JPH0571231B2 - - Google Patents
Info
- Publication number
- JPH0571231B2 JPH0571231B2 JP864186A JP418686A JPH0571231B2 JP H0571231 B2 JPH0571231 B2 JP H0571231B2 JP 864186 A JP864186 A JP 864186A JP 418686 A JP418686 A JP 418686A JP H0571231 B2 JPH0571231 B2 JP H0571231B2
- Authority
- JP
- Japan
- Prior art keywords
- spf10
- reaction
- antitumor component
- culture
- antitumor
- 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
Links
- 230000000259 anti-tumor effect Effects 0.000 claims description 49
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 31
- 238000006243 chemical reaction Methods 0.000 claims description 29
- 238000000034 method Methods 0.000 claims description 22
- 239000000126 substance Substances 0.000 claims description 21
- 241000194017 Streptococcus Species 0.000 claims description 16
- MYKOKMFESWKQRX-UHFFFAOYSA-N 10h-anthracen-9-one;sulfuric acid Chemical compound OS(O)(=O)=O.C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 MYKOKMFESWKQRX-UHFFFAOYSA-N 0.000 claims description 14
- 210000004748 cultured cell Anatomy 0.000 claims description 12
- 238000000862 absorption spectrum Methods 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 8
- 230000012010 growth Effects 0.000 claims description 8
- 229930182555 Penicillin Natural products 0.000 claims description 7
- 229940049954 penicillin Drugs 0.000 claims description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 4
- 238000010521 absorption reaction Methods 0.000 claims description 4
- 230000002378 acidificating effect Effects 0.000 claims description 4
- 230000002401 inhibitory effect Effects 0.000 claims description 4
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 4
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- 238000000921 elemental analysis Methods 0.000 claims description 3
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 2
- 235000018417 cysteine Nutrition 0.000 claims description 2
- 238000000354 decomposition reaction Methods 0.000 claims description 2
- 230000007935 neutral effect Effects 0.000 claims description 2
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 claims description 2
- 238000005199 ultracentrifugation Methods 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 description 34
- 239000000243 solution Substances 0.000 description 25
- 239000002609 medium Substances 0.000 description 22
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 21
- 239000000706 filtrate Substances 0.000 description 15
- 241000193996 Streptococcus pyogenes Species 0.000 description 13
- 229940056360 penicillin g Drugs 0.000 description 12
- 238000010828 elution Methods 0.000 description 11
- 230000000694 effects Effects 0.000 description 10
- 239000008363 phosphate buffer Substances 0.000 description 10
- 239000011780 sodium chloride Substances 0.000 description 10
- 206010028980 Neoplasm Diseases 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 7
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
- 239000012980 RPMI-1640 medium Substances 0.000 description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 5
- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 description 5
- 229940041514 candida albicans extract Drugs 0.000 description 5
- 230000002949 hemolytic effect Effects 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000012138 yeast extract Substances 0.000 description 5
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 4
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 239000004480 active ingredient Substances 0.000 description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 4
- 235000011130 ammonium sulphate Nutrition 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 229930027917 kanamycin Natural products 0.000 description 4
- 229960000318 kanamycin Drugs 0.000 description 4
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 4
- 229930182823 kanamycin A Natural products 0.000 description 4
- 235000013372 meat Nutrition 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000000108 ultra-filtration Methods 0.000 description 4
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 229920005654 Sephadex Polymers 0.000 description 3
- 239000012507 Sephadex™ Substances 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 235000019341 magnesium sulphate Nutrition 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 208000006268 Sarcoma 180 Diseases 0.000 description 2
- 241000194022 Streptococcus sp. Species 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 2
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 230000009036 growth inhibition Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 229940111688 monobasic potassium phosphate Drugs 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 230000004660 morphological change Effects 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- HOKIDJSKDBPKTQ-GLXFQSAKSA-N Cephalosporin C Natural products S1CC(COC(=O)C)=C(C(O)=O)N2C(=O)[C@@H](NC(=O)CCC[C@@H](N)C(O)=O)[C@@H]12 HOKIDJSKDBPKTQ-GLXFQSAKSA-N 0.000 description 1
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 239000007621 bhi medium Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- HOKIDJSKDBPKTQ-GLXFQSAKSA-M cephalosporin C(1-) Chemical compound S1CC(COC(=O)C)=C(C([O-])=O)N2C(=O)[C@@H](NC(=O)CCC[C@@H]([NH3+])C([O-])=O)[C@@H]12 HOKIDJSKDBPKTQ-GLXFQSAKSA-M 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 238000003134 dye exclusion method Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000005918 in vitro anti-tumor Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 210000003200 peritoneal cavity Anatomy 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Description
本発明は、新規な抗腫瘍性成分SPF10−11及び
その製法に関するものである。
従来、溶血性連鎖状球菌(以下溶連菌という)
の生菌体を弱毒化して製剤化したものは、すでに
制癌剤として使用されている。
また、溶連菌の菌体を破砕後水または塩類溶液
で有効成分を抽出し、有機溶媒を加えて、抗腫瘍
性成分を沈澱として、回収する方法(特公昭38−
1647)、溶連菌を溶菌酵素リゾチーム、セルラー
ゼまたは蛋白質分解酵素により、溶菌し、活性画
分を水溶性区分として分画する方法(英国特許
1163865号)、溶連菌の菌体を破砕後水不溶性物質
を採取し、核酸分解酵素および蛋白分解酵素で処
理する方法(特開昭55−7014)などが知られてい
る。
このように、ストレプトコツカス属細菌そのも
のもしくはその菌体成分に抗腫瘍性があることは
広く知られているのであるが、従来知られたもの
は、菌体もしくは水溶性もしくは水不溶性高分子
細胞構成物質であるに過ぎなかつた。菌体もしく
は菌体内から有効成分を単離しようとすれば、菌
体を溶菌したり、機械的に破砕したりして全体を
分画しなければならなかつた。
このような処理では、精製は複雑となり、有効
成分の単離はきわめて困難であつた。実際に分離
し、有効成分として測定された例では分子量
200000の蛋白質(特公昭48−43841、特開昭51−
44617)及び分子量150000の糖蛋白質(特開昭58
−22026)が知られている程度である。
本発明者らは、先に溶連菌の培養液中に抗腫瘍
性成分を溶出させる方法を鋭意研究したところ、
培養中にペニシリン又はその関連物質を添加する
ことによつて抗腫瘍性成分が培養液中に溶出する
ことを見出し(特開昭60−30677号)、培養液中か
ら生理活性物質SPF−1を分離するに至つたので
ある。(特開昭60−30689号)
本発明者らは、更に、溶連菌の培養濾液中から
より有効な成分を分離する目的で研究したとこ
ろ、本発明において、癌化白血球培養細胞L1210
(以下培養細胞L1210という)の生育を阻害し、
かつ、アンスロン硫酸法による呈色反応陽性の画
分であることにより特徴づけられる抗腫瘍性成分
SPF10−11を分離するに至つた。
本発明の抗腫瘍性成分SPF10−11は元素分析、
呈色反応等から糖を主成分とし、一部ペプチドを
含む物質と考えられるが、紫外線吸収スペクトル
および赤外線吸収スペクトルから、既知の抗腫瘍
性物質とは、相違する新規な物質と認められるも
のである。
本発明は、ストレプトコツカス属に属する抗腫
瘍性成分SPF10−11生産菌を培養し、培養物から
抗腫瘍性成分SPF10−11を採取することを特徴と
する抗腫瘍性成分SPF10−11の製造法を包含する
ものである。
本発明においては、ストレプトコツカス属に属
する抗腫瘍性成分SPF10−11生産菌が広く使用で
きる。次に抗腫瘍性成分SPF10−11生産菌を記載
する。
ストレプトコツカス・ピオゲネス
(Streptococcus pyogenes) ATCC21060
ストレプトコツカス・エスピー
(Streptococcus sp.) ATCC21597
ストレプトコツカス・ピオゲネス
(Streptococcus pyogens) ATCC21546
ストレプトコツカス・ピオゲネス
(Streptococcus pyogenes) ATCC21547
ストレプトコツカス・ピオゲネス
(Streptococcus pyogenes) ATCC21548
培養液は、肉エキス培地、酵母エキス培地、ブ
レイン・ハート・インフユージヨン培地(BHI
培地)等の天然培地がよく用いられるが、ストレ
プトコツカス属細菌の生育に適した培地であれば
任意の培地を使用できる。
培養は、PH5.0〜8.0、好ましくは、6.1〜7.2で
あり、培養温度は、30〜40℃、好ましくは、35〜
37℃であり、嫌気的に静置培養または、撹拌培養
を行なうことができる。
本発明においては、培養中に適当な時期に、ペ
ニシリンまたは、その関連物質を添加すること
が、抗腫瘍性成分SPF10−11の採取に重要な役割
をはたすことになる。
ペニシリンまたは、その関連物質の添加時期
は、35〜37℃の培養で、対数増殖期にかかつて
後、3〜20時間の間、特に5〜10時間が好まし
い。その後1〜20時間、好ましくは3〜15時間培
養を継続することにより、培養中に抗腫瘍性成分
SPF10−11を多量蓄積させることができる。
ペニシリンまたはその関連物質としては、すで
に知られたペニシリンと類似の作用をもつ関連物
質であればいかなるものでもよいが、ペニシリン
Gが普通用いられる。添加量は、ペニシリンGで
100〜7000単位/ml、好ましくは、300〜5000単
位/ml培養液程度で十分である。
ストレプトコツカス属細菌のペニシリンまた
は、その関連物質の添加培養によつて得られた培
養液は遠心分離によつて菌体を除去し、濾液を得
る。濾液は、硫安を添加し、50〜90%飽和度の画
分を分取するか、もしくは、濾液は、限外濾過膜
を用いて濃縮する。
得られた抗腫瘍性成分SPF10−11を含む硫安塩
析物は凍結状態で保存することもできる。
この硫安塩析物から抗腫瘍性成分SPF10−11を
抽出するには、塩析物を緩衝液に溶解して用い
る。又、限外濾過膜を用いて得た濃縮液を、その
まま用いることもできる。この水溶液をDEAEセ
ルロースカラムに吸着させ、燐酸緩衝液を用いて
段階的に溶出させ、培養細胞L1210の生育を阻害
する活性画分を分取し、この活性画分をDEAEセ
フアデツクスA−25カラムに吸着させ、燐酸緩衝
液中の塩化ナトリウム濃度を直線的に上昇させつ
つ溶出し、その非吸着部分及び塩化ナトリウム低
濃度部分を分取し、この分画部分を緩衝液に対し
て透析し、ゲル濾過材トヨパールHW−50Fカラ
ムに吸着させ、0.1M NaClを含む燐酸緩衝液で
溶出させ、アンスロン硫酸法による呈色反応及び
培養細胞L1210に対する活性を調べる。ここで培
養細胞L1210に対して強い活性を示す画分、すな
わちトヨパールHW50Fの排除限界(以下Voと略
す)付近のアンスロン硫酸法による呈色反応でピ
ークを形成する画分を得る。
この画分を濃縮し、トヨパールHW60Fカラム
に吸着させ、0.1M NaClを含む燐酸緩衝液で溶
出させる。
SPF10−11は、トヨパールHW60Fカラムクロ
マトグラフイーにおいてVo近くの280nmの吸収
がごく微弱でアンスロン硫酸法に強い呈色を示す
画分として得ることが出来る。
次に、実施例1で得られた抗腫瘍性成分SPF10
−11の凍結乾燥標品は、次の理化学的性質を示
す。
1 元素元析 C 37.2〜39.4%
H 5.6〜6.5%
N 0.1〜2.3%
O 56.9〜49.3%
Ash 0.2〜2.5%
2 分解点 本物質は165℃で褐変し240℃になる
と黒色となり分解する。
3 比旋光度〔α〕20 D=+95゜〜+130゜(C=1.00)
4 紫外線吸収スペクトル 本物質0.1%の水溶
液の紫外線吸収スペクトルは第1図に示され
る。
5 赤外線吸収スペクトル 第2図に示される。
840cm-1に吸収が認められ特徴的である。
6 塩基性、酸性、中性の区別 本物質0.1%の
水溶液のPHは6.2〜6.8である。
7 物質の色 白色(凍結乾燥品)
8 呈色反応
ローリー反応 +
ビユーレツト反応 +
ニンヒドリン反応 +
アンスロン硫酸反応 +
モーリツシユ反応 +
システイン硫酸反応 +
オルシン塩酸反応 −
9 分子量 Reyleigh干渉光学系を用いた超遠
心法により20×104以上と測定される。
10 溶剤に対する溶解性 水に可溶であるが、メ
タノール、エタノール、n−プロパノール、ア
セトン、エチルエーテル、n−ヘキサン、クロ
ロホルム、酢酸エチル等の溶剤には難溶又は不
溶である。
次にインビトロにおける培養細胞L1210に対す
る抗腫瘍性活性の測定方法及びアンスロン硫酸法
による呈色反応の方法を示す。
抗腫瘍活性の測定方法
抗腫瘍活性の測定は、培養細胞L1210の生育阻
止率(IR%)の測定により行つた。
L1210細胞を10%FBS添加RPMI1640培地(5
mg/カナマイシン含有)に懸濁した。この培養
液0.5mlをフアルコン2058チユーブに注加し、細
胞数が1×105cells/tubeになるようにした。次
いでこの培養液に所定量の標品(抗腫瘍性成分
SPF10−11)を目的濃度になるように培養液に溶
解した0.5mlを注加して、37℃で5%CO2存在下
に培養した。標品を添加して48時間後にトリパン
ブルーによる染色をおこない、次式によりIR
(%)を算出した。
IR(%)=(A)−各実験群の生細胞数/対照群の生細
胞数×100
ここで(A)とは対照群の生細胞数を示す。
対照は標品を含まない培養液0.5mlを用い同時
に行つた。
アンスロン硫酸法による呈色反応
脱イオン水に溶解し目的濃度にした標品1mlに
2mlのアンスロン試薬(0.20grのアンスロンを
100mlの濃硫酸に溶解したもの)を注加し、混合
30分後、標品1mlに代えて脱イオン水1mlに2ml
のアンスロン試薬を注加した液を対照として
620nmで吸収を測定する。定量値はグルコースを
用いて同様に測定して得た検量式より求める。
次に本発明の実施例を示す。
実施例 1
ストレプトコツカス・ピオゲネス
(Streptococcus pyogenes)ATCC21060を
BHI培地100mlに接種して37℃、8時間静置培養
をおこなつて得た種培養液を第1表に示す培地
A1lに接種し、種培養と同一条件で嫌気的に前培
養を行つた。
第1表 培地A
マルトース 0.25%
肉エキス 1.0%
ポリペプトン 1.0%
酵母エキス 0.25%
酸性第一燐酸カリウム 0.1%
硫酸マグネシウム 0.05%
PH=6.8
10ジヤーフアーメンターに培地A8を投入
して120℃、10分間加熱殺菌後、37℃まで冷却し
て、前培養液1を接種し、37℃、15.5時間、PH
6.8、300回転/分で撹拌しながら嫌気的に培養す
る。次いでペニシリンG1000単位/ml培養液にな
るように添加して、培養を更に5時間継続した。
得られた培養液を遠心分離にかけて菌体を除去し
た。
培養濾液は、分画分子量10000の限外濾過膜
(米国、ロミコン社)を用いて、1.5に縮濃し
た。この濃縮液を、DEAEセルロースカラム(5
×70cm)に吸着させた後、10mM、PHを7.0の燐
酸緩衝液を用いて、洗浄し、次いで0.3M塩化ナ
トリウムを含む上記燐酸緩衝液を用いて、段階的
に溶出させ、アンスロン硫酸法による呈色反応陽
性でかつ培養細胞L1210の生育を阻害する活性画
分を分取した。
この活性画分をDEAEセフアデツクスA−25カ
ラム(7.5×50cm)に吸着させ、次いで上記燐酸
緩衝液で洗浄後塩化ナトリウム濃度を直線的に上
昇させて溶出を行い、培養細胞L1210に対して活
性のある部分を分取する。この溶出曲線は第3図
に示される。第3図において点線部分は非吸着部
分で、実線部分は塩化ナトリウム添加部分であ
る。Aは培養細胞L1210生育阻害活性画分で、
MP−2(特開昭60−30689)生育阻害非活性画分
であり、Bは培養細胞L1210生育阻害非活性画分
で、MP−2生育阻害活性画分である。
Aの活性画分の溶出液を分画分子量10000の限
外濾過膜(ミリポア社)で30mlに濃縮する。この
濃縮液をトヨパールHW50Fカラム(5.0×100cm)
に吸着させ、次いで、上記燐酸緩衝液中に塩化ナ
トリウム0.1Mを含む溶液で溶出させ、アンスロ
ン硫酸法による呈色反応陽性の画分を分取した。
この溶出曲線は第4図に示される。第4図におい
て実線は全体の溶出曲線を示し、点線はアンスロ
ン硫酸法による呈色反応陽性部分の溶出曲線を示
している。ここで、Cはアンスロン硫酸法による
呈色反応陽性画分であるが、培養細胞L1210に対
する生育阻止活性を調べ活性の強いVo付近の、
アンスロン硫酸法でピークを形成するD画分を得
る。D画分は、凍結乾燥する。この凍結乾燥標品
を、燐酸緩衝液に溶解し、トヨパールHW60Fカ
ラム(2.6×100cm)に吸着させ次いでトヨパール
HW50Fの場合と同じ溶出液で溶出する。
この溶出曲線は第5図に示される。第5図にお
いて実線は全体の溶出曲線を示し、黒丸付の実線
はアンスロン硫酸法による呈色反応陽性部分の溶
出曲線を示している。ここでアンスロン硫酸法に
よる呈色反応で強い呈色を示すFの画分をSPF10
−11とし、Gの画分をSPF10−12とした。
Fの画分を凍結乾燥した。この凍結乾燥標品は
抗腫瘍性成分SPF10−11であり、0.32grを得た。
実施例 2
ストレプトコツカス・ピオゲネスATCC21060
を第1表に示した培地Aを用いて実施例1と同様
に培養した。この場合、ペニシリンGは300単
位/ml培養液となるように添加し、更に培養を10
時間継続した。この培養濾液に硫安を添加し50〜
90%飽和度の画分を分取する。透析により硫安を
除き実施例1と同様に精製して抗腫瘍性成分
SPF10−11 0.15grを得た。
実施例 3
ストレプトコツカス・ピオゲネスATCC21060
を第1表に示した培地Aを用いて実施例1と同様
に培養した。この場合、ペニシリンGは3000単
位/ml培養液となるように添加し、更に培養を3
時間継続した。この培養濾液を実施例1と同様に
精製して抗腫瘍性成分SPF10−11 0.37grを得た。
実施例 4
ストレプトコツカス・ピオゲネスATCC21060
を第2表に示した培地Bを用いて実施例1と同様
に培養した。この場合、ペニシリンGは1000単
位/ml培養液となるように添加し、更に培養を5
時間継続した。この培養濾液を実施例1と同様に
精製して抗腫瘍性成分SPF10−11 0.20grを得た。
第2表 培地B
マルトース 0.1%
肉エキス 0.5%
ポリペプトン 1.0%
酵母エキス 0.25%
塩化ナトリウム 0.1%
PH=7.2
実施例 5
ストレプトコツカス・ピオゲネスATCC21060
を第3表に示した培地Cを用いて実施例1と同様
に35℃で培養した。この場合、ペニシリンGは
1000単位/ml培養液となるように添加し、更に培
養を5時間継続した。この培養濾液を実施例1と
同様に精製して抗腫瘍性成分SPF10−11 0.21gr
を得た。
第3表 培地C
マルトース 0.1%
肉エキス 0.5%
ポリペプトン 0.5%
カザミノ酸 0.3%
酵母エキス 0.5%
酸性第一燐酸カリウム 0.1%
硫酸マグネシウム 0.05%
PH=6.5
実施例 6
ストレプトコツカス・ピオゲネスATCC21060
を第4表に示した培地Dを用いて実施例1と同様
に培養した。この場合、ペニシリンGは1000単
位/ml培養液となるように添加し、更に培養を5
時間継続した。この培養濾液を実施例1と同様に
精製して抗腫瘍性成分SPF10−11 0.12grを得た。
第4表 培地D
マルトース 0.25%
カザミノ酸 0.3%
酵母エキス 1.0%
酸性第一燐酸カリウム 0.1%
硫酸マグネシウム 0.05%
PH=6.9
実施例 7
ストレプトコツカス・エスピーATCC21597を
第1表に示した培地Aを用いて実施例1と同様に
培養した。この場合、ペニシリンGは1000単位/
ml培養液となるように添加し、更に培養を5時間
継続した。この培養濾液を実施例1と同様に精製
して抗腫瘍性成分SPF10−11 0.27grを得た。
実施例 8
ストレプトコツカス・ピオゲネスATCC21546
を第1表に示した培地Aを用いて実施例1と同様
に培養した。この場合、ペニシリンGは1000単
位/ml培養液となるように添加し、更に培養を5
時間継続した。この培養濾液を実施例1と同様に
精製して抗腫瘍性成分SPF10−11 0.29grを得た。
実施例 9
ストレプトコツカス・ピオゲネスATCC21547
を第1表に示した培地Aを用いて実施例1と同様
に培養した。この場合、ペニシリンGは1000単
位/ml培養液となるように添加し、更に培養を5
時間継続した。この培養濾液を実施例1と同様に
精製して抗腫瘍性成分SPF10−11 0.26grを得た。
実施例 10
ストレプトコツカス・ピオゲネスATCC21548
を第1表に示した培地Aを用いて実施例1と同様
に培養した。この場合、ペニシリンGは1000単
位/ml培養液となるように添加し、更に培養を5
時間継続した。この培養濾液を実施例1と同様に
精製して抗腫瘍性成分SPF10−11 0.23grを得た。
実施例 11
ストレプトコツカス・ピオゲネスATCC21060
を第1表に示した培地Aを用いて実施例1と同様
に培養した。この場合、セフアロスポリンCは
600μg/ml培養液となるように添加し、更に培
養を5時間継続した。この培養濾液を実施例1と
同様に精製して抗腫瘍性成分SPF10−11 0.28gr
を得た。
実験例 1
実施例1で得られた抗腫瘍性成分SPF10−11の
インビトロにおける抗腫瘍活性試験は、本文中に
記載した抗腫瘍活性の測定方法により行い、その
結果を第5表に示す。
第5表 SPF10−11の抗腫瘍活性の結果
SPF10−11(mg/ml) IR%
1.0 53.4
0.5 21.3
0.25 8.5
実験例 2
実施例1で得られた抗腫瘍性成分SPF10−11の
インビボにおける抗腫瘍活性試験は、Crj;CD−
1(ICR系、雌性7週齢)マウスを使用して実施
した。腫瘍細胞としては、Sarcoma−180腹水癌
細胞を用いこれを生理食塩水に浮遊させ、マウス
の腹腔内に5×105cells/マウス接種した。
癌細胞接種24時間後から1日1回5日間連続し
てSPF10−11を腹腔内に投与してその生存数を観
察しその結果を第6表に示す。
The present invention relates to a novel antitumor component SPF10-11 and a method for producing the same. Conventionally, hemolytic streptococcus (hereinafter referred to as streptococcus)
Attenuated viable bacterial cells have been prepared and are already being used as anticancer agents. In addition, after crushing the cells of hemolytic streptococcus, the active ingredients are extracted with water or a saline solution, an organic solvent is added, and the antitumor ingredients are precipitated and recovered.
1647), a method of lysing hemolytic streptococci with the lytic enzyme lysozyme, cellulase or proteolytic enzyme, and fractionating the active fraction as a water-soluble fraction (British patent
1163865), and a method in which water-insoluble substances are collected after crushing the cells of hemolytic streptococcus and treated with a nuclease and a protease (Japanese Patent Application Laid-Open No. 7014-1982). As described above, it is widely known that Streptococcus bacteria themselves or their bacterial cell components have antitumor properties. It was nothing more than a constituent substance. In order to isolate the active ingredient from the bacterial cells or inside the bacterial cells, it was necessary to lyse the bacterial cells or mechanically crush them to fractionate the whole. Such treatments complicate purification and isolation of active ingredients is extremely difficult. In cases where the active ingredient was actually separated and measured, the molecular weight
200,000 proteins (Special Publication No. 43841, No. 43841, No. 43841, No. 43841)
44,617) and a glycoprotein with a molecular weight of 150,000 (Unexamined Japanese Patent Publication No. 1983
−22026) is known. The present inventors previously conducted extensive research on a method for eluating antitumor components into the culture solution of streptococcus, and found that
It was discovered that antitumor components were eluted into the culture medium by adding penicillin or related substances during culture (Japanese Patent Application Laid-open No. 60-30677), and the physiologically active substance SPF-1 was extracted from the culture medium. This led to the separation. (Japanese Unexamined Patent Publication No. 60-30689) The present inventors further conducted research for the purpose of isolating a more effective component from the culture filtrate of hemolytic streptococcus, and found that in the present invention, cancerous white blood cell culture cells L121
(hereinafter referred to as cultured cell L1210),
and an antitumor component characterized by being a positive color reaction fraction by the Anthrone sulfuric acid method.
We were able to isolate SPF10-11. The antitumor component SPF10-11 of the present invention is determined by elemental analysis,
Although it is thought to be a substance whose main component is sugar and some peptides based on the color reaction, etc., the ultraviolet absorption spectrum and infrared absorption spectrum indicate that it is a new substance that is different from known antitumor substances. be. The present invention relates to the production of antitumor component SPF10-11, which is characterized by culturing antitumor component SPF10-11-producing bacteria belonging to the genus Streptococcus and collecting antitumor component SPF10-11 from the culture. It encompasses the law. In the present invention, bacteria that produce the antitumor component SPF10-11 belonging to the genus Streptococcus can be widely used. Next, the bacteria producing the antitumor component SPF10-11 will be described. Streptococcus pyogenes ATCC21060 Streptococcus sp. ATCC21597 Streptococcus pyogens ATCC21546 Streptococcus pyogenes ATCC21547 Streptococcus pyogenes pyogenes) ATCC21548 Culture media include meat extract medium, yeast extract medium, brain heart infusion medium (BHI
A natural medium such as Streptococcus spp. is often used, but any medium can be used as long as it is suitable for the growth of Streptococcus bacteria. The pH of the culture is 5.0 to 8.0, preferably 6.1 to 7.2, and the culture temperature is 30 to 40°C, preferably 35 to 7.2.
The temperature is 37°C, and static culture or stirring culture can be performed anaerobically. In the present invention, adding penicillin or its related substances at an appropriate time during culture plays an important role in collecting the antitumor component SPF10-11. The timing of addition of penicillin or its related substances is preferably 3 to 20 hours, particularly 5 to 10 hours after the culture reaches the logarithmic growth phase at 35 to 37°C. After that, by continuing the culture for 1 to 20 hours, preferably 3 to 15 hours, antitumor components are added during the culture.
A large amount of SPF10-11 can be accumulated. Penicillin or its related substances may be any known related substances that have similar effects to penicillin, but penicillin G is commonly used. The amount added is penicillin G.
A concentration of 100 to 7000 units/ml, preferably 300 to 5000 units/ml of culture solution is sufficient. A culture solution obtained by culturing Streptococcus bacteria with the addition of penicillin or its related substances is centrifuged to remove bacterial cells to obtain a filtrate. Ammonium sulfate is added to the filtrate and a fraction having a saturation level of 50 to 90% is collected, or the filtrate is concentrated using an ultrafiltration membrane. The obtained ammonium sulfate precipitate containing the antitumor component SPF10-11 can also be stored in a frozen state. To extract the antitumor component SPF10-11 from this ammonium sulfate salt precipitate, the salt precipitate is dissolved in a buffer solution and used. Further, a concentrated solution obtained using an ultrafiltration membrane can also be used as it is. This aqueous solution was adsorbed onto a DEAE cellulose column and eluted stepwise using phosphate buffer to collect the active fraction that inhibits the growth of cultured cells L1210.This active fraction was transferred to a DEAE Sephadex A-25 column. adsorb and elute while linearly increasing the sodium chloride concentration in the phosphate buffer, separate the non-adsorbed portion and the low sodium chloride concentration portion, dialyze this fractionated portion against the buffer solution, and gel. Adsorb onto a Toyopearl HW-50F filter column, elute with phosphate buffer containing 0.1M NaCl, and examine color reaction and activity against cultured cells L1210 using the Anthrone sulfuric acid method. Here, we obtain a fraction that exhibits strong activity against cultured cells L1210, that is, a fraction that forms a peak in the color reaction by the Anthrone sulfuric acid method near the exclusion limit (hereinafter abbreviated as Vo) of Toyopearl HW50F. This fraction is concentrated, adsorbed onto a Toyopearl HW60F column, and eluted with a phosphate buffer containing 0.1M NaCl. SPF10-11 can be obtained as a fraction that has very weak absorption at 280 nm near Vo in Toyopearl HW60F column chromatography and shows strong coloration in the Anthrone sulfuric acid method. Next, the antitumor component SPF10 obtained in Example 1
The freeze-dried specimen of -11 exhibits the following physical and chemical properties. 1 Elemental analysis C 37.2-39.4% H 5.6-6.5% N 0.1-2.3% O 56.9-49.3% Ash 0.2-2.5% 2 Decomposition point This substance turns brown at 165°C and turns black at 240°C and decomposes. 3 Specific rotation [α] 20 D = +95° to +130° (C = 1.00) 4 Ultraviolet absorption spectrum The ultraviolet absorption spectrum of a 0.1% aqueous solution of this substance is shown in FIG. 5 Infrared absorption spectrum Shown in Figure 2.
Absorption is observed at 840 cm -1, which is characteristic. 6. Distinction between basic, acidic, and neutral The pH of a 0.1% aqueous solution of this substance is 6.2 to 6.8. 7 Color of substance White (freeze-dried product) 8 Color reaction Lowry reaction + Buillet reaction + Ninhydrin reaction + Anthrone sulfuric acid reaction + Mauritsch reaction + Cysteine sulfuric acid reaction + Orsine hydrochloric acid reaction - 9 Molecular weight Ultracentrifugation using Reyleigh interference optics It is measured as 20×10 4 or more. 10 Solubility in solvents It is soluble in water, but poorly soluble or insoluble in solvents such as methanol, ethanol, n-propanol, acetone, ethyl ether, n-hexane, chloroform, and ethyl acetate. Next, a method for measuring antitumor activity against cultured cells L1210 in vitro and a color reaction method using the anthrone sulfate method will be shown. Method for measuring antitumor activity Antitumor activity was measured by measuring the growth inhibition rate (IR%) of cultured cells L1210. L1210 cells were grown in RPMI1640 medium (5%) supplemented with 10% FBS.
mg/kanamycin). 0.5 ml of this culture solution was poured into a Falcon 2058 tube so that the number of cells was 1×10 5 cells/tube. Next, add a predetermined amount of the preparation (antitumor component) to this culture solution.
0.5 ml of SPF10-11) dissolved in the culture medium to the desired concentration was added and cultured at 37°C in the presence of 5% CO2 . 48 hours after adding the standard, staining with trypan blue was performed, and IR was determined using the following formula.
(%) was calculated. IR (%) = (A) - Number of viable cells in each experimental group/Number of viable cells in control group x 100 Here, (A) indicates the number of viable cells in the control group. A control was performed at the same time using 0.5 ml of culture solution that did not contain the standard. Color reaction using the Anthrone sulfuric acid method Add 2 ml of Anthrone reagent (0.20 gr of Anthrone to 1 ml of the sample dissolved in deionized water to the desired concentration).
(dissolved in 100ml of concentrated sulfuric acid) and mix.
After 30 minutes, replace 1 ml of standard with 2 ml in 1 ml of deionized water.
The solution containing Anthrone reagent was used as a control.
Measure absorption at 620nm. The quantitative value is obtained from a calibration formula obtained by measuring in the same manner using glucose. Next, examples of the present invention will be shown. Example 1 Streptococcus pyogenes ATCC21060
The seed culture solution obtained by inoculating 100ml of BHI medium and statically culturing at 37℃ for 8 hours is used as the medium shown in Table 1.
A1l was inoculated and precultured anaerobically under the same conditions as the seed culture. Table 1 Medium A Maltose 0.25% Meat extract 1.0% Polypeptone 1.0% Yeast extract 0.25% Acidic monobasic potassium phosphate 0.1% Magnesium sulfate 0.05% PH=6.8 10 Pour medium A8 into a jar fermenter and heat at 120℃ for 10 minutes After heat sterilization, cool to 37℃, inoculate with preculture solution 1, and incubate at 37℃ for 15.5 hours at PH.
6.8. Incubate anaerobically with stirring at 300 rpm. Then, penicillin G was added at 1000 units/ml culture solution, and the culture was continued for an additional 5 hours.
The resulting culture solution was centrifuged to remove bacterial cells. The culture filtrate was concentrated to 1.5 using an ultrafiltration membrane with a molecular weight cutoff of 10,000 (Romicon, USA). This concentrated solution was applied to a DEAE cellulose column (5
x 70cm), washed with a 10mM phosphate buffer with a pH of 7.0, and then eluted stepwise using the above phosphate buffer containing 0.3M sodium chloride, followed by the Anthrone sulfate method. An active fraction that showed a positive color reaction and inhibited the growth of cultured cells L1210 was collected. This active fraction was adsorbed on a DEAE Sephadex A-25 column (7.5 x 50 cm), and then washed with the above phosphate buffer and eluted by linearly increasing the sodium chloride concentration. Separate a certain portion. This elution curve is shown in FIG. In FIG. 3, the dotted line portion is the non-adsorbed portion, and the solid line portion is the sodium chloride added portion. A is a fraction with activity to inhibit cultured cell L1210 growth;
B is the non-active fraction inhibiting the growth of MP-2 (JP-A-60-30689); B is the non-active fraction inhibiting the growth of cultured cells L1210; B is the active fraction inhibiting MP-2 growth. The eluate of the active fraction of A was concentrated to 30 ml using an ultrafiltration membrane with a molecular weight cutoff of 10,000 (Millipore). Transfer this concentrated solution to a Toyopearl HW50F column (5.0 x 100cm).
Then, the mixture was eluted with a solution containing 0.1M sodium chloride in the above phosphate buffer, and a fraction showing a positive color reaction by the Anthrone sulfuric acid method was collected.
This elution curve is shown in FIG. In FIG. 4, the solid line shows the overall elution curve, and the dotted line shows the elution curve of the positive color reaction portion by the Anthrone sulfuric acid method. Here, C is a positive color reaction fraction by the Anthrone sulfuric acid method, but the growth inhibition activity against cultured cells L1210 was investigated, and the fraction near Vo, which has strong activity,
A D fraction that forms a peak is obtained by the Anthrone sulfuric acid method. Fraction D is lyophilized. This freeze-dried sample was dissolved in phosphate buffer, adsorbed on a Toyopearl HW60F column (2.6 x 100cm), and then
Elute with the same eluent as for HW50F. This elution curve is shown in FIG. In FIG. 5, the solid line shows the overall elution curve, and the solid line with black circles shows the elution curve of the positive color reaction portion by the Anthrone sulfuric acid method. Here, the fraction of F that shows strong coloration in the color reaction using the Anthrone sulfuric acid method was selected using SPF10.
-11, and the G fraction was set as SPF 10-12. The F fraction was lyophilized. This freeze-dried specimen had an antitumor component SPF 10-11, and yielded 0.32 gr. Example 2 Streptococcus pyogenes ATCC21060
were cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G was added at 300 units/ml culture solution, and the culture was further increased by 10
Lasted for an hour. Add ammonium sulfate to this culture filtrate and
Collect the fraction at 90% saturation. The antitumor component was purified in the same manner as in Example 1 by removing ammonium sulfate by dialysis.
Obtained SPF10−11 0.15gr. Example 3 Streptococcus pyogenes ATCC21060
were cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G was added at a concentration of 3000 units/ml culture solution, and the culture was further incubated for 3
Lasted for an hour. This culture filtrate was purified in the same manner as in Example 1 to obtain 0.37 gr of antitumor component SPF10-11. Example 4 Streptococcus pyogenes ATCC21060
were cultured in the same manner as in Example 1 using medium B shown in Table 2. In this case, penicillin G is added at 1000 units/ml culture solution, and the culture is further
Lasted for an hour. This culture filtrate was purified in the same manner as in Example 1 to obtain 0.20 gr of antitumor component SPF10-11. Table 2 Medium B Maltose 0.1% Meat extract 0.5% Polypeptone 1.0% Yeast extract 0.25% Sodium chloride 0.1% PH=7.2 Example 5 Streptococcus pyogenes ATCC21060
were cultured at 35°C in the same manner as in Example 1 using medium C shown in Table 3. In this case, penicillin G
The mixture was added at a concentration of 1000 units/ml culture solution, and the culture was continued for an additional 5 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain 0.21 gr of antitumor component SPF10-11.
I got it. Table 3 Medium C Maltose 0.1% Meat extract 0.5% Polypeptone 0.5% Casamino acids 0.3% Yeast extract 0.5% Acidic potassium monophosphate 0.1% Magnesium sulfate 0.05% PH=6.5 Example 6 Streptococcus pyogenes ATCC21060
were cultured in the same manner as in Example 1 using medium D shown in Table 4. In this case, penicillin G is added at 1000 units/ml culture solution, and the culture is further
Lasted for an hour. This culture filtrate was purified in the same manner as in Example 1 to obtain 0.12 gr of antitumor component SPF10-11. Table 4 Medium D Maltose 0.25% Casamino acids 0.3% Yeast extract 1.0% Acid monobasic potassium phosphate 0.1% Magnesium sulfate 0.05% PH=6.9 Example 7 Streptococcus sp. ATCC21597 was grown using medium A shown in Table 1. The cells were cultured in the same manner as in Example 1. In this case, penicillin G is 1000 units/
ml culture solution, and culture was continued for an additional 5 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain 0.27 gr of antitumor component SPF10-11. Example 8 Streptococcus pyogenes ATCC21546
were cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G is added at 1000 units/ml culture solution, and the culture is further
Lasted for an hour. This culture filtrate was purified in the same manner as in Example 1 to obtain 0.29 gr of antitumor component SPF10-11. Example 9 Streptococcus pyogenes ATCC21547
were cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G is added at 1000 units/ml culture solution, and the culture is further
Lasted for an hour. This culture filtrate was purified in the same manner as in Example 1 to obtain 0.26 gr of antitumor component SPF10-11. Example 10 Streptococcus pyogenes ATCC21548
were cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G is added at 1000 units/ml culture solution, and the culture is further
Lasted for an hour. This culture filtrate was purified in the same manner as in Example 1 to obtain 0.23 gr of antitumor component SPF10-11. Example 11 Streptococcus pyogenes ATCC21060
were cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, cephalosporin C is
The mixture was added at a concentration of 600 μg/ml culture solution, and the culture was continued for an additional 5 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain 0.28 gr of antitumor component SPF10-11.
I got it. Experimental Example 1 An in vitro antitumor activity test of the antitumor component SPF10-11 obtained in Example 1 was conducted using the method for measuring antitumor activity described in the text, and the results are shown in Table 5. Table 5 Results of antitumor activity of SPF10-11 SPF10-11 (mg/ml) IR% 1.0 53.4 0.5 21.3 0.25 8.5 Experimental example 2 Antitumor activity of antitumor component SPF10-11 obtained in Example 1 in vivo The activity test is Crj;CD-
1 (ICR strain, female, 7 weeks old) mice were used. Sarcoma-180 ascites cancer cells were used as tumor cells, suspended in physiological saline, and inoculated intraperitoneally into mice at 5×10 5 cells/mouse. SPF10-11 was administered intraperitoneally once a day for 5 consecutive days starting 24 hours after cancer cell inoculation, and the number of survivors was observed. The results are shown in Table 6.
【表】
実験例 3
実施例2で得られたSPF10−11の固形癌に対す
る抗腫瘍活性試験は、Crj;CD−1(ICR系雌性
5週齢)マウスを使用し実施した。
固形癌細胞としては、Sarcoma180を用い、マ
ウス左大腿部皮下に5×105cells/マウス接種し
た。
SPF10−11は、癌細胞接種24時間後から1日1
回5日間連続して腹腔内に投与し、癌細胞接種32
日目に、固形癌を切り取り、その重量を測定し
た。その重量の平均値を第7表に示す。[Table] Experimental Example 3 The antitumor activity test of SPF10-11 obtained in Example 2 against solid tumors was carried out using Crj;CD-1 (ICR female, 5 weeks old) mice. Sarcoma180 was used as solid tumor cells and 5×10 5 cells/mouse were inoculated subcutaneously into the left thigh of the mouse. SPF10-11 is applied once a day starting 24 hours after cancer cell inoculation.
Administer intraperitoneally for 5 consecutive days to inoculate cancer cells 32 times
On the day, solid tumors were excised and their weights were measured. The average weight values are shown in Table 7.
【表】
実験例 4
実施例1で得られたSPF10−11のEhrlich細胞
の形態変化に与える影響は、下記の通り行つた。
Ehrlich細胞は、Crj;CD−1(ICR系雌性7週
齢)の腹腔内で1週間増殖させたのち、採取し、
10%FBS添加RPMI1640培地(5mg/カナマイ
シン含有)に懸濁し、1×106cells/mlに調整後、
その0.5mlをフアルコン2058チユーブに注加する。
SPF10−11は、上記培地に溶解し、0.45μmの
メンブランフイルターで濾過し、その0.5mlを上
記細胞と混合した後37℃5%CO2存在下培養を20
時間行う。形態変化の測定は、顕微鏡下、コント
ロールに比較し、顕著な膨化細胞、細胞内の空洞
形成および細胞内成分の漏出する細胞などを計数
した。
その結果は、第8表に示す。[Table] Experimental Example 4 The influence of SPF10-11 obtained in Example 1 on the morphological changes of Ehrlich cells was determined as follows.
Ehrlich cells were grown in the peritoneal cavity of Crj; CD-1 (ICR female, 7 weeks old) for one week, and then harvested.
After suspending in RPMI1640 medium (containing 5 mg/kanamycin) supplemented with 10% FBS and adjusting to 1 × 10 6 cells/ml,
Pour 0.5ml into the Falcon 2058 tube. SPF10-11 was dissolved in the above medium, filtered with a 0.45 μm membrane filter, 0.5 ml of it was mixed with the above cells, and cultured at 37°C in the presence of 5% CO2 for 20 days.
Do time. Morphological changes were measured under a microscope by comparing cells with significant swelling, forming intracellular cavities, and counting cells leaking intracellular components. The results are shown in Table 8.
【表】
実験例 5
実施例2で得られたSPF10−11のTNF誘導活
性の測定は、原中勝征;“TNF”日本医事新報社
(1984)を参考にして、下記の通り行つた。
マウス(Crj;CD−1、ICR系、雌性7週齢)
の腹腔にBCG(日本ビーシージー製造協会)5mg
を接種し、(1次感作)、2週間後、SPF10−11を
尾静脈より投与し(2次感作)、SPF10−11投与
2時間後心臓から採血し、血清を調整する。
検定細胞は10%FBS添加RPMI1640培地(5
mg/カナマイシン含有)で3〜4日間培養した
L929細胞を用いる。
L929細胞は、1×105cells/mlの懸濁液0.5mlを
フアルコン24wellsに注加する。
ここに、上記血清を上記RPMI1640培地で20倍
希釈しその0.5mlを注加混合後、37℃ 5%
CO2存在下48時間培養する。
TNF誘導活性の測定は、L929細胞をトリパン
ブルーで染色し、色素排除法で生細胞数を計測し
た。その結果を第9表に示す。[Table] Experimental Example 5 The TNF-inducing activity of SPF10-11 obtained in Example 2 was measured as follows, with reference to Katsuyuki Haranaka; "TNF" Nihon Iji Shinposha (1984). Mouse (Crj; CD-1, ICR strain, female, 7 weeks old)
BCG (Japan BCG Manufacturing Association) 5 mg in the abdominal cavity of
Two weeks later, SPF10-11 is administered through the tail vein (secondary sensitization). Two hours after SPF10-11 administration, blood is collected from the heart and serum is adjusted. Test cells were grown in RPMI1640 medium (5% FBS-supplemented).
mg/kanamycin) for 3 to 4 days.
Use L929 cells. For L929 cells, 0.5 ml of a suspension of 1×10 5 cells/ml is injected into 24 wells of Falcon. Here, the above serum was diluted 20 times with the above RPMI1640 medium, 0.5 ml of it was added and mixed, and the mixture was heated to 5% at 37°C.
Incubate for 48 hours in the presence of CO2 . To measure TNF-induced activity, L929 cells were stained with trypan blue, and the number of viable cells was counted using a dye exclusion method. The results are shown in Table 9.
【表】
実験例 6
実施例1で得られたSPF10−11のクロフアージ
の培養細胞J−774−1(以下、J−774−1と略
す)に対する変形活性の測定は、下記の通り行つ
た。
J−774−1は10%FBS添加RPMI1640培地
(5mg/カナマイシン含有)で3〜4日培養後、
10×104コ/mlに調整する。
SPF10−11は、生理食塩水で5mg/mlとし、
0.22μmの除菌フイルターで濾過し、10μずつ96
穴のwellに2倍階段希釈法により分注する。
各wellに上記J−774−1懸濁液100μ注加
し、96時間37℃ 5%CO2条件下で培養する。
変形活性の測定は、各wellを倒立顕微鏡下観察
し、形態変化(球状から伸展し細長くなる)を誘
起する最少濃度を求めた。その結果を第10表に示
す。
第10表 SPF10−11のJ−774−1に対する変
形誘起最少濃度
変形誘起最少濃度
SPF10−11 4.0μg/ml[Table] Experimental Example 6 The deformation activity of cultured cells J-774-1 (hereinafter abbreviated as J-774-1) of SPF 10-11 of SPF10-11 obtained in Example 1 was measured as follows. After culturing J-774-1 for 3 to 4 days in RPMI1640 medium (containing 5 mg/kanamycin) supplemented with 10% FBS,
Adjust to 10× 104 /ml. SPF10-11 is 5mg/ml with physiological saline,
Filter with a 0.22μm sterilization filter, 10μm each96
Dispense into wells using the 2-fold serial dilution method. 100μ of the above J-774-1 suspension is added to each well, and cultured for 96 hours at 37°C and 5% CO2 . To measure the deformation activity, each well was observed under an inverted microscope, and the minimum concentration that induced a change in morphology (from spherical to elongated and elongated) was determined. The results are shown in Table 10. Table 10 Minimum concentration to induce deformation of SPF10-11 for J-774-1 Minimum concentration to induce deformation SPF10-11 4.0μg/ml
第1図は抗腫瘍性成分SPF10−11の紫外線吸収
スペクトルを示し、第2図は同じく赤外線吸収ス
ペクトルを示す。第3図は実施例1における活性
画分のDEAEセフアデツクスA−25カラムの溶出
曲線を示す図で、第4図はこのA画分をトヨパー
ルHW50Fカラムに吸着させ、0.1M NaClを含む
燐酸緩衝液による溶出曲線を示す図で、第5図は
このD画分をトヨパールHW60Fカラムに吸着さ
せ、0.1M NaCl含有燐酸緩衝液による溶出曲線
を示す。
FIG. 1 shows the ultraviolet absorption spectrum of the antitumor component SPF10-11, and FIG. 2 similarly shows the infrared absorption spectrum. Fig. 3 shows the elution curve of the active fraction in Example 1 on the DEAE Sephadex A-25 column, and Fig. 4 shows the elution curve of the active fraction in Example 1, and Fig. 4 shows the elution curve of the active fraction in Example 1. Figure 5 shows the elution curve using a phosphate buffer containing 0.1M NaCl after this D fraction was adsorbed onto a Toyopearl HW60F column.
Claims (1)
SPF10−11。 1 元素元析 C 37.2〜39.4% H 5.6〜6.5% N 0.1〜2.3% O 56.9〜49.3% Ash 0.2〜2.5% 2 分解点 本物質は165℃で褐変し240℃になる
と黒色となり分解する。 3 比旋光度〔α〕20 D=+95゜〜+130゜(C=1.00) 4 紫外線吸収スペクトル 本物質0.1%の水溶
液の紫外線吸収スペクトルは第1図に示され
る。 5 赤外線吸収スペクトル 第2図に示される。
840cm-1に吸収が認められ特徴的である。 6 塩基性、酸性、中性の区別 本物質0.1%の
水溶液のPHは6.2〜6.8である。 7 物質の色 白色(凍結乾燥品) 8 呈色反応 ローリー反応 + ビユーレツト反応 + ニンヒドリン反応 + アンスロン硫酸反応 + モーリツシユ反応 + システイン硫酸反応 + オルシン塩酸反応 − 9 分子量 Reyleigh干渉光学系を用いた超遠
心法により20×104以上と測定される。 10 溶剤に対する溶解性 水に可溶であるが、メ
タノール、エタノール、n−プロパノール、ア
セトン、エチルエーテル、n−ヘキサン、クロ
ロホルム、酢酸エチル等の溶剤には、難溶又は
不溶である。 2 ストレプトコツカス属に属する抗腫瘍性成分
SPF10−11生産菌を培養し、培養物から抗腫瘍性
成分SPF10−11を採取することを特徴とする抗腫
瘍性成分SPF10−11の製法。 3 ストレプトコツカス属に属する抗腫瘍性成分
SPF10−11生産菌を培養するに際し、培養中の適
当な時期にペニシリン又はその関連物質を添加し
て培養することを特徴とする特許請求の範囲第2
項に記載の抗腫瘍性成分SPF10−11の製法。 4 ストレプトコツカス属に属する抗腫瘍性成分
SPF10−11生産菌を培養し、培養物から抗腫瘍性
成分SPF10−11を採取するに当たり、培養細胞
L1210の生育を阻害し、又は/及びアンスロン硫
酸法による呈色反応陽性の画分を分取することを
特徴とする特許請求の範囲第2項に記載の抗腫瘍
性成分SPF10−11の製法。[Claims] 1. Antitumor component having the following physicochemical properties:
SPF10−11. 1 Elemental analysis C 37.2-39.4% H 5.6-6.5% N 0.1-2.3% O 56.9-49.3% Ash 0.2-2.5% 2 Decomposition point This substance turns brown at 165°C and turns black at 240°C and decomposes. 3 Specific rotation [α] 20 D = +95° to +130° (C = 1.00) 4 Ultraviolet absorption spectrum The ultraviolet absorption spectrum of a 0.1% aqueous solution of this substance is shown in FIG. 5 Infrared absorption spectrum Shown in Figure 2.
Absorption is observed at 840 cm -1, which is characteristic. 6. Distinction between basic, acidic, and neutral The pH of a 0.1% aqueous solution of this substance is 6.2 to 6.8. 7 Color of substance White (freeze-dried product) 8 Color reaction Lowry reaction + Buillet reaction + Ninhydrin reaction + Anthrone sulfuric acid reaction + Mauritsch reaction + Cysteine sulfuric acid reaction + Orsine hydrochloric acid reaction - 9 Molecular weight Ultracentrifugation using Reyleigh interference optics It is measured as 20×10 4 or more. 10 Solubility in solvents It is soluble in water, but poorly soluble or insoluble in solvents such as methanol, ethanol, n-propanol, acetone, ethyl ether, n-hexane, chloroform, and ethyl acetate. 2 Antitumor component belonging to the genus Streptococcus
A method for producing an antitumor component SPF10-11, which comprises culturing SPF10-11-producing bacteria and collecting the antitumor component SPF10-11 from the culture. 3 Antitumor component belonging to the genus Streptococcus
Claim 2, characterized in that when culturing SPF10-11 producing bacteria, penicillin or a related substance is added at an appropriate time during the culturing.
The method for producing the antitumor component SPF10-11 described in Section 1. 4 Antitumor component belonging to the genus Streptococcus
When culturing SPF10-11-producing bacteria and collecting the antitumor component SPF10-11 from the culture, cultured cells
The method for producing the antitumor component SPF10-11 according to claim 2, which comprises inhibiting the growth of L1210 and/or separating a fraction that is positive for a color reaction by the anthrone sulfuric acid method.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP61004186A JPS62164628A (en) | 1986-01-14 | 1986-01-14 | Spf 10-11: antitumor component and its preparation |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP61004186A JPS62164628A (en) | 1986-01-14 | 1986-01-14 | Spf 10-11: antitumor component and its preparation |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS62164628A JPS62164628A (en) | 1987-07-21 |
JPH0571231B2 true JPH0571231B2 (en) | 1993-10-06 |
Family
ID=11577672
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP61004186A Granted JPS62164628A (en) | 1986-01-14 | 1986-01-14 | Spf 10-11: antitumor component and its preparation |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS62164628A (en) |
-
1986
- 1986-01-14 JP JP61004186A patent/JPS62164628A/en active Granted
Also Published As
Publication number | Publication date |
---|---|
JPS62164628A (en) | 1987-07-21 |
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