JPH0394693A - Novel substance dc116 - Google Patents

Novel substance dc116

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Publication number
JPH0394693A
JPH0394693A JP1233342A JP23334289A JPH0394693A JP H0394693 A JPH0394693 A JP H0394693A JP 1233342 A JP1233342 A JP 1233342A JP 23334289 A JP23334289 A JP 23334289A JP H0394693 A JPH0394693 A JP H0394693A
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Japan
Prior art keywords
culture
substance
properties
strain
color
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.)
Granted
Application number
JP1233342A
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Japanese (ja)
Other versions
JP2786267B2 (en
Inventor
Hirofumi Nakano
洋文 中野
Mitsunobu Hara
光信 原
Isao Kawamoto
勲 川本
Yoichi Uosaki
宇於崎 洋一
Toshimitsu Takiguchi
滝口 利光
Tadashi Ashizawa
芦沢 忠
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KH Neochem Co Ltd
Original Assignee
Kyowa Hakko Kogyo Co Ltd
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Priority to JP1233342A priority Critical patent/JP2786267B2/en
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    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Plural Heterocyclic Compounds (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

NEW MATERIAL:A compound expressed by the formula. Besides, said compound exhibits following physicochemical properties. Molecular weight: 462; molecular formula: C25H18O9; optical rotation: ]alpha] =+270 deg.C (c=0.012, in CHCl3); solubility: soluble in chloroform, dimethyl sulfoxide, methanol, ethyl acetate and acetone, hardly soluble in water on n-hexane; color reaction: positive to iodine reagent; color and properties of the substance: red acidic substance. USE:Useful as medicine having antibacterial and antitumor activity. PREPARATION:Fungus belonging to genus Streptomyces such as FERM BP-2559 is cultured at 25-32 deg.C and at pH6-8 for 1-7 days by a deep-stirring culturing method and separated from the cultured solution and fungal cells, and then purified.

Description

【発明の詳細な説明】 産業上の利用分野 本発明はアンスラキノンピラン構造を有する新規抗腫瘍
抗生物質DC116およびその製法に関する。
DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a novel antitumor antibiotic DC116 having an anthraquinone pyran structure and a method for producing the same.

従来の技術 従来、キノン骨格を持つ抗腫瘍抗生物質として、CH3 DH DH CH. で表わす。〉で表わされるDC−928あるいはDC9
2D(特公昭62−504380号公報)下記式(II
) 課題を解決するための手段 本発明者らは、東京都町田市の土壌から分離した微生物
(以下、DO−116株という。)を培地に培養して得
られた培養物の中に抗腫瘍活性を有する抗生物質が生産
されることを見出した。
BACKGROUND OF THE INVENTION Conventionally, as an antitumor antibiotic having a quinone skeleton, CH3 DH DH CH. It is expressed as > DC-928 or DC9
2D (Japanese Patent Publication No. 62-504380) The following formula (II
) Means for Solving the Problems The present inventors cultivated a microorganism isolated from the soil of Machida City, Tokyo (hereinafter referred to as strain DO-116) in a medium, and the resulting culture contained antitumor substances. It has been found that an active antibiotic is produced.

この物質を単離、精製し、理化学的性質を調べた結果、
新規物質であることがわかり、DC 116と命名した
As a result of isolating and purifying this substance and examining its physical and chemical properties,
It was found to be a new substance and was named DC 116.

本発明によれば、下記の一般式 U で表わされるSF2587 (特開平1−193265
号公報)などがあるが、OCll6はこれらの物質とは
明らかに異なる新規な物質である。
According to the present invention, SF2587 (JP-A-1-193265
However, OCll6 is a new substance that is clearly different from these substances.

発明が解決しようとする課題 本発明の目的は、優れた抗生物質、抗腫瘍性化合物を提
供することにある。
Problems to be Solved by the Invention An object of the present invention is to provide an excellent antibiotic and antitumor compound.

で表わされる抗腫瘍作用および抗菌活性を有する新規物
質DC116が提供される。
A novel substance DC116 having antitumor and antibacterial activity is provided.

この化合物はストレプトマイセス属に属する微生驕を培
養することによって得られる。
This compound is obtained by culturing microorganisms belonging to the genus Streptomyces.

以下に本発明を詳細に説明する。The present invention will be explained in detail below.

以下にDC116の理化学的性質を示す。The physical and chemical properties of DC116 are shown below.

■分子量=462 ■分子式:CzsH+a○, ■質量分析: SIMS 465(+J+3)3. 4
64 (M+2) ” , 463 (M+1) ”2
5 ■比旋光度: 〔α〕,・↓270゜(c=0. 01
2. CHCj! 3)■紫外線吸収スペクトル: (
メタノール中で測定)λ...  :  242nm 
(E =63,000),264nm (sh,  e
 =29,000),380nm (ε=5,700>
. 506n+++ ( e = 11, 000>,■赤
外線吸収スペクトル: (KBr錠剤法で測定)(cm
−’)3700−2400. 3430. 1755.
 1?34,  1654,  1629,1585,
 1454. 1224. ■PMRスペクトル: (CD([ .−CD30D中
で測定,内部標準TMS) H−NMR (500MHz)’δ(ppm)8.18
 (IH. s), 7.39 (IH, d, J=
9JHz),7J4 (LH, d, J=9.3Hz
), 6.47 (IH, s),5.86 (1N,
 ddq, J=11.2. 7.1, 1.0Hz)
.5.15 (IH, ddq, J=ll.2. 8
.3, 1.8Hz).4.40 (IH,  d. 
 J=16.6Hz),  4.36 (IH,  d
,J=16.6flz),  3.96  (IH, 
 d,  J=8.6Hz),1.99 (3H,  
s),  1.85 (LH.  d,  J・7, 
l.  1. 8Hz),■CMRスペクトル:  (
CDCIl,−CD.0D中、内部標準TMS) ”C−NMR  (125Ml{Z}; δ  (pp
m)185.30,  185.28,   178.
8,   172.5,166.7,   157.8
9,   157.88,  156,7,144.1
,   137.0.   134.7,   130
.8.129.1,   126.9,   126.
5,   122.6,121.8,   113.4
,   112.5,   110.9,62.2. 
  61.3.    42.1,    19.9,
13. 8. ■ 溶解性:クロロホルム、ジメチルスルホキシド(D
MS○)、メタノール、酢酸エチルおよびアセトンに可
溶、水およびn−ヘキサンには難溶。
■Molecular weight = 462 ■Molecular formula: CzsH+a○, ■Mass spectrometry: SIMS 465 (+J+3)3. 4
64 (M+2) ”, 463 (M+1) ”2
5 ■Specific optical rotation: [α],・↓270° (c=0.01
2. CHCj! 3) ■ Ultraviolet absorption spectrum: (
(measured in methanol) λ. .. .. : 242nm
(E = 63,000), 264 nm (sh, e
=29,000), 380nm (ε=5,700>
.. 506n+++ (e = 11,000>, ■Infrared absorption spectrum: (measured by KBr tablet method) (cm
-') 3700-2400. 3430. 1755.
1?34, 1654, 1629, 1585,
1454. 1224. ■PMR spectrum: (CD ([.-measured in CD30D, internal standard TMS) H-NMR (500MHz)'δ (ppm) 8.18
(IH. s), 7.39 (IH, d, J=
9JHz), 7J4 (LH, d, J=9.3Hz
), 6.47 (IH, s), 5.86 (1N,
ddq, J=11.2. 7.1, 1.0Hz)
.. 5.15 (IH, ddq, J=ll.2.8
.. 3, 1.8Hz). 4.40 (IH, d.
J=16.6Hz), 4.36 (IH, d
, J=16.6flz), 3.96 (IH,
d, J=8.6Hz), 1.99 (3H,
s), 1.85 (LH. d, J.7,
l. 1. 8Hz), ■CMR spectrum: (
CDCIl,-CD. 0D, internal standard TMS) "C-NMR (125Ml{Z}; δ (pp
m) 185.30, 185.28, 178.
8, 172.5, 166.7, 157.8
9, 157.88, 156,7, 144.1
, 137.0. 134.7, 130
.. 8.129.1, 126.9, 126.
5, 122.6, 121.8, 113.4
, 112.5, 110.9, 62.2.
61.3. 42.1, 19.9,
13. 8. ■ Solubility: Chloroform, dimethyl sulfoxide (D
MS○), soluble in methanol, ethyl acetate and acetone, sparingly soluble in water and n-hexane.

■ 呈色反応:ヨード試薬に陽性。■ Color reaction: Positive for iodine reagent.

0 物質の色、性質:赤色の酸性物質 ■ 薄層クロマトグラフィー:シリカゲル薄層(HPT
Lc plate Art、15647 E. Mer
ck社製)トルエン:アセトン:酢酸(7:2:0.1
v/v/v )の展開溶媒でRf0.60。
0 Color and properties of substance: Red acidic substance ■ Thin layer chromatography: Thin layer of silica gel (HPT
Lc plate Art, 15647 E. Mer
(manufactured by CK) Toluene: Acetone: Acetic acid (7:2:0.1
Rf0.60 with developing solvent (v/v/v).

C11CL:酢酸(100:lv/v)でRfO.30
C11CL: RfO. with acetic acid (100:lv/v). 30
.

展開後DC116のスポットは、バチルス ズブチリス
(Bacillus subtilis)を用いるノイ
イオアッセイ、熱硫酸および紫外部吸収により検出でき
る。
After development, DC116 spots can be detected by neuioassay using Bacillus subtilis, hot sulfuric acid, and ultraviolet absorption.

次にDC l 1 6の生物活性について説明する。Next, the biological activity of DC l 1 6 will be explained.

(A)抗菌作用 各種、細菌に対する最小生育阻止濃度(M,IC)を寒
天希釈法(pH1. 0 )により測定した。その結果
を第1表に示す。
(A) Antibacterial activity The minimum inhibitory concentration (M, IC) against various bacteria was measured by the agar dilution method (pH 1.0). The results are shown in Table 1.

(B)急性毒性(LDs。) マウスへの静脈内投与では6.9■/kgであった。(B) Acute toxicity (LDs.) When administered intravenously to mice, the dose was 6.9 μ/kg.

(C)抗腫瘍作用 ■ リンホサイティック・リューケミアP−388腫瘍
に対する治療効果 体重約22gのCDF.雄マウス1群5匹に、リンホサ
イティック・り二−ケミア(Lymphocyt+cL
eukemia) P−388腫瘍細胞IXIO’個を
腹腔内移植した。移植後24時間目にDC116を含む
リン酸緩衝液生理食塩水(PBS)溶液0.2mlを1
回腹腔内に投与した。PBSの組成はNaCf  O.
8g/di, KCj! 0.02 g/d1、NaJ
PO− 1. 15g / d1、κH.PO. 0.
02g/d1、p87. 2である。比較例として、腫
瘍細胞移植後24時間目にマイトマイシンCを含むPB
S溶液Q.2mlを腹腔内投与した群を設けた。移植後
の延命効果をT/C CT :試験例の平均生存日数、
C:対照(PBS溶液0.2rrtlを復腔内投与した
もの〉の平均生存日数〕として第2表に示す。
(C) Antitumor action■ Treatment effect on Lymphocytic Leukemia P-388 tumor CDF weighing approximately 22 g. Lymphocytic Rhinichemia (Lymphocyt+cL) was administered to 5 male mice per group.
Eukemia) P-388 tumor cells IXIO' were implanted intraperitoneally. 24 hours after transplantation, 0.2 ml of phosphate buffered saline (PBS) solution containing DC116 was added to
It was administered intraperitoneally. The composition of PBS was NaCfO.
8g/di, KCj! 0.02 g/d1, NaJ
PO-1. 15g/d1, κH. P.O. 0.
02g/d1, p87. It is 2. As a comparative example, PB containing mitomycin C was administered 24 hours after tumor cell implantation.
S solution Q. A group was established in which 2 ml was administered intraperitoneally. T/C CT: Average survival days of test cases;
C: Mean survival days of control (0.2 rrtl of PBS solution administered intracavitally) is shown in Table 2.

第    2    表 に投与した群を設けた。Table 2 A group was set up in which patients were given the same treatment.

移植lO日後のT/C [:T :試験例の平均腫瘍体
積(叩3)、C:対照(PBS溶液0.2−を腹腔内投
与したもの)の平均腫瘍体積(mm3)]を測定した。
10 days after transplantation, T/C [:T: average tumor volume of test example (3), C: average tumor volume (mm3) of control (intraperitoneal administration of PBS solution 0.2-)] was measured. .

その結果を第3表に示す。The results are shown in Table 3.

第   3   表 マイトマイシン C 4 l.80 ■ サルコーマ180腫瘍に対する治療効果体重約20
gのacty雄マウス1群6匹にサルコーマ180腫瘍
細胞5X10’個を腋窩部皮下に移植した。移植後1日
目から5日目まで24時間毎に5回第2表に示す濃度の
DC116を含むPBS溶液0,2−を復腔内に投与し
た。
Table 3 Mitomycin C 4 l. 80 ■ Treatment effect on Sarcoma 180 tumor Weight approx. 20
5 x 10' Sarcoma 180 tumor cells were subcutaneously implanted into the axillary region of each group of 6 male mice. From day 1 to day 5 after transplantation, a PBS solution 0,2- containing DC116 at the concentration shown in Table 2 was administered intravenously 5 times every 24 hours.

比較例として腫瘍細胞移植後24時間目にマイトマイシ
ンCを含むP B 3 0. 2 m&を腹腔内次にD
C 1 1 6の製造法について説明する。
As a comparative example, PB30 containing mitomycin C was used 24 hours after tumor cell transplantation. 2 m & intraperitoneally then D
A method for producing C 1 1 6 will be explained.

DC116はストレブトマイセス属に属し、DC116
を生産する能力を有する微生物を培地に培養し、培養物
中にDC l l 6を生成M積させ、該培養物からD
C 1 1 6を採取することによって得ることができ
る。
DC116 belongs to the genus Strebtomyces, and DC116
A microorganism having the ability to produce D is cultured in a medium, and DCl 6 is produced in the culture, and D is produced from the culture.
It can be obtained by collecting C 1 1 6.

DC116生産菌株としてはストレプトマイセス属に属
し、DC 1 1 6生産能を有する菌株であればいず
れの菌株でも用いることができる。またこれらの菌株の
人工的変異方法、たとえば紫外線照射、X線照射、変異
誘起剤処理などによって変異させた変異株あるいは自然
的に変異した変異株でもDC116を生産するものであ
れば本発明に用いることができる。代表的菌株としてD
O−116株があげられる。
As the DC116-producing strain, any strain belonging to the genus Streptomyces and having the ability to produce DC116 can be used. In addition, mutant strains mutated by artificial mutation methods of these strains, such as ultraviolet irradiation, X-ray irradiation, treatment with mutagens, etc., or naturally mutated mutant strains, can be used in the present invention if they produce DC116. be able to. D as a representative strain
O-116 strain is mentioned.

Do−116株の菌学的性質について以下に述べる。該
性質の決定は、国際ストレプトマイセス(Strept
omyces)プロジェクト(ISP)がストレブトマ
イセス種の特性決定のために推奨する方法〔巳.B.シ
エIJング(巳.B,Shirling)およびD.ゴ
ットリープ(D, Gottl ieb)らのインター
ナショナル・ジャーナル・システマティック・バクテリ
オロジ−(Int, J.Syst,Bacterio
l.) 16. 313〜340 (1966))に従
った。全細胞の加水分解物中のジアミノピメリン酸の異
性体はビー・ベツカー(B. Beaker)らの方式
〔アプライド・ミクロバイオロジー(Appl, Mi
crobiol.H2巻: 421 〜423(196
4) ]によって確認した。形態学的検討は、光学顕微
鏡を用いておこない、とくに胞子表面の形態については
走査型電子顕微鏡によった。色の名称の割当てには、カ
ラー・ハーモニー・マニsアル(Color flar
mony Manual)  Cコンティナー・コーポ
レーション・オブ・アメリカ(Container C
orporation  of America)、第
4版195B]を使用した。Do−116株の菌学的性
質は次の通りである。
The mycological properties of strain Do-116 will be described below. The determination of the properties is based on Streptomyces international
Methods recommended by the Strebtomyces project (ISP) for the characterization of Strebtomyces species. B. Shirling, Shirling and D. International Journal of Systematic Bacteriology (Int. J. Syst, Bacteriology) by Gottlieb et al.
l. ) 16. 313-340 (1966)). Isomers of diaminopimelic acid in whole cell hydrolysates were determined using the method of B. Beaker et al. [Applied Microbiology (Appl, Mi
crobiol. Volume H2: 421-423 (196
4)] was confirmed. Morphological examination was performed using an optical microscope, and in particular, the morphology of the spore surface was examined using a scanning electron microscope. The Color Harmony Manual is used to assign color names.
mony Manual) C Container Corporation of America (Container C
organization of America), 4th edition 195B] was used. The mycological properties of strain Do-116 are as follows.

(1)形態 気菌糸二分技する。(1) Form Aerial mycelium bisection technique.

基生菌糸二分技するが、分断はない。The basal hyphae bisect, but there is no division.

抱子:気菌糸上に分節胞子(10個から30個もしくは
さらに多数)の長い連鎖として 着生する。連鎖の形態は屈曲状もしく はループ状。
Clutches: Settle on aerial hyphae as long chains of segmented spores (10 to 30 or even more). The form of the chain is bent or looped.

胞子の表面:平滑(Smooth) 胞子の運動性:なし 胞子の形・大きさ:楕円形(0. 5X 0. 7μm
〉なお、菌核、胞子のうは観察されない。
Spore surface: Smooth Spore motility: None Spore shape/size: Oval (0.5 x 0.7 μm
〉No sclerotia or sporangia were observed.

(2)色調 気菌糸:灰色 基生閑糸:淡黄色〜黄茶色 可溶性色素:茶色 メラニン色素:なし (3)細胞壁の化学組成 ジアミノピメリン酸の立体型:LL型 (4)生理的性質 炭素源の同化性: 同化する:グルコース、キシロース、 イノシトール、マンニトール、 アラビノース、ラムノース、 ラフィノース、ラクトース シュークロース、サリシン、 ガラクトース 同化しない:なし ゼラチンの肢化:陰性 スターチの加水分解:陽性 脱脂牛乳の凝固:陰性 I 脱脂牛乳のベブトン化:陽性 繊維素の分解:陽性 生育温度範囲:16〜37℃(至適28〜32℃)なお
、生育温度範囲については2日後、ゼラチン、脱脂牛乳
および繊維素に対する作用については、28℃、1ケ月
後の結果を、そのほかの性質は28℃2週間後の結果を
示した。
(2) Color tone: Aerial hyphae: Gray base, blank thread: Pale yellow to yellowish brown Soluble pigment: Brown Melanin pigment: None (3) Chemical composition of cell wall Stereotype of diaminopimelic acid: LL type (4) Physiological properties Carbon source Anabolic properties: Assimilated: glucose, xylose, inositol, mannitol, arabinose, rhamnose, raffinose, lactose sucrose, salicin, galactose Not assimilated: None Gelatin limbification: Negative Starch hydrolysis: Positive Skimmed milk coagulation: Negative I Bebutonization of skim milk: Positive Decomposition of cellulose: Positive Growth temperature range: 16-37℃ (optimum 28-32℃) The growth temperature range is after 2 days, and the effect on gelatin, skim milk and cellulin is The results are shown after one month at 28°C, and the results for other properties are shown after two weeks at 28°C.

(5)各種寒天培地における生育状態 各種寒天培地テ28℃、28日間、Do−116株を培
養した結果を第4表に示す。
(5) Growth status on various agar media Table 4 shows the results of culturing strain Do-116 on various agar media at 28°C for 28 days.

なお、略号は次のとおり、 G:生育の程度、 AM=気菌糸の着生度および色調、 SM二基生菌糸の色調、 P:可溶性色素の色調。The abbreviations are as follows. G: Degree of growth; AM = degree of attachment and color tone of aerial mycelium; SM bibasic hyphae color tone, P: Color tone of soluble pigment.

(6)  Do−116株の同定 Do−116株はLL型のジアミノピメリン酸が検出さ
れることから、エム・ピー・レチェハリエとエイチ・エ
ー・レチェバリエ(M. P.Lechevalier
 and H,^. Lechevalier)による
放線菌の分類〔インターナショナル・ジャーナル・シス
テマティック・バクテリオロジ−(Int. J. S
yst.Bacteriol,> 20巻、435−4
43 (1970))によると細胞壁I型と分類される
。さらに該菌株の形態学的特徴を組み合わせると、この
菌株をストレプトマイセス・属に帰属させるのが適当で
ある。
(6) Identification of strain Do-116 Since LL-type diaminopimelic acid was detected in strain Do-116, M.P. Lechevalier and M.P. Lechevalier
and H, ^. Classification of actinomycetes by Lechevalier [International Journal Systematic Bacteriology (Int. J.S.
yst. Bacteriol, > vol. 20, 435-4
43 (1970)), it is classified as cell wall type I. Furthermore, in combination with the morphological characteristics of the strain, it is appropriate to assign this strain to the genus Streptomyces.

本属における種の同定においては、灰色系の気菌糸、屈
曲状もしくはループ状の胞子連鎖、平滑な胞子表面、メ
ラニン様色素非産生、可溶性色素の産土および炭素源の
資化パターンなどの本菌株の特徴をもとに、細菌学名承
認リスト〔ヴイー・ビー・ディー・スカーマン(V.B
.D. Skerman)らの、Inf, J,−Sy
st.ロacterio1. 30巻、 2  2  
5 〜4  2  0  (1980)]で承認されて
いる種名まり、分類的特徴が類似している種をISPの
記載[1nt.J.Syst,Bacteriol, 
18巻、69〜189、(1968)、同誌、18巻、
279〜392、(1968)、同誌、l9巻、391
〜512、(1969)、同誌、22巻、265〜39
4(1972) Jよびアール・イー・ブカナン(R,
 B, Buchanan)とエヌ・イー・ギボンズ(
N.E. Gibbons)編、バージーズ・マニュア
ル・オブ・デターミネイティブ・バクテリオロジイ(B
ergey s Manual ofロeter−mi
nat+veBacteriology)第8版〕で検
索した。
In order to identify species in this genus, this strain is characterized by its gray aerial hyphae, bent or looped spore chains, smooth spore surface, non-production of melanin-like pigments, soluble pigment production, and carbon source utilization pattern. An approved list of bacterial scientific names [V.B.D. Scarman (V.B.
.. D. Inf, J, -Sy of Skerman et al.
st. Roacterio1. Volume 30, 2 2
5-420 (1980)], species with similar taxonomic characteristics are listed in the ISP [1nt. J. Syst, Bacteriol,
18, 69-189, (1968), same magazine, 18,
279-392, (1968), same magazine, volume l9, 391
~512, (1969), same magazine, vol. 22, 265-39
4 (1972) J. and R.E. Buchanan (R,
B, Buchanan) and N.E. Gibbons (
N. E. Gibbons), Burgess Manual of Determinative Bacteriology (B
Ergey's Manual of Roeter-mi
nat+veBacteriology) 8th edition].

検索の結果、ストレプトマイセス・ニグラ(Strep
tomyces nigra)がD○−116株の近縁
の種として挙げられたが、種を特定することは困難であ
り、ストレプトマイセス・エスピー(Streptom
yces sp.) D○−116と命名した。
As a result of the search, Streptomyces nigra (Strep
tomyces nigra) was mentioned as a species closely related to strain D○-116, but it is difficult to identify the species, and Streptomyces sp.
yces sp. ) It was named D○-116.

該菌株はブダペスト条約に基づき、工業技術院微生物工
業技術研究所に微工研条寄第2559号(FERM  
OP−2559)として寄託されている(原寄託日:平
成元年8月22日)。
Based on the Budapest Treaty, the strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the FERM Report No. 2559 (FERM
OP-2559) (original deposit date: August 22, 1989).

次に培養法について述べる。Next, we will discuss the culture method.

本発明の培養においては通常の放線菌の培養法が一般に
用いられる。培地としては資化可能な炭素源、窒素源、
無機物および必要な生育、生産促進物質を程よく含有す
る培地であれば合或培地、天然培地いずれでも使用可能
である。
In the cultivation of the present invention, ordinary methods for culturing actinomycetes are generally used. As a medium, assimilable carbon sources, nitrogen sources,
Either a synthetic medium or a natural medium can be used as long as it contains appropriate amounts of inorganic substances and necessary growth and production promoting substances.

炭素源としてはグルコース、澱粉、デキストリン、マン
ノース、フラクトース、シュクロース、ラクトース、キ
シロース、アラビノース、マンニトール、糖蜜などを単
独または組み合わせて用いられる。さらに、菌の資化能
によっては炭化水素、アルコール類、有機酸なども用い
られる。
As the carbon source, glucose, starch, dextrin, mannose, fructose, sucrose, lactose, xylose, arabinose, mannitol, molasses, etc. are used alone or in combination. Furthermore, depending on the assimilation ability of the bacteria, hydrocarbons, alcohols, organic acids, etc. may also be used.

窒素源としては塩化アンモニウム、硫酸アンモニウム、
硝酸アンモニウム、硝酸ナトリウム、尿素、ベプトン、
肉エキス、酵母エキス、乾燥酵母、コーン・スチーブ・
リカー大豆扮、カザミノ酸などが単独または組み合わせ
て用いられる。
Ammonium chloride, ammonium sulfate,
Ammonium nitrate, sodium nitrate, urea, beptone,
Meat extract, yeast extract, dry yeast, corn/steve/
Soybean liquor, casamino acids, etc. are used alone or in combination.

そのほか、必要に応じて食塩、塩化カリウム、硫酸マグ
ネシウム、炭酸カルシウム、リン酸二水素カリウム、リ
ン酸水素二カリウム、硫酸第一鉄、塩化カルシウム、硫
酸マンガン、硫酸亜鉛、硫酸銅などの無機塩類を加える
In addition, inorganic salts such as table salt, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate, calcium chloride, manganese sulfate, zinc sulfate, copper sulfate, etc. Add.

さらに使用菌の生育やDC116の生産を促進する微量
戊分を適当に添加することができる。
Furthermore, a trace amount of fertilization which promotes the growth of the bacteria used and the production of DC116 can be appropriately added.

培養法としては、液体培養法、とくに深部攪拌培養法が
もっとも適している。培養は、温度16〜37℃、好ま
しくは25〜32℃、pH4〜10、好ましくは6〜8
でおこなわれ、通常1〜7日で完了し、目的物質DCl
16が培養液中および菌体中に生戒蓄積される。
The most suitable culture method is a liquid culture method, especially a deep agitation culture method. The culture is carried out at a temperature of 16 to 37°C, preferably 25 to 32°C, and a pH of 4 to 10, preferably 6 to 8.
It is usually completed in 1 to 7 days, and the target substance DCl
16 is accumulated in the culture solution and in the bacterial cells.

培地のpH.1m整にはアンモニア水や炭酸アンモニウ
ム溶液などが用いられる。
Medium pH. Ammonia water, ammonium carbonate solution, etc. are used for 1m adjustment.

培養物中の生成量が最大に達したときに培養を停止する
The culture is stopped when the maximum amount of production in the culture is reached.

培養物からDC116の単離精製は、微生物代謝生産物
をその培養物から単離精製するために常用される方法に
従っておこなわれる。
Isolation and purification of DC116 from the culture is carried out according to methods commonly used for isolating and purifying microbial metabolic products from the culture.

たとえば培養物を一過により培養枦液と菌体に分け、菌
体をクロロホルム、アセトンなどで抽出する。ついで、
抽出液と培養枦液とを合わせて、ポリスチレン系吸着剤
たとえばダイヤイオンHP20(三菱化成社製〉などに
通塔して活性或分を吸着させ、ついで酢酸エチル、アセ
トンなどで溶出する。溶出液を濃縮し、シリカゲルによ
るカラムクロマトグラフィー、高速液体クロマトグラフ
ィーなどにより、DC 1 1 6の赤色粉末を得る。
For example, a culture is separated into culture fluid and bacterial cells by passing, and the bacterial cells are extracted with chloroform, acetone, or the like. Then,
The extract and culture solution are combined and passed through a polystyrene adsorbent such as Diaion HP20 (manufactured by Mitsubishi Kasei Corporation) to adsorb the active fraction, and then eluted with ethyl acetate, acetone, etc. Eluate is concentrated and subjected to column chromatography using silica gel, high performance liquid chromatography, etc. to obtain a red powder of DC 1 16.

なお、培養、精製摸作中のDC116の勤向はバチルス
・ズブチリスNal0707を用いるパイ才アッセイ、
または薄層クロマトグラフィーによるDC116の紫外
線吸収を目安として追跡することができる。
In addition, the concentration of DC116 during culture and purification was performed using a pie assay using Bacillus subtilis Nal0707.
Alternatively, it can be tracked using DC116 ultraviolet absorption by thin layer chromatography as a guide.

以下に本発明の実施例および参考例を示す。Examples and reference examples of the present invention are shown below.

実施例1. 種菌としてストレプトマイセス・エスビーD○116を
用いる。該菌株を21容量の三角フラスコ中のバクト・
トリブトン(Difco社製)5g/C酵母エキス5g
/j!,肉エキス3gz!,可溶性澱粉10g/j!,
グルコース10g/1、炭酸カルシウム5g/I!の組
或からなる種培地(殺菌前pH7. 2)300mlに
植菌し、30℃で48時間振盪(200rpm)培養し
た。
Example 1. Streptomyces SB D○116 is used as the inoculum. The strain was transferred to Bacto in a 21 volume Erlenmeyer flask.
Tributone (manufactured by Difco) 5g/C yeast extract 5g
/j! , Meat extract 3gz! , Soluble starch 10g/j! ,
Glucose 10g/1, calcium carbonate 5g/I! The cells were inoculated into 300 ml of a seed medium (pH 7.2 before sterilization) consisting of a combination of the above and cultured at 30° C. for 48 hours with shaking (200 rpm).

得られた種培養液を301l容培養槽中の下記組或から
なる発酵培地15AにlO%{容量}の割合で移し、2
8℃で通気攪拌方式(回転数200rpm,通気1tl
5j’/min)により培養をおこなった。
The obtained seed culture solution was transferred to a fermentation medium 15A consisting of the following composition in a 301-liter culture tank at a ratio of 10% {volume}, and
Aeration stirring method (rotation speed 200 rpm, aeration 1 tl) at 8℃
Culture was performed at a rate of 5j'/min).

発酵培地組戊:グリセロール25g/Cグルコース25
g/R,乾燥酵母1 5 g / R , KH2P0
40. 5 g / f ,  MgSL・7H20 
 0. 5 g/ l ,炭酸カルシウム5g/l(殺
菌前pH7.0、Na叶で調整〉培養中、培地のρHは
とくに制御しないで、150時間培養した。培養液にn
−プロバノール15jl!を添加し攪拌した後、培養物
から菌体および沈殿口物を枦別し、ρ液284’を得た
Fermentation medium composition: Glycerol 25g/C glucose 25
g/R, dry yeast 15 g/R, KH2P0
40. 5 g/f, MgSL・7H20
0. 5 g/l, calcium carbonate 5 g/l (pH 7.0 before sterilization, adjusted with Na leaf) During cultivation, the ρH of the medium was not particularly controlled, and the culture was carried out for 150 hours.
-Probanol 15jl! After adding and stirring, the bacterial cells and precipitate were separated from the culture to obtain ρ liquid 284'.

枦液を濃縮後、水で希釈しポリスチレン系吸着剤ダイヤ
イオンHP20(IOIl)のカラムに通塔して活性物
質を吸着させた。
After concentrating the honeycomb solution, it was diluted with water and passed through a column of polystyrene adsorbent Diaion HP20 (IOIl) to adsorb the active substance.

脱イオン水および50%メタノールで不純物を溶出後酢
酸エチルで活性物質を溶出した。活性画分を為縮後、水
を加えダイヤイオン}IP20SS (2 J )のカ
ラムに通塔し、活性物質を吸着させた。
Impurities were eluted with deionized water and 50% methanol, and then the active material was eluted with ethyl acetate. After condensing the active fraction, water was added and the mixture was passed through a Diaion IP20SS (2 J) column to adsorb the active substance.

50%メタノールで不純物を溶出後90%メタノールで
活性物質を溶出させた。活性画分を濃縮後、酢酸エチル
で抽出した。
After eluting impurities with 50% methanol, the active substance was eluted with 90% methanol. The active fraction was concentrated and then extracted with ethyl acetate.

酢酸エチル層を濃縮後、セファデックスL}120にか
けメタノールで溶出させた。活性物質を含む画分を濃縮
し、得られた物質を次に逆相系シリカゲル(YMC O
DS SH−363−5、ワイエムシー社製)を用い、
80%メタノールを溶媒として高速液体クロマトグラフ
ィーをおこなった。DC116を含む両分をクロロホル
ムで抽出後、濃縮乾固し、DC116の赤色粉末30m
gを得た。
After concentrating the ethyl acetate layer, it was applied to Sephadex L}120 and eluted with methanol. The fractions containing the active substance were concentrated, and the resulting material was then purified on reversed-phase silica gel (YMCO
DS SH-363-5, manufactured by YMC Corporation),
High performance liquid chromatography was performed using 80% methanol as a solvent. After extracting both parts containing DC116 with chloroform, they were concentrated to dryness to obtain 30ml of red powder of DC116.
I got g.

発明の効果 本発明により抗菌、抗腫瘍活性を有する新規発酵生産物
Dell6が提供される。
Effects of the Invention The present invention provides a novel fermentation product Dell6 having antibacterial and antitumor activities.

Claims (1)

【特許請求の範囲】[Claims] 下記の一般式で示される新規物質DC116▲数式、化
学式、表等があります▼
New substance DC116 shown by the general formula below ▲ Numerical formulas, chemical formulas, tables, etc. are available ▼
JP1233342A 1989-09-08 1989-09-08 New substance DC116 Expired - Fee Related JP2786267B2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP1233342A JP2786267B2 (en) 1989-09-08 1989-09-08 New substance DC116

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP1233342A JP2786267B2 (en) 1989-09-08 1989-09-08 New substance DC116

Publications (2)

Publication Number Publication Date
JPH0394693A true JPH0394693A (en) 1991-04-19
JP2786267B2 JP2786267B2 (en) 1998-08-13

Family

ID=16953648

Family Applications (1)

Application Number Title Priority Date Filing Date
JP1233342A Expired - Fee Related JP2786267B2 (en) 1989-09-08 1989-09-08 New substance DC116

Country Status (1)

Country Link
JP (1) JP2786267B2 (en)

Also Published As

Publication number Publication date
JP2786267B2 (en) 1998-08-13

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