecular weight of 150,000 or less. Each fraction obtained in this experiment was dialyzed against distilled water for two days and nights, and lyophilized to give 0.18 g of S-A, 0.35 g of S-B, and 0.35 g of S-B.
0.30g of C was obtained in each case. This specimen S-A, S-
When the antitumor properties of B and S-C were investigated against Sarcoma 180 sarcoma nodular type, as shown in the text, the S-B and S-C fractions showed strong antitumor properties. It was found that the fraction of S-A with a molecular weight of 300,000 or more did not contain any strong antitumor activity. Example 5 SB and S that showed strong antitumor activity in Example 4
210 mg of S-B fraction at the ratio in which these were obtained.
and 180 mg of S-C fraction were mixed and dissolved in distilled water.
Lyophilized. Thus, 0.39g of powder standard (4f)
Obtained. The various physicochemical properties of the antitumor substance comprising the water-soluble glycoprotein of the present invention shown in the text were measured using this powder sample (4f). Example 6 Sarcoma of the powder specimen (4f) obtained in Example 5
The antitumor effect against 180 sarcoma nodular type is shown in the following table.
【表】
本実験で粉末標品（4f）は腫瘍内投与によつて
腫瘍を著しく縮退させる効果があり、適当な投与
量で完治させることができる。
実施例 ７
実施例４で得られた粉末標品Ｓ−Ｂ，Ｓ−Ｃ及
び実施例５で得られた粉末標品（4f）の直接細胞
毒性を次の方法で調べたが毒性は認められない。
マウス白血病（L5178YLymphoma）細胞を組
織培養培地イーグルMEM（仔牛血清15％含有）
に５×105個／mlに懸濁させ、検定試料を添加し
て５％炭酸ガス含有空気流下、37℃に置く。48時
間後、細胞の生死を公知の方法に従いトリバンブ
ルーによる染色によつて判定する。対照区として
生理食塩水加区及び公知の抗腫瘍剤であるマイト
マイシンＣ添加区を設けた。次表に抗腫瘍性物質
の直接細胞毒性を示す。[Table] In this experiment, the powder preparation (4f) had the effect of significantly reducing the tumor when administered intratumorally, and complete cure could be achieved with an appropriate dose. Example 7 The direct cytotoxicity of the powder samples S-B and S-C obtained in Example 4 and the powder sample (4f) obtained in Example 5 was investigated using the following method, but no toxicity was observed. do not have. Mouse leukemia (L5178YLymphoma) cells were grown in tissue culture medium Eagle MEM (containing 15% calf serum).
The suspension was suspended at 5 x 10 5 cells/ml, added with a test sample, and placed at 37°C under a flow of air containing 5% carbon dioxide. After 48 hours, the viability of the cells is determined by staining with trivan blue according to a known method. As control groups, a group added with physiological saline and a group added with mitomycin C, a known antitumor agent, were provided. The following table shows the direct cytotoxicity of antitumor substances.
【表】
実施例 ８
本実施例では、これまでと同様な方法で同様の
腫瘍細胞を使つて腫瘍細胞を移植し、固形腫瘍が
形成されていることを確認後、実施例５で得た粉
末標品4fを所定の濃度となるように生理食塩水に
溶解しマウスの尾の静脈に１〜３回注射（iv投
与）した。そして腫瘍移植５週間後の固形腫瘍を
摘出し、前述の抗腫瘍活性の検定方法と同様にし
て検定した。その結果は下表の通りで、本物質は
静脈内注射による投与でも効果がある。[Table] Example 8 In this example, tumor cells were transplanted using the same method as before, and after confirming that a solid tumor had been formed, the powder obtained in Example 5 was transplanted. Specimen 4f was dissolved in physiological saline to a predetermined concentration and injected (iv) into the tail vein of mice 1 to 3 times. Then, 5 weeks after tumor implantation, solid tumors were excised and assayed in the same manner as the above-mentioned method for assaying antitumor activity. The results are shown in the table below, and this substance is effective even when administered by intravenous injection.
【表】
実施例 ９
抗腫瘍活性の検定方法の中、検定試料の投与方
法をマウスの腹腔中へ投与する（ip投与）方法に
変更する他は前述の検定方法と全く同様にして実
施例５で得た粉末標品4fをマウス１匹当り２mgを
４回投与した。その結果は下表のとおりである。[Table] Example 9 In the method for testing antitumor activity, Example 5 was carried out in exactly the same manner as the above testing method, except that the method of administering the test sample was changed to intraperitoneal administration (IP administration) of mice. 2 mg of the powder preparation 4f obtained in step 4 was administered to each mouse four times. The results are shown in the table below.
【表】
この実験で標品4fは腹腔内投与でも抗腫瘍効果
があり、特に５匹中２匹が完治することは注目に
値する。
実施例 10
５週令のICR系雌のマウスの腹腔内にサルコー
マ180腫瘍細胞を接種し、１週間後に増殖した腫
瘍細胞を腹水と共に抜き取り、この細胞４×106
個を他の５週令のICR系雌マウスの腋下皮下に移
植する。腫瘍移植後１，３，５日目に実施例５で
得た粉末標品4fの検定試料を、腫瘍細胞を移植し
た反対側の腋下皮下（sc投与）に投与した。腫瘍
移植３週間後の固形腫瘍を摘出し、その重量を試
料の代りに生理食塩水を投与した対照群の場合と
比較して、抗腫瘍活性の検定方法と同様に腫瘍阻
止率（％）を求めた。[Table] In this experiment, it is noteworthy that standard 4f had an antitumor effect even when administered intraperitoneally, and that 2 out of 5 animals were completely cured. Example 10 Sarcoma 180 tumor cells were intraperitoneally inoculated into a 5-week-old ICR female mouse, and after 1 week, the proliferated tumor cells were extracted together with ascites fluid, and 4 x 10 6 of these cells were collected.
The mouse is subcutaneously transplanted into the axilla of another 5-week-old ICR female mouse. On days 1, 3, and 5 after tumor transplantation, a test sample of the powder preparation 4f obtained in Example 5 was administered subcutaneously (sc administration) under the armpit on the opposite side to which the tumor cells were transplanted. Solid tumors were excised 3 weeks after tumor implantation, and their weight was compared with that of a control group in which physiological saline was administered instead of the sample, and the tumor inhibition rate (%) was determined in the same way as the antitumor activity assay method. I asked for it.
【表】
この結果、標品4fは皮下投与によつても抗腫瘍
効果があり、８匹中１匹は完治することは注目に
値する。
実施例 11
実施例５の粉末標品4fについて抗腫瘍活性をサ
ルコーマ180固形腫瘍以外の各種固形腫瘍に対す
る抗腫瘍効果を検討した。すなわち、エールリツ
チ癌、白血性腹水腫瘍（SN−36）、NTF細網細
胞肉腫（NTF reticulum cell Sarcoma）、線維
肉腫（Fibrosarcoma）をそれぞれ106個を他の６
週令のマウスをそけい部皮下に移植し、１週間後
の固形腫瘍に対して実施例５の標品4fを所定の濃
度となるように生理食塩水に溶解し4fとして５
mg／マウス×３回、隔日に投与し、腫瘍移植５週
間後の固形腫瘍を摘出し、その重量を試料の代り
に生理食塩水を投与した対照群と比較した。[Table] As a result, it is noteworthy that the standard 4f has an antitumor effect even when administered subcutaneously, and one out of eight animals was completely cured. Example 11 The antitumor activity of the powder preparation 4f of Example 5 against various solid tumors other than Sarcoma 180 solid tumor was examined. That is, 10 each of Ehrlitsu's carcinoma, leukemic ascites tumor (SN-36), NTF reticulum cell sarcoma, and fibrosarcoma, and 6 of the other 6.
A week-old mouse was implanted subcutaneously in the groin area, and one week later, the preparation 4f of Example 5 was dissolved in physiological saline to a predetermined concentration and applied to the solid tumor as 4f.
mg/mouse x 3 times every other day, solid tumors were excised 5 weeks after tumor implantation, and their weights were compared with a control group in which physiological saline was administered instead of the sample.
【表】
実施例 12
サザエを使い、実施例１〜５と同様にして白色
粉末を得た。本標品（12x）の抗腫瘍活性は400
mg／Kg×３回投与で腫瘍阻止率65％であつた。
実施例 13
アワビを使い、実施例１〜５と同様にして白色
粉末を得た。本標品（13y）の抗腫瘍活性は、
400mg／Kg×３回投与で腫瘍阻止率73％であつた。
実施例 14
ハマグリを使い、実施例１〜５と同様にして白
色粉末を得た。本標品（14z）の抗腫瘍活性は400
mg／Kg×３回投与で腫瘍阻止率67％であつた。
実施例 15
ホタテ貝煮汁の乾燥粉末として実施例１で得た
粉末1cの代りに1aを使用した以外は実施例２〜
５と同様にして分離精製し、実施例５で得られた
抗腫瘍性物質とほとんど同じ理化学的性状および
抗腫瘍活性を有する抗腫瘍性物質を得た。
実施例 16
ホタテ貝煮汁の乾燥粉末として実施例１で得た
粉末1cの代りに1bを使用した以外は実施例２〜
５と同様にして分離精製し、実施例５で得られた
抗腫瘍性物質とほとんど同じ理化学的性状および
抗腫瘍活性を有する抗腫瘍性物質を得た。[Table] Example 12 A white powder was obtained in the same manner as in Examples 1 to 5 using turban shell. The antitumor activity of this preparation (12x) is 400
The tumor inhibition rate was 65% after administration of mg/Kg x 3 times. Example 13 A white powder was obtained in the same manner as in Examples 1 to 5 using abalone. The antitumor activity of this preparation (13y) is
The tumor inhibition rate was 73% after administration of 400 mg/Kg x 3 times. Example 14 A white powder was obtained in the same manner as in Examples 1 to 5 using clams. The antitumor activity of this preparation (14z) is 400
The tumor inhibition rate was 67% after administration of mg/Kg x 3 times. Example 15 Example 2~ except that 1a was used instead of powder 1c obtained in Example 1 as the dry powder of scallop broth.
The antitumor substance was separated and purified in the same manner as in Example 5 to obtain an antitumor substance having almost the same physicochemical properties and antitumor activity as the antitumor substance obtained in Example 5. Example 16 Example 2~ except that 1b was used instead of the powder 1c obtained in Example 1 as the dry powder of scallop broth.
The antitumor substance was separated and purified in the same manner as in Example 5 to obtain an antitumor substance having almost the same physicochemical properties and antitumor activity as the antitumor substance obtained in Example 5.
第１図、第２図は本発明方法において、予備処
理したホタテ貝煮汁粉末の水溶液をイオン交換ク
ロマトグラフイーに付して塩化ナトリウム溶液で
溶出した場合の溶出パターンを示し、第３図は第
１図のＣ画分をゲル過クロマトグラフイーに付
した場合の溶出パターンを示す。第４図は本発明
の水溶性含糖蛋白質性抗腫瘍性物質の0.4mg／ml
水溶液の紫外線吸収スペクトル図を示し、第５図
は同物質の赤外吸収スペクトル図（KBr錠）を
示す。
Figures 1 and 2 show the elution pattern when an aqueous solution of pretreated scallop broth powder is subjected to ion exchange chromatography and eluted with a sodium chloride solution in the method of the present invention, and Figure 3 shows the elution pattern. The elution pattern when fraction C in Figure 1 was subjected to gel permeation chromatography is shown. Figure 4 shows 0.4 mg/ml of the water-soluble glycoprotein antitumor substance of the present invention.
FIG. 5 shows an ultraviolet absorption spectrum of an aqueous solution, and FIG. 5 shows an infrared absorption spectrum of the same substance (KBr tablet).
Claims (1)
イ科及びマルスダレガイ科に属する貝類の煮汁粉
末又は濃縮物から得た、次の特性： 帯微褐白色ないしは白色の固状； 水に易溶性でメタノール、エタノール、アセ
トンなどの有機溶媒に不溶； 両性電解質； KBrペレツトによる赤外線吸収スペクトル
で3500〜3300cm-1、1650cm-1、1540cm-1及び
1400cm-1附近に主ピークを示す； ビウレツト反応、キサントプロテイン反応、
フエノール試薬による反応が陽性； アンスロン硫酸反応、フエノール硫酸反応が
陽性でシステイン硫酸反応が偽陽性； 紫外線吸収スペクトルにおいてλH2O nax278nmに
吸収をもつ； 分子量範囲として10000〜300000のものを含
む； 明確な融点を示さず、235℃以上で分解しは
じめる； 加水分解物中のアミノ酸としてアスパラギン
酸、ハイドロプロリン、スレオニン、セリン、
グルタミン酸、プロリン、グリシン、アラニ
ン、システイン、バリン、メチオニン、イソロ
イシン、ロイシン、チロシン、フエニルアラニ
ン、リジン、アルギニン、ヒスチジン、ハイド
ロキシリジンが認められる； xi 加水分解物中の糖としてフルクトース、マン
ノース、フコース、イノシトール、ガラクトー
スが認められる； を有する水溶性含糖蛋白質よりなる抗腫瘍性物
質。 ２ イタヤガイ科、リユウテンサザエ科、ミミガ
イ科およびマルスダレガイ科に属する貝類の可食
部分を熱水性溶媒で蒸煮もしくは加熱加工する際
に得られる煮汁の乾燥粉末又は濃縮物を原料と
し、これを塩基性陰イオン交換体を使用するイオ
ン交換クロマトグラフイー及びゲル過ゲルを用
いるゲル過に付して、分子量10000〜300000の
範囲内の水溶性含糖蛋白質からなる抗腫瘍性物質
を分取することを特徴とする、次の特性： 帯微褐白色ないしは白色の固状； 水に易溶性でメタノール、エタノール、アセ
トンなどの有機溶媒に不溶； 両性電解質； KBrペレツトによる赤外線吸収スペクトル
で3500〜3300cm-1、1650cm-1、1540cm-1及び
1400cm-1附近に主ピークを示す； ビウレツト反応、キサントプロテイン反応、
フエノール試薬による反応が陽性； アンスロン硫酸反応、フエノール硫酸反応が
陽性でシステイン硫酸反応が偽陽性； 紫外線吸収スペクトルにおいてλH2O nax278nmに
吸収をもつ； 分子量範囲として10000〜300000のものを含
む； 明確な融点を示さず、235℃以上で分解しは
じめる； ｉ 加水分解物中のアミノ酸としてアスパラギ
ン酸、ハイドロプロリン、スレオニン、セリ
ン、グルタミン酸、プロリン、グリシン、アラ
ニン、システイン、バリン、メチオニン、イソ
ロイシン、ロイシン、チロシン、フエニルアラ
ニン、リジン、アルギニン、ヒスチジン、ハイ
ドロキシリジンが認められる； xi 加水分解物中の糖としてフルクトース、マン
ノース、フコース、イノシトール、ガラクトー
スが認められる； を有する水溶性含糖蛋白質よりなる抗腫瘍性物質
の製造法。 ３ イオン交換クロマトグラフイーにより処理し
た後にゲル過を行なう特許請求の範囲第２項記
載の製造法。 ４ ゲル過により処理した後にイオン交換クロ
マトグラフイーを行なう特許請求の範囲第２項記
載の製造法。[Scope of Claims] 1 Obtained from the boiled liquid powder or concentrate of shellfish belonging to the family Apocalypidae, the family Apocalypidae, the family Apothecidae, and the family Apocalypidae, with the following properties: Slightly brownish white or white solid; Easily soluble in water. Soluble and insoluble in organic solvents such as methanol, ethanol, and acetone; Ampholyte; Infrared absorption spectrum with KBr pellets shows 3500 to 3300 cm -1 , 1650 cm -1 , 1540 cm -1 and
Shows main peak around 1400 cm -1 ; Biuretz reaction, xanthoprotein reaction,
Reaction with phenol reagent is positive; Anthrone sulfuric acid reaction and phenol sulfuric acid reaction are positive, cysteine sulfuric acid reaction is false positive; Ultraviolet absorption spectrum has absorption at λ H2O nax 278 nm; Includes molecular weight range of 10,000 to 300,000; Clear It has no melting point and begins to decompose above 235°C; amino acids in the hydrolyzate include aspartic acid, hydroproline, threonine, serine,
Glutamic acid, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, arginine, histidine, and hydroxylidine are recognized; xi Sugars in the hydrolyzate include fructose, mannose, fucose, An antitumor substance consisting of a water-soluble glycoprotein containing inositol and galactose. 2 The raw material is a dry powder or concentrate of the broth obtained when the edible parts of shellfish belonging to the family Asteraceae, the family Aperaceae, the family Asteridae, and the family Amaranthidae are steamed or heated in a hydrothermal solvent, and this is used as a base material. ion exchange chromatography using a neutral anion exchanger and gel filtration using a gel permeation gel to separate an antitumor substance consisting of a water-soluble glycoprotein with a molecular weight within the range of 10,000 to 300,000. It is characterized by the following properties: Slightly brownish white or white solid; Easily soluble in water and insoluble in organic solvents such as methanol, ethanol, and acetone; Ampholyte; 3500-3300 cm - in the infrared absorption spectrum of KBr pellets. 1 , 1650cm -1 , 1540cm -1 and
Shows main peak around 1400 cm -1 ; Biuretz reaction, xanthoprotein reaction,
Reaction with phenol reagent is positive; Anthrone sulfuric acid reaction and phenol sulfuric acid reaction are positive, cysteine sulfuric acid reaction is false positive; Ultraviolet absorption spectrum has absorption at λ H2O nax 278 nm; Includes molecular weight range of 10,000 to 300,000; Clear Does not show a melting point and begins to decompose above 235°C; i Amino acids in the hydrolyzate include aspartic acid, hydroproline, threonine, serine, glutamic acid, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, and tyrosine. , phenylalanine, lysine, arginine, histidine, and hydroxylidine are observed; xi Fructose, mannose, fucose, inositol, and galactose are observed as sugars in the hydrolyzate; Method of manufacturing a substance. 3. The production method according to claim 2, wherein gel filtration is performed after treatment by ion exchange chromatography. 4. The production method according to claim 2, wherein ion exchange chromatography is performed after gel filtration treatment.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP57137208A JPS5927829A (en) | 1982-08-09 | 1982-08-09 | Antitumor agent and preparation thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP57137208A JPS5927829A (en) | 1982-08-09 | 1982-08-09 | Antitumor agent and preparation thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5927829A JPS5927829A (en) | 1984-02-14 |
| JPH0433764B2 true JPH0433764B2 (en) | 1992-06-04 |
Family
ID=15193311
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP57137208A Granted JPS5927829A (en) | 1982-08-09 | 1982-08-09 | Antitumor agent and preparation thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5927829A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101749548B1 (en) * | 2015-10-19 | 2017-06-21 | 대한민국 | Antimicrobial peptide derived from abalone lipopolysaccharide and beta-glucan binding protein, nucleic acid encoding the peptide and uses thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS52122612A (en) * | 1976-04-05 | 1977-10-15 | Microbial Chem Res Found | Anti-tumor substance and its preparation |
| JPS5441315A (en) * | 1977-09-02 | 1979-04-02 | Microbial Chem Res Found | Anti-tumor substance and its preparation |
| JPS57130918A (en) * | 1981-02-09 | 1982-08-13 | Cosmo Co Ltd | Preparation of antitumor agent |
-
1982
- 1982-08-09 JP JP57137208A patent/JPS5927829A/en active Granted
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS52122612A (en) * | 1976-04-05 | 1977-10-15 | Microbial Chem Res Found | Anti-tumor substance and its preparation |
| JPS5441315A (en) * | 1977-09-02 | 1979-04-02 | Microbial Chem Res Found | Anti-tumor substance and its preparation |
| JPS57130918A (en) * | 1981-02-09 | 1982-08-13 | Cosmo Co Ltd | Preparation of antitumor agent |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5927829A (en) | 1984-02-14 |
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